dna polymerase  (New England Biolabs)


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    Name:
    DNA Polymerase I E coli
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    DNA Polymerase I E coli 2 500 units
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    m0209l
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    2 500 units
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    DNA Polymerases
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    New England Biolabs dna polymerase
    DNA Polymerase I E coli
    DNA Polymerase I E coli 2 500 units
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    Average 95 stars, based on 964 article reviews
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    dna polymerase - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.
    Figure Legend Snippet: End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Techniques Used: Incubation, Labeling, Recombinant, Agarose Gel Electrophoresis, Autoradiography

    2) Product Images from "Nucleic acid evolution and minimization by nonhomologous random recombination"

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    Journal: Nature biotechnology

    doi: 10.1038/nbt736

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Figure Legend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Techniques Used: Ligation, Binding Assay

    3) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    4) Product Images from "Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69"

    Article Title: Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69

    Journal: Journal of Bacteriology

    doi:

    Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.
    Figure Legend Snippet: Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.

    Techniques Used: Library Screening, Labeling, Recombinant, Generated, Purification, Polymerase Chain Reaction, Amplification

    Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).
    Figure Legend Snippet: Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).

    Techniques Used: Plasmid Preparation, Lysis

    Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.
    Figure Legend Snippet: Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Amplification, SDS Page

    5) Product Images from "Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69"

    Article Title: Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69

    Journal: Journal of Bacteriology

    doi:

    Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.
    Figure Legend Snippet: Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.

    Techniques Used: Library Screening, Labeling, Recombinant, Generated, Purification, Polymerase Chain Reaction, Amplification

    Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).
    Figure Legend Snippet: Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).

    Techniques Used: Plasmid Preparation, Lysis

    Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.
    Figure Legend Snippet: Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Amplification, SDS Page

    6) Product Images from "Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification"

    Article Title: Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.5.2339-2344.2005

    (A) RCA detection using 20 pmol of circularizable probe and 10 11 artificial DNA and RNA template. Six of seven probes were shown to detect their corresponding artificial template (Table ), the negative signal (OCP-E) could be due to
    Figure Legend Snippet: (A) RCA detection using 20 pmol of circularizable probe and 10 11 artificial DNA and RNA template. Six of seven probes were shown to detect their corresponding artificial template (Table ), the negative signal (OCP-E) could be due to

    Techniques Used:

    Pictorial representation of the RCA method. (A) Padlock probe containing target-complementary segment hybridization to a target DNA or RNA sequence. (B) The padlock probe can be circularized by DNA ligase. (C) Ligated probe and binding of complementary
    Figure Legend Snippet: Pictorial representation of the RCA method. (A) Padlock probe containing target-complementary segment hybridization to a target DNA or RNA sequence. (B) The padlock probe can be circularized by DNA ligase. (C) Ligated probe and binding of complementary

    Techniques Used: Hybridization, Sequencing, Binding Assay

    7) Product Images from "Enhanced Protocol for Determining the 3? Terminus of Hepatitis C Virus"

    Article Title: Enhanced Protocol for Determining the 3? Terminus of Hepatitis C Virus

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2010.03.030

    Amplification of the HCV 3′UTR by ligation-mediated RT-PCR. Three linkers were estimated for their efficiency in the ligation and subsequent RT-PCR. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M1, 100 bp DNA ladder (Invitrogen). M2, 123 bp DNA ladder (Invitrogen).
    Figure Legend Snippet: Amplification of the HCV 3′UTR by ligation-mediated RT-PCR. Three linkers were estimated for their efficiency in the ligation and subsequent RT-PCR. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M1, 100 bp DNA ladder (Invitrogen). M2, 123 bp DNA ladder (Invitrogen).

    Techniques Used: Amplification, Ligation, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Estimation of processing activity and fidelity of 5 DNA polymerases in the amplification of ~130 bp poly(T/TG) domain. The plasmid p90H/FL pol- consisting of the full-length HCV genome was used as the PCR template. A series of 1:10 dilution of the plasmid template, starting at 4 ng, was applied and matched to lanes 1, 2, 3 and 4, respectively. Lane 5, negative control. M, 123 DNA ladder (Invitrogen).
    Figure Legend Snippet: Estimation of processing activity and fidelity of 5 DNA polymerases in the amplification of ~130 bp poly(T/TG) domain. The plasmid p90H/FL pol- consisting of the full-length HCV genome was used as the PCR template. A series of 1:10 dilution of the plasmid template, starting at 4 ng, was applied and matched to lanes 1, 2, 3 and 4, respectively. Lane 5, negative control. M, 123 DNA ladder (Invitrogen).

    Techniques Used: Activity Assay, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Negative Control

    Amplification of the HCV poly(U/UC) tract and 3′ X tail. Three reverse primers, located at different sites of 3′ X tail, were used for RT and the first round PCR. While the RT-PCR with either 3HBVR1UTR2 or 3HBVR1UTR3 generated DNA bands with expected sizes, ~300 and 324 bp, respectively, the primer 3HBVR1UTR1, located on the uttermost part of 3′ X tail, gave a much smaller product (~123 bp), indicating the existence of potential replication slippage. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M, 123 bp DNA ladder (Invitrogen).
    Figure Legend Snippet: Amplification of the HCV poly(U/UC) tract and 3′ X tail. Three reverse primers, located at different sites of 3′ X tail, were used for RT and the first round PCR. While the RT-PCR with either 3HBVR1UTR2 or 3HBVR1UTR3 generated DNA bands with expected sizes, ~300 and 324 bp, respectively, the primer 3HBVR1UTR1, located on the uttermost part of 3′ X tail, gave a much smaller product (~123 bp), indicating the existence of potential replication slippage. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M, 123 bp DNA ladder (Invitrogen).

    Techniques Used: Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Generated, Negative Control

    8) Product Images from "DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide"

    Article Title: DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150713111

    DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]
    Figure Legend Snippet: DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]

    Techniques Used: Generated, Nick Translation

    9) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Techniques Used: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Techniques Used: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    10) Product Images from "Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs"

    Article Title: Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm017

    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
    Figure Legend Snippet: Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

    Techniques Used: Synthesized, Molecular Weight, Clone Assay

    11) Product Images from "Functional roles for the GerE-family carboxyl-terminal domains of nitrate response regulators NarL and NarP of Escherichia coli K-12"

    Article Title: Functional roles for the GerE-family carboxyl-terminal domains of nitrate response regulators NarL and NarP of Escherichia coli K-12

    Journal: Microbiology

    doi: 10.1099/mic.0.040469-0

    NarL and TraR CTD sequences. The four α -helices include the central HTH element. Results for TraR are from Qin et al. (2009) and White Winans (2005) . Boxed residues indicate phenotypes for Ala substitutions: black, PC; white, functional; bold type and outline, deficient. Grey-shaded boxes indicate positions where substitution with residues other than Ala results in the PC phenotype. Residues in bold type are implicated in direct recognition of DNA ( Maris et al. , 2005 ; White Winans, 2007 ). The TraR-CTD sequence shown is from plasmid pTiR10 ( White Winans, 2005 ); the TraR-CTD sequence from plasmid pTiC58 differs at three positions (Val-168, Met-189 and Val-194) ( Qin et al. , 2009 ).
    Figure Legend Snippet: NarL and TraR CTD sequences. The four α -helices include the central HTH element. Results for TraR are from Qin et al. (2009) and White Winans (2005) . Boxed residues indicate phenotypes for Ala substitutions: black, PC; white, functional; bold type and outline, deficient. Grey-shaded boxes indicate positions where substitution with residues other than Ala results in the PC phenotype. Residues in bold type are implicated in direct recognition of DNA ( Maris et al. , 2005 ; White Winans, 2007 ). The TraR-CTD sequence shown is from plasmid pTiR10 ( White Winans, 2005 ); the TraR-CTD sequence from plasmid pTiC58 differs at three positions (Val-168, Met-189 and Val-194) ( Qin et al. , 2009 ).

    Techniques Used: Functional Assay, Sequencing, Plasmid Preparation

    12) Product Images from "Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance"

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn575

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Figure Legend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    13) Product Images from "Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences"

    Article Title: Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences

    Journal:

    doi: 10.1080/14653240802192636

    Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic DNA HeLa cell clone was subjected to FLEA-PCR using T7 DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each
    Figure Legend Snippet: Effects of blocking oligonucleotides on FLEA-PCR-amplified libraries. A genomic DNA HeLa cell clone was subjected to FLEA-PCR using T7 DNA polymerase with and without addition of internal sequences blocking oligonucleotide. A plasmid library for each

    Techniques Used: Blocking Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    14) Product Images from "DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes"

    Article Title: DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg863

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.
    Figure Legend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.

    Techniques Used: Activity Assay, Inhibition, DNA Synthesis

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.
    Figure Legend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.

    Techniques Used: Activity Assay

    15) Product Images from "Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs"

    Article Title: Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1092

    ( A ) The recognition sequences of BtsCI and related REases are aligned at the common TG bases (underlined). The common 2-base 3′ overhangs are highlighted. Arrowheads indicate the cleavage sites. ( B ) Amino acid sequence alignments of the two catalytic sites of BtsCI and related REases. Identical residues are shown as white on black. The identity indicates the percentage of amino acid sequence identity between BtsCI and related REases. The DNA sequence of BtsCI R-M system has been deposited in GenBank with accession no GQ449683.
    Figure Legend Snippet: ( A ) The recognition sequences of BtsCI and related REases are aligned at the common TG bases (underlined). The common 2-base 3′ overhangs are highlighted. Arrowheads indicate the cleavage sites. ( B ) Amino acid sequence alignments of the two catalytic sites of BtsCI and related REases. Identical residues are shown as white on black. The identity indicates the percentage of amino acid sequence identity between BtsCI and related REases. The DNA sequence of BtsCI R-M system has been deposited in GenBank with accession no GQ449683.

    Techniques Used: Sequencing

    DNA nicking activity of top-strand nicking BtsCI variant. Two-fold serial dilutions of the cell extract of BtsCI nicking variant E128F were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.
    Figure Legend Snippet: DNA nicking activity of top-strand nicking BtsCI variant. Two-fold serial dilutions of the cell extract of BtsCI nicking variant E128F were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.

    Techniques Used: Activity Assay, Variant Assay, Incubation, Agarose Gel Electrophoresis

    Run-off sequencing of nicked products by BtsI nicking variants. The cleavage product of mutants E128F ( A ) and D388A/E403A/E405A ( B ) were gel-purified and subjected to Sanger sequencing on both strands. The recognition sequence is bolded and the cleavage site was indicated by arrows. The extra A peak at the end of run-off sequence was added by the template independent terminal transferase activity of Taq DNA polymerase during sequencing. The extra A peak and drop off in peak signal indicate the nicking site (cleaved template).
    Figure Legend Snippet: Run-off sequencing of nicked products by BtsI nicking variants. The cleavage product of mutants E128F ( A ) and D388A/E403A/E405A ( B ) were gel-purified and subjected to Sanger sequencing on both strands. The recognition sequence is bolded and the cleavage site was indicated by arrows. The extra A peak at the end of run-off sequence was added by the template independent terminal transferase activity of Taq DNA polymerase during sequencing. The extra A peak and drop off in peak signal indicate the nicking site (cleaved template).

    Techniques Used: Sequencing, Purification, Activity Assay

    DNA nicking activity of the bottom-strand nicking BtsCI variant. Two-fold serial dilutions of the crude extract of BtsCI nicking variant (D388A/E403A/E405A) were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.
    Figure Legend Snippet: DNA nicking activity of the bottom-strand nicking BtsCI variant. Two-fold serial dilutions of the crude extract of BtsCI nicking variant (D388A/E403A/E405A) were incubated with pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled.

    Techniques Used: Activity Assay, Variant Assay, Incubation, Agarose Gel Electrophoresis

    DNA nicking activity of BtsCI mutants. Two-fold serial dilutions of the clarified cell extracts of E. coli cultures that expressed the indicated BtsCI mutants were incubated with 0.5 µg of pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled; −, no cleavage; +, pUC19 nicked by Nt.BsmAI.
    Figure Legend Snippet: DNA nicking activity of BtsCI mutants. Two-fold serial dilutions of the clarified cell extracts of E. coli cultures that expressed the indicated BtsCI mutants were incubated with 0.5 µg of pUC19 as described in ‘Materials and Methods’ section. The cleavage products were analyzed on a 1% agarose gel. OC, open circle; SC, supercoiled; −, no cleavage; +, pUC19 nicked by Nt.BsmAI.

    Techniques Used: Activity Assay, Incubation, Agarose Gel Electrophoresis

    ( A ) Generation of overhangs. Cleavage sites for BtsCI, FokI and a combination of FokI/Nt.BtsCI and FokI/Nb.BtsCI are indicated. For BtsCI, a 2-nt 5′ recessive end is generated. For FokI, a 4-nt 3′ recessive end is generated. For FokI/Nt.BtsCI, an 11-nt 3′ recessive end is generated. For FokI/Nb.BtsCI, a 9-nt 5′ recessive end is generated. Grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites. ( B ) Annealing of oligonucleotides to the long overhangs for PCR. Plasmid pUC19 was cleaved by the indicated enzyme(s) and then ligated to a 100-bp long oligonucleotide as described in ‘Materials and Methods’ section. The ligation products were used as template for PCR that specifically detect the ligated DNA. Only the cleavage product of FokI/Nt.BtsCI can be annealed to the 11-nt 3′ recessive end oligonucleotide (left panel), whereas only the cleavage product of FokI/Nb.BtsCI can be annealed to the 9-nt 5′ recessive end oligonucleotide (right panel).
    Figure Legend Snippet: ( A ) Generation of overhangs. Cleavage sites for BtsCI, FokI and a combination of FokI/Nt.BtsCI and FokI/Nb.BtsCI are indicated. For BtsCI, a 2-nt 5′ recessive end is generated. For FokI, a 4-nt 3′ recessive end is generated. For FokI/Nt.BtsCI, an 11-nt 3′ recessive end is generated. For FokI/Nb.BtsCI, a 9-nt 5′ recessive end is generated. Grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites. ( B ) Annealing of oligonucleotides to the long overhangs for PCR. Plasmid pUC19 was cleaved by the indicated enzyme(s) and then ligated to a 100-bp long oligonucleotide as described in ‘Materials and Methods’ section. The ligation products were used as template for PCR that specifically detect the ligated DNA. Only the cleavage product of FokI/Nt.BtsCI can be annealed to the 11-nt 3′ recessive end oligonucleotide (left panel), whereas only the cleavage product of FokI/Nb.BtsCI can be annealed to the 9-nt 5′ recessive end oligonucleotide (right panel).

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation, Ligation

    DNA labeling by end filling of the 11-nt 3′ recessive end. The 11-nt 3′ recessive end was generated by FokI/Nt.BtsCI. Strong fluorescent signal detected at the smaller DNA fragment (lower band) digested by FokI/Nt.BtsCI. The expected cleavage products and the 3′ recessive ends that can be labeled by end filling are illustrated at the lower panel. Green blocks, fluorescent labeled region; grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites.
    Figure Legend Snippet: DNA labeling by end filling of the 11-nt 3′ recessive end. The 11-nt 3′ recessive end was generated by FokI/Nt.BtsCI. Strong fluorescent signal detected at the smaller DNA fragment (lower band) digested by FokI/Nt.BtsCI. The expected cleavage products and the 3′ recessive ends that can be labeled by end filling are illustrated at the lower panel. Green blocks, fluorescent labeled region; grey arrows, BtsCI top-strand cleavage site; white arrows, BtsCI bottom-strand cleavage site; black arrows, FokI cleavage sites.

    Techniques Used: DNA Labeling, Generated, Labeling

    16) Product Images from "The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection"

    Article Title: The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp534

    Determination of RpaB5I recognition site. ( A ) An agarose gel showing the products of RpaB5I digestion of various DNAs. The putative recognition sequence was derived from the positions of the cut sites and analysis of the pBR322 and pBC4 DNA sequences as described in ‘Materials and Methods’ section. Digestion of lambda, T7 and T3 phage DNAs served to verify the predicted specificity. Lane 1: lambda DNA, lane 2: T7 DNA, lane 3: T3 DNA. Lanes 4–8: pBR322 DNA cut by RpaB5I and: lane 4: RpaB5I only, lane 5: EcoRV, lane 6: BsmI, lane 7: NdeI, lane 8: PstI. Lanes 9–15: pBC4 DNA cut by RpaB5I and: lane 9: RpaB5I only, lane 10: NdeI, lane 11: AvrII, lane 12: PmeI, lane 13: AscI, lane 14: SpeI, lane 15: EcoRV. Lanes M are HindIII-lambda and HaeIII-PhiX174 DNA size standards. ( B ) Computer generated digestion patterns for cleavage at the predicted RpaB5I recognition sequence (5′-CGRGGAC-3′). Lane 1: lambda DNA, lane 2: T7 DNA, lane 3: T3 DNA.
    Figure Legend Snippet: Determination of RpaB5I recognition site. ( A ) An agarose gel showing the products of RpaB5I digestion of various DNAs. The putative recognition sequence was derived from the positions of the cut sites and analysis of the pBR322 and pBC4 DNA sequences as described in ‘Materials and Methods’ section. Digestion of lambda, T7 and T3 phage DNAs served to verify the predicted specificity. Lane 1: lambda DNA, lane 2: T7 DNA, lane 3: T3 DNA. Lanes 4–8: pBR322 DNA cut by RpaB5I and: lane 4: RpaB5I only, lane 5: EcoRV, lane 6: BsmI, lane 7: NdeI, lane 8: PstI. Lanes 9–15: pBC4 DNA cut by RpaB5I and: lane 9: RpaB5I only, lane 10: NdeI, lane 11: AvrII, lane 12: PmeI, lane 13: AscI, lane 14: SpeI, lane 15: EcoRV. Lanes M are HindIII-lambda and HaeIII-PhiX174 DNA size standards. ( B ) Computer generated digestion patterns for cleavage at the predicted RpaB5I recognition sequence (5′-CGRGGAC-3′). Lane 1: lambda DNA, lane 2: T7 DNA, lane 3: T3 DNA.

    Techniques Used: Agarose Gel Electrophoresis, Sequencing, Derivative Assay, Lambda DNA Preparation, Generated

    PspOMII digestion of DNAs containing one strand from native genomic P. pseudomonas DNA and one newly synthesized (unmodified) strand. Lanes 2–5 are newly synthesized top strand with genomic bottom strand, while lanes 6–10 are the newly synthesized bottom strand with genomic top strand. Lane 2: uncut, lane 3: four units PspOMII, lane 4: two units PspOMII, lane 5: 10 units BanII. Lane 6: uncut, lane 7: four units PspOMII, lane 8: two units PspOMII, lane 9: four units PspOMII digestion of mixed newly synthesized bottom strand and top strand DNA (as a positive control for PspOMII activity), lane 10: 10 units BanII. Lanes 1 and 11: PhiX174-HaeIII size standard. PspOMII cuts the DNA containing a genomic P. species OM2164 bottom strand and an unmodified top strand (lanes 3, 4 and 9), but not the DNA containing a genomic P. species OM2164 top strand and an unmodified bottom strand (lanes 7, 8 and 9). The native host DNA from P. species OM2164 is thus modified to prevent PspOMII cleavage only in the top DNA strand (5′-CGCCCAR-3′') of the PspOMII recognition sequence.
    Figure Legend Snippet: PspOMII digestion of DNAs containing one strand from native genomic P. pseudomonas DNA and one newly synthesized (unmodified) strand. Lanes 2–5 are newly synthesized top strand with genomic bottom strand, while lanes 6–10 are the newly synthesized bottom strand with genomic top strand. Lane 2: uncut, lane 3: four units PspOMII, lane 4: two units PspOMII, lane 5: 10 units BanII. Lane 6: uncut, lane 7: four units PspOMII, lane 8: two units PspOMII, lane 9: four units PspOMII digestion of mixed newly synthesized bottom strand and top strand DNA (as a positive control for PspOMII activity), lane 10: 10 units BanII. Lanes 1 and 11: PhiX174-HaeIII size standard. PspOMII cuts the DNA containing a genomic P. species OM2164 bottom strand and an unmodified top strand (lanes 3, 4 and 9), but not the DNA containing a genomic P. species OM2164 top strand and an unmodified bottom strand (lanes 7, 8 and 9). The native host DNA from P. species OM2164 is thus modified to prevent PspOMII cleavage only in the top DNA strand (5′-CGCCCAR-3′') of the PspOMII recognition sequence.

    Techniques Used: Synthesized, Positive Control, Activity Assay, Modification, Sequencing

    Cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing a specific recognition site. ( A ) No in trans DNA. RpaB5I digestion in a 2-fold serial dilution from 2 units/µg DNA to 0.25 units/µg DNA on pBR322 DNA previously linearized by digestion with PstI. ( B ) Forty nanomolar in trans DNA containing an RpaB5I recognition site. The same reaction conditions as (A) supplemented with 40 nM of a 30 bp in trans DNA containing the RpaB5I recognition site. ( C ) 2-fold dilution series of the in trans DNA containing the RpaB5I recognition site, from 40 nM to 0.625 nM, in reactions containing two units RpaB5I and 1 µg PstI-linearized pBR322 per 50 µl reaction (7 nM RpaB5I sites). ( D ) The same reaction conditions as (A) supplemented with 40 nM of a 30 bp in trans DNA lacking the RpaB5I recognition site.
    Figure Legend Snippet: Cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing a specific recognition site. ( A ) No in trans DNA. RpaB5I digestion in a 2-fold serial dilution from 2 units/µg DNA to 0.25 units/µg DNA on pBR322 DNA previously linearized by digestion with PstI. ( B ) Forty nanomolar in trans DNA containing an RpaB5I recognition site. The same reaction conditions as (A) supplemented with 40 nM of a 30 bp in trans DNA containing the RpaB5I recognition site. ( C ) 2-fold dilution series of the in trans DNA containing the RpaB5I recognition site, from 40 nM to 0.625 nM, in reactions containing two units RpaB5I and 1 µg PstI-linearized pBR322 per 50 µl reaction (7 nM RpaB5I sites). ( D ) The same reaction conditions as (A) supplemented with 40 nM of a 30 bp in trans DNA lacking the RpaB5I recognition site.

    Techniques Used: Serial Dilution

    Cleavage of a multiple site substrate by NmeAIII. ( A ) 2-fold serial dilution series of NmeAIII digestion of pBR322 DNA (three NmeAIII sites), from 32 units per 50 µl reaction to 0.5 units per 50 µl reaction. ( B ) 2-fold dilution series of an in trans DNA containing the NmeAIII recognition site, from 640 nM to 20 nM, in reactions containing 16 units NmeAIII and 1 µg pBR322 per 50 µl reaction (21 nM NmeAIIII sites).
    Figure Legend Snippet: Cleavage of a multiple site substrate by NmeAIII. ( A ) 2-fold serial dilution series of NmeAIII digestion of pBR322 DNA (three NmeAIII sites), from 32 units per 50 µl reaction to 0.5 units per 50 µl reaction. ( B ) 2-fold dilution series of an in trans DNA containing the NmeAIII recognition site, from 640 nM to 20 nM, in reactions containing 16 units NmeAIII and 1 µg pBR322 per 50 µl reaction (21 nM NmeAIIII sites).

    Techniques Used: Serial Dilution

    17) Product Images from "Site-specific strand breaks in RNA produced by 125I radiodecay"

    Article Title: Site-specific strand breaks in RNA produced by 125I radiodecay

    Journal: Nucleic Acids Research

    doi:

    Binding of antisense oligonucleotides to target RNA and DNA analyzed by 12% native PAGE. Lanes 3 and 7, [ 32 P]DNA and [ 32 P]RNA targets, respectively; lanes 1, 2, 5 and 6, [ 32 P]DNA and [ 32 P]RNA targets incubated with [ 125 I]AO-I (lanes 2 and 6 with DMSO); lanes 4 and 8, [ 32 P]DNA and [ 32 P]RNA targets incubated with excess cold AO-I.
    Figure Legend Snippet: Binding of antisense oligonucleotides to target RNA and DNA analyzed by 12% native PAGE. Lanes 3 and 7, [ 32 P]DNA and [ 32 P]RNA targets, respectively; lanes 1, 2, 5 and 6, [ 32 P]DNA and [ 32 P]RNA targets incubated with [ 125 I]AO-I (lanes 2 and 6 with DMSO); lanes 4 and 8, [ 32 P]DNA and [ 32 P]RNA targets incubated with excess cold AO-I.

    Techniques Used: Binding Assay, Clear Native PAGE, Incubation

    Analysis of break distribution in target DNA and RNA. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]-AO-II; lane 3, [ 32 P]DNA with [ 125 I]AO-I; lane 4, Maxam–Gilbert G sequencing line of [ 32 P]DNA; lane 5, [ 32 P]RNA; lane 6, [ 32 P]RNA with 125 I-AO-II; lane 7, [ 32 P]RNA with [ 125 I]-AO-I; lane 8, RNase T1 G sequencing line of [ 32 P]RNA. ( B ) Percentages of breaks at individual bases of the target molecules calculated from the data of the gel in (A) for DNA (open squares) and RNA (filled circles).
    Figure Legend Snippet: Analysis of break distribution in target DNA and RNA. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]-AO-II; lane 3, [ 32 P]DNA with [ 125 I]AO-I; lane 4, Maxam–Gilbert G sequencing line of [ 32 P]DNA; lane 5, [ 32 P]RNA; lane 6, [ 32 P]RNA with 125 I-AO-II; lane 7, [ 32 P]RNA with [ 125 I]-AO-I; lane 8, RNase T1 G sequencing line of [ 32 P]RNA. ( B ) Percentages of breaks at individual bases of the target molecules calculated from the data of the gel in (A) for DNA (open squares) and RNA (filled circles).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing

    Strand breaks in target DNA and RNA from two 125 I atoms incorporated in antisense oligonucleotides. ( A ) Scheme of the target and antisense oligonucleotides. ( B ) Autoradiogram of the 12% urea–PAGE showing RNA fragments after incubation of duplexes at –70°C (lanes 1–8) and at +5°C (lanes 9–12). Lanes 1, 3, 5, 7, 9 and 11, RNA targets without [ 125 I]oligonucleotides; lanes 2, 4, 6, 8, 10 and 12, RNA targets with [ 125 I]oligonucleotides, complementary AO-II (lanes 2, 4, 10 and 12) and non-complimentary AO-N (lanes 6 and 8). Samples in lanes 3, 4, 7, 8, 11 and 12 contained 10% DMSO. Lane 13, [ 32 P]DNA marker, 8–32 nt.
    Figure Legend Snippet: Strand breaks in target DNA and RNA from two 125 I atoms incorporated in antisense oligonucleotides. ( A ) Scheme of the target and antisense oligonucleotides. ( B ) Autoradiogram of the 12% urea–PAGE showing RNA fragments after incubation of duplexes at –70°C (lanes 1–8) and at +5°C (lanes 9–12). Lanes 1, 3, 5, 7, 9 and 11, RNA targets without [ 125 I]oligonucleotides; lanes 2, 4, 6, 8, 10 and 12, RNA targets with [ 125 I]oligonucleotides, complementary AO-II (lanes 2, 4, 10 and 12) and non-complimentary AO-N (lanes 6 and 8). Samples in lanes 3, 4, 7, 8, 11 and 12 contained 10% DMSO. Lane 13, [ 32 P]DNA marker, 8–32 nt.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Marker

    Analysis of breaks in RNA and DNA with extended antisense oligonucleotide AO-L. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]AO-L; lane 3, [ 32 P]RNA; lane 4, [ 32 P]RNA with [ 125 I]AO-L; lane 5, Maxam–Gilbert G sequencing line of [ 32 P]DNA. ( B ) Percentages of breaks at individual bases of the target DNA (open squares) and RNA (filled circles) calculated from the data of the gel in (A). Error bars show standard deviations calculated from three independent measurements.
    Figure Legend Snippet: Analysis of breaks in RNA and DNA with extended antisense oligonucleotide AO-L. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]AO-L; lane 3, [ 32 P]RNA; lane 4, [ 32 P]RNA with [ 125 I]AO-L; lane 5, Maxam–Gilbert G sequencing line of [ 32 P]DNA. ( B ) Percentages of breaks at individual bases of the target DNA (open squares) and RNA (filled circles) calculated from the data of the gel in (A). Error bars show standard deviations calculated from three independent measurements.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing

    18) Product Images from "An Intermediate Pluripotent State Controlled by microRNAs is Required for the Naïve to Primed Stem Cell Transition"

    Article Title: An Intermediate Pluripotent State Controlled by microRNAs is Required for the Naïve to Primed Stem Cell Transition

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2018.04.021

    ISY1 establishes poised pluripotency through a subset of miRNAs (A) Reduced representation bisulfite sequencing (RRBS) was performed to measure genome-wide DNA methylation profiles in naïve and poised cells, and violin plots was used to measure the relative methylation level. (B) Genome-wide splicing analysis based on RNA-seq data in naïve and poised cells. Splicing junctions was identified through Tophat software and number is normalized to total reads number. (C) Western blot of lysates prepared from Dox-inducible Flag-ISY1 KH2 mouse ESCs (left) or indicated siRNA knockdown (right) analyzed using the indicated antibodies. (D) Heat map of expression of mature miRNAs dependent on ISY1 based on small RNA-seq data, and the corresponding pri-miRNAs. ( E ) q.RT-PCR analysis of pri-miRNAs in the ESCs in (B). Pri-miR-25~93 was used as a control. (F) PCA of the indicated cells by all expressed miRNAs based on small RNA-seq data. Orange, gray and brown region indicated the naïve, poised and primed pluripotency state, respectively. (G) (Top) Overlap of ISY1 dependent miRNAs in (C) and poised cell enriched miRNAs. (Bottom) Representative miRNA families containing the overlapping miRNAs (green) and miRNAs appeared in either individual group. (H) Number of predicted target sites of miRNAs from different miRNA families for poised downregulated and for naïve genes. Target prediction is from Targetscan database. miR-32 was used as a control. (I) Scatter plots were used to compare the expression level of target genes of different miRNA families by comparing poised (+Dox) with naïve (−Dox) cells. (J) q.RT-PCR analysis of the indicated genes in KH2-ISY1 ESCs treated with or without Dox, and transfected with the miRNA mimics. All the above q.RT-PCR data are normalized to ACTIN and represented as mean +/− SEM from three biological repeat.
    Figure Legend Snippet: ISY1 establishes poised pluripotency through a subset of miRNAs (A) Reduced representation bisulfite sequencing (RRBS) was performed to measure genome-wide DNA methylation profiles in naïve and poised cells, and violin plots was used to measure the relative methylation level. (B) Genome-wide splicing analysis based on RNA-seq data in naïve and poised cells. Splicing junctions was identified through Tophat software and number is normalized to total reads number. (C) Western blot of lysates prepared from Dox-inducible Flag-ISY1 KH2 mouse ESCs (left) or indicated siRNA knockdown (right) analyzed using the indicated antibodies. (D) Heat map of expression of mature miRNAs dependent on ISY1 based on small RNA-seq data, and the corresponding pri-miRNAs. ( E ) q.RT-PCR analysis of pri-miRNAs in the ESCs in (B). Pri-miR-25~93 was used as a control. (F) PCA of the indicated cells by all expressed miRNAs based on small RNA-seq data. Orange, gray and brown region indicated the naïve, poised and primed pluripotency state, respectively. (G) (Top) Overlap of ISY1 dependent miRNAs in (C) and poised cell enriched miRNAs. (Bottom) Representative miRNA families containing the overlapping miRNAs (green) and miRNAs appeared in either individual group. (H) Number of predicted target sites of miRNAs from different miRNA families for poised downregulated and for naïve genes. Target prediction is from Targetscan database. miR-32 was used as a control. (I) Scatter plots were used to compare the expression level of target genes of different miRNA families by comparing poised (+Dox) with naïve (−Dox) cells. (J) q.RT-PCR analysis of the indicated genes in KH2-ISY1 ESCs treated with or without Dox, and transfected with the miRNA mimics. All the above q.RT-PCR data are normalized to ACTIN and represented as mean +/− SEM from three biological repeat.

    Techniques Used: Methylation Sequencing, Genome Wide, DNA Methylation Assay, Methylation, RNA Sequencing Assay, Software, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    19) Product Images from "High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients"

    Article Title: High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsv010

    Sequencing error rates for the target regions. Substitution error rates with (black) and without (gray) barcode tags. Q5, single strand labelling with Q5 DNA polymerase for PCR amplification; Pt, single strand labelling with the Platinum Taq DNA polymerase High Fidelity kit for PCR amplification; DS, double strand labelling. Thirty nanograms of genomic DNA were used. The calculations were based on the sequence data from the seven (Q5, Pt) or five (except TK102 and TK103U for DS) regions obtained using an Ion Proton sequencer. Ninety-five per cent confidence intervals of the error rates are as follows: Q5 tag+, 2.8 × 10 −6 – 8.8 × 10 −6 ; Pt tag+, 6.9 × 10 −6 – 1.3 × 10 −5 ; DS tag+, 3.3 × 10 −6 – 1.6 × 10 −5 ; Q5 tag−, 9.0 × 10 −5 – 9.3 × 10 −5 ; Pt tag−, 5.7 × 10 −4 –5.7 × 10 −4 ; DS tag−, 3.7 × 10 −4 – 3.7 × 10 −4 .
    Figure Legend Snippet: Sequencing error rates for the target regions. Substitution error rates with (black) and without (gray) barcode tags. Q5, single strand labelling with Q5 DNA polymerase for PCR amplification; Pt, single strand labelling with the Platinum Taq DNA polymerase High Fidelity kit for PCR amplification; DS, double strand labelling. Thirty nanograms of genomic DNA were used. The calculations were based on the sequence data from the seven (Q5, Pt) or five (except TK102 and TK103U for DS) regions obtained using an Ion Proton sequencer. Ninety-five per cent confidence intervals of the error rates are as follows: Q5 tag+, 2.8 × 10 −6 – 8.8 × 10 −6 ; Pt tag+, 6.9 × 10 −6 – 1.3 × 10 −5 ; DS tag+, 3.3 × 10 −6 – 1.6 × 10 −5 ; Q5 tag−, 9.0 × 10 −5 – 9.3 × 10 −5 ; Pt tag−, 5.7 × 10 −4 –5.7 × 10 −4 ; DS tag−, 3.7 × 10 −4 – 3.7 × 10 −4 .

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

    20) Product Images from "New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase"

    Article Title: New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37513-7

    Processivity assay of ϕ29 DNA polymerase mutant Y101A. The assay was carried out as described in Materials and Methods by using a 5′ labelled sp1/sp1c + 18 (15/33 mer) depicted at the top of the figure as substrate, in the presence of the indicated concentrations of wild-type or mutant ϕ29 DNA polymerases. As a control of non-processive elongation, the Klenow DNA polymerase (units) was used. Asterisk indicates the 5′- 32 P-labelled end of the primer strand. c: control DNA.
    Figure Legend Snippet: Processivity assay of ϕ29 DNA polymerase mutant Y101A. The assay was carried out as described in Materials and Methods by using a 5′ labelled sp1/sp1c + 18 (15/33 mer) depicted at the top of the figure as substrate, in the presence of the indicated concentrations of wild-type or mutant ϕ29 DNA polymerases. As a control of non-processive elongation, the Klenow DNA polymerase (units) was used. Asterisk indicates the 5′- 32 P-labelled end of the primer strand. c: control DNA.

    Techniques Used: Mutagenesis

    21) Product Images from "Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage"

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni058

    Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).
    Figure Legend Snippet: Synthesis of a functional chloramphenicol acetyltransferase gene with changed codon composition. The ratio r of ‘active clones’ to ‘analyzed clones’ as described in the text is shown for different gene synthesis methods with or without an EMC step. A significant increase of r can be observed only in the cases where EMC is combined with an exonuclease activity present in the reaction or in the later amplification reaction. Prolonged incubation with E.coli endonuclease V results in no detectable product after the amplification steps (ss, single-stranded synthesis, ds, double-stranded synthesis; VII, T4 endonuclease VII; V, E.coli endonuclease V; T, Taq DNA polymerase; and Vn, Vent DNA polymerase).

    Techniques Used: Functional Assay, Clone Assay, Activity Assay, Amplification, Incubation

    22) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    Effects of a caged thymidine nucleobase on the DNA polymerization by mesophilic DNA polymerases. ( A ) Products resulting from T4 DNA Polymerase extension. When using the non-caged template full-length product (38 nt) is obtained; however, using either D1 or D2 caged templates, polymerization is halted, leading to truncated product (26 or 22 nt, respectively). ( B ) Products resulting from T7 DNA Polymerase extension. Similar truncations are observed as with T4 DNA Polymerase. ( C ) Products resulting from DNA Polymerase I extension, demonstrating a polymerase read-through to yield full-length product (38 nt) in all cases.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the DNA polymerization by mesophilic DNA polymerases. ( A ) Products resulting from T4 DNA Polymerase extension. When using the non-caged template full-length product (38 nt) is obtained; however, using either D1 or D2 caged templates, polymerization is halted, leading to truncated product (26 or 22 nt, respectively). ( B ) Products resulting from T7 DNA Polymerase extension. Similar truncations are observed as with T4 DNA Polymerase. ( C ) Products resulting from DNA Polymerase I extension, demonstrating a polymerase read-through to yield full-length product (38 nt) in all cases.

    Techniques Used:

    DNA polymerization through extension of a primer using a 32 nt template with a caged thymidine (blue square) 17 or 21 nt from the 3′ end of the template. A single caging group blocked polymerization by T4 and T7 DNA polymerase.
    Figure Legend Snippet: DNA polymerization through extension of a primer using a 32 nt template with a caged thymidine (blue square) 17 or 21 nt from the 3′ end of the template. A single caging group blocked polymerization by T4 and T7 DNA polymerase.

    Techniques Used:

    23) Product Images from "Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering"

    Article Title: Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1497

    APE-MITOMI applied to ZF TF module combinatorics. ( A ) Cartoon model of canonical Cys 2 His 2 ZF TF binding to DNA with residues −1, 2, 3 and 6 of the recognition helix primarily encoding DNA specificity. Residue 2 makes a cross-strand contact, which creates ‘context dependent’ effects. ( B ) Schematic of the APE solid-phase gene assembly technique, showing assembly through the first two extension steps. ( C ) Process timeline from gene assembly to protein characterization. ( D ) Comparison of APE error rate with values from previously published gene assembly techniques. A line between two points indicates a range of error rates from different experimental conditions. ( E ) Overview of experimental results obtained from combinatoric assembly of ZF TFs demonstrating protein expression and functional DNA binding success rates. ( F ) Heatmap of relative binding affinities for each assembled ZF TF ( y -axis) to 64 predicted consensus DNA targets ( x -axis). Protein naming convention indicates ZF domain from C-to-N (F3 to F1), where AAA (A f3 A f2 A f1 ) = Zif268, BBB = 37–12, CCC = 92–1, DDD = 158–2 ( 14 ); for example, protein ABC = F3 from Zif268, F2 from 37–12, F1 from 92–1; target ABC = Zif268 F3 binding consensus triplet GCG, 37–12 F2 binding consensus GAC, 92–1 F1 binding consensus triplet GCC (5′-GCG GAC GCC). The values represent averages of multiple measurements and the precise number of technical repeats and a histogram thereof are shown in Supplementary Figures S7 and S8, respectively. Oligomer assembly and target sequences are given in Supplementary Tables S7 and S8.
    Figure Legend Snippet: APE-MITOMI applied to ZF TF module combinatorics. ( A ) Cartoon model of canonical Cys 2 His 2 ZF TF binding to DNA with residues −1, 2, 3 and 6 of the recognition helix primarily encoding DNA specificity. Residue 2 makes a cross-strand contact, which creates ‘context dependent’ effects. ( B ) Schematic of the APE solid-phase gene assembly technique, showing assembly through the first two extension steps. ( C ) Process timeline from gene assembly to protein characterization. ( D ) Comparison of APE error rate with values from previously published gene assembly techniques. A line between two points indicates a range of error rates from different experimental conditions. ( E ) Overview of experimental results obtained from combinatoric assembly of ZF TFs demonstrating protein expression and functional DNA binding success rates. ( F ) Heatmap of relative binding affinities for each assembled ZF TF ( y -axis) to 64 predicted consensus DNA targets ( x -axis). Protein naming convention indicates ZF domain from C-to-N (F3 to F1), where AAA (A f3 A f2 A f1 ) = Zif268, BBB = 37–12, CCC = 92–1, DDD = 158–2 ( 14 ); for example, protein ABC = F3 from Zif268, F2 from 37–12, F1 from 92–1; target ABC = Zif268 F3 binding consensus triplet GCG, 37–12 F2 binding consensus GAC, 92–1 F1 binding consensus triplet GCC (5′-GCG GAC GCC). The values represent averages of multiple measurements and the precise number of technical repeats and a histogram thereof are shown in Supplementary Figures S7 and S8, respectively. Oligomer assembly and target sequences are given in Supplementary Tables S7 and S8.

    Techniques Used: Binding Assay, Expressing, Functional Assay, Countercurrent Chromatography

    24) Product Images from "Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells"

    Article Title: Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-018-0465-4

    Identification of chromosome breaks in BA-treated TWO4 cells. Genomic DNA was extracted from BA-treated TWO4 cells for nested IPCR as described in “Methods” section. a Representative gel picture showing the AF9 gene cleavages in BA-treated TWO4 cells detected within: ( a i ) SAR region ( a ii ) Non-SAR region. TWO4 cells were left untreated (Lanes 1–6) or treated for 3 h with 0.5 mM of BA at pH 7.4 (Lanes 7–12) and pH 5.8 (Lanes 13–18). The IPCR bands derived from the AF9 cleaved fragments were indicated by the side bracket. M: 100 bp DNA ladder. N: Negative control for IPCR. b The average number of AF9 gene cleavages detected by IPCR. Data represents means and SDs of three independent experiments. Each experiment consisted of at least two sets of IPCR assays performed in five to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p
    Figure Legend Snippet: Identification of chromosome breaks in BA-treated TWO4 cells. Genomic DNA was extracted from BA-treated TWO4 cells for nested IPCR as described in “Methods” section. a Representative gel picture showing the AF9 gene cleavages in BA-treated TWO4 cells detected within: ( a i ) SAR region ( a ii ) Non-SAR region. TWO4 cells were left untreated (Lanes 1–6) or treated for 3 h with 0.5 mM of BA at pH 7.4 (Lanes 7–12) and pH 5.8 (Lanes 13–18). The IPCR bands derived from the AF9 cleaved fragments were indicated by the side bracket. M: 100 bp DNA ladder. N: Negative control for IPCR. b The average number of AF9 gene cleavages detected by IPCR. Data represents means and SDs of three independent experiments. Each experiment consisted of at least two sets of IPCR assays performed in five to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p

    Techniques Used: Derivative Assay, Negative Control

    Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p
    Figure Legend Snippet: Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p

    Techniques Used: DNA Extraction, Derivative Assay, Marker, Negative Control

    25) Product Images from "Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance"

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn575

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Figure Legend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    26) Product Images from "Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro"

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    Journal: Nucleic Acids Research

    doi:

    Intermediates in the ligation-based protocol. The plasmid pBR322 was used to illustrate the individual steps in the protocol for two reasons. Its smaller size (4361 bp) makes it easier to see small changes in molecular weight, and the large distance separating Eco RI and Bam HI (377 bp) is useful to illustrate the excision of such a fragment. Lane 1, 100 ng pBR322, which is primarily supercoiled (band A) after CsCl banding but contains a small amount of nicked circle (band E). Lane 2, treatment with 2 U Bam HI/µg DNA yields a linear plasmid (band B). Lane 3, addition of a 40-fold excess of heteroduplex oligo ends and T4 DNA ligase (100 U/µg plasmid DNA) yields band D. Lane 4, digestion with Eco RI, followed by size-exclusion chromatography on Sephacryl S500 resin (band C). An equivalent fraction of the total volume from each step was loaded, so that the relative staining intensities reflect relative yield.
    Figure Legend Snippet: Intermediates in the ligation-based protocol. The plasmid pBR322 was used to illustrate the individual steps in the protocol for two reasons. Its smaller size (4361 bp) makes it easier to see small changes in molecular weight, and the large distance separating Eco RI and Bam HI (377 bp) is useful to illustrate the excision of such a fragment. Lane 1, 100 ng pBR322, which is primarily supercoiled (band A) after CsCl banding but contains a small amount of nicked circle (band E). Lane 2, treatment with 2 U Bam HI/µg DNA yields a linear plasmid (band B). Lane 3, addition of a 40-fold excess of heteroduplex oligo ends and T4 DNA ligase (100 U/µg plasmid DNA) yields band D. Lane 4, digestion with Eco RI, followed by size-exclusion chromatography on Sephacryl S500 resin (band C). An equivalent fraction of the total volume from each step was loaded, so that the relative staining intensities reflect relative yield.

    Techniques Used: Ligation, Plasmid Preparation, Molecular Weight, Size-exclusion Chromatography, Staining

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    Figure Legend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Techniques Used: Plasmid Preparation, Ligation, Generated

    Optimization of the ring closure ligation. A modified pET11aΔH intermediate (Fig. 1, structure 4) was incubated with T4 DNA ligase using DNA concentrations decreasing from 100 to 10 ng/µl (see Materials and Methods for conditions). This LS has one DNA end generated by Eco RI digestion and one contributed by the T·G-10H oligonucleotide heteroduplex. The supercoiled (SC) pET11aΔH plasmid loaded in the first and last lanes contains a small amount of NC molecules that serves as a close marker for the mobility of the desired product. At 100 ng/µl DNA, the predominant products are multimers that migrate more slowly. At 10 ng/µl template, the desired NC heteroduplex represents up to 30% of the products, and the formation of linear multimers is reduced. For each lane representing a sample of a ligation reaction, an equivalent sample was treated with Exonuclease V, which degrades linear molecules and reveals the circular products. Note that a trace amount of CD is formed in this experiment.
    Figure Legend Snippet: Optimization of the ring closure ligation. A modified pET11aΔH intermediate (Fig. 1, structure 4) was incubated with T4 DNA ligase using DNA concentrations decreasing from 100 to 10 ng/µl (see Materials and Methods for conditions). This LS has one DNA end generated by Eco RI digestion and one contributed by the T·G-10H oligonucleotide heteroduplex. The supercoiled (SC) pET11aΔH plasmid loaded in the first and last lanes contains a small amount of NC molecules that serves as a close marker for the mobility of the desired product. At 100 ng/µl DNA, the predominant products are multimers that migrate more slowly. At 10 ng/µl template, the desired NC heteroduplex represents up to 30% of the products, and the formation of linear multimers is reduced. For each lane representing a sample of a ligation reaction, an equivalent sample was treated with Exonuclease V, which degrades linear molecules and reveals the circular products. Note that a trace amount of CD is formed in this experiment.

    Techniques Used: Ligation, Modification, Incubation, Generated, Plasmid Preparation, Marker

    27) Product Images from "Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor"

    Article Title: Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

    Journal: Journal of Bacteriology

    doi:

    Schematic representations (a) and results of PCR analysis (b) on wild-type (WT) C. acetobutylicum ATCC 824 and solR mutants B and H using primers (a) designed to amplify the junction between the vector portion of pO1X and the solR gene. For each gel in panel b, lane 1 contains Hin dIII-digested lambda marker, lane 2 contains the WT genomic template, lane 3 contains the solR mutant B template, and lane 4 contains the solR mutant H as the template. (Gel A) Extralong PCR using primers solR453 and solR1361 designed to amplify the complete insert. In lane 2, WT DNA shows an expected ∼0.9-kb band. This band is also seen, but much weaker, in both lanes 3 and 4. In addition, lane 4 contains a band with an apparent size of ∼7 kb (marked with an arrow), consistent with the presence of one insert of pO1X into solR of mutant H. (Gel B) PCR results using primers solR453 and Tc238. A band of ∼1.2 kb can be seen in lanes 3 and 4 with no product in lane 2. (Gel C) PCR results using primers Em373 and solR1361. A band of ∼2.1 kb is visible in lanes 3 and 4. Again, no product was observed with WT DNA (lane 2).
    Figure Legend Snippet: Schematic representations (a) and results of PCR analysis (b) on wild-type (WT) C. acetobutylicum ATCC 824 and solR mutants B and H using primers (a) designed to amplify the junction between the vector portion of pO1X and the solR gene. For each gel in panel b, lane 1 contains Hin dIII-digested lambda marker, lane 2 contains the WT genomic template, lane 3 contains the solR mutant B template, and lane 4 contains the solR mutant H as the template. (Gel A) Extralong PCR using primers solR453 and solR1361 designed to amplify the complete insert. In lane 2, WT DNA shows an expected ∼0.9-kb band. This band is also seen, but much weaker, in both lanes 3 and 4. In addition, lane 4 contains a band with an apparent size of ∼7 kb (marked with an arrow), consistent with the presence of one insert of pO1X into solR of mutant H. (Gel B) PCR results using primers solR453 and Tc238. A band of ∼1.2 kb can be seen in lanes 3 and 4 with no product in lane 2. (Gel C) PCR results using primers Em373 and solR1361. A band of ∼2.1 kb is visible in lanes 3 and 4. Again, no product was observed with WT DNA (lane 2).

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker, Mutagenesis

    28) Product Images from "Improvement of ?29 DNA polymerase amplification performance by fusion of DNA binding motifs"

    Article Title: Improvement of ?29 DNA polymerase amplification performance by fusion of DNA binding motifs

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1011428107

    , in the presence 1 ng of genomic DNA from B. subtilis and 50 nM of each polymerase. After the indicated reaction times, 1 μl of each sample was loaded in a Etd bromide-containing 0.7% agarose gel ( A ). At the left, linear DNA fragments obtained after digesting φ29 DNA with Hind . The specific amplification factors are represented as Mean ± SD in B .
    Figure Legend Snippet: , in the presence 1 ng of genomic DNA from B. subtilis and 50 nM of each polymerase. After the indicated reaction times, 1 μl of each sample was loaded in a Etd bromide-containing 0.7% agarose gel ( A ). At the left, linear DNA fragments obtained after digesting φ29 DNA with Hind . The specific amplification factors are represented as Mean ± SD in B .

    Techniques Used: Agarose Gel Electrophoresis, Amplification

    Chimerical DNA polymerases show an enhanced RCR efficiency. ( A using 60 nM of φ29 wild-type or chimerical DNA polymerases. The position of unit-length M13 DNA is shown at the right. ( B , in the presence of 250 ng of multiply primed M13 DNA as input and 60 nM of either φ29 DNA polymerase or the indicated chimerical DNA polymerase. Data are represented as Mean ± SD. ( C ) Processive synthesis by φ29 DNA polymerase and chimerical DNA polymerases. The assay was performed as described in the text in the presence of 250 ng of singly primed M13 DNA and decreasing concentrations of the indicated DNA polymerase. After incubation at 30 °C for 20 min, samples were processed as described in A .
    Figure Legend Snippet: Chimerical DNA polymerases show an enhanced RCR efficiency. ( A using 60 nM of φ29 wild-type or chimerical DNA polymerases. The position of unit-length M13 DNA is shown at the right. ( B , in the presence of 250 ng of multiply primed M13 DNA as input and 60 nM of either φ29 DNA polymerase or the indicated chimerical DNA polymerase. Data are represented as Mean ± SD. ( C ) Processive synthesis by φ29 DNA polymerase and chimerical DNA polymerases. The assay was performed as described in the text in the presence of 250 ng of singly primed M13 DNA and decreasing concentrations of the indicated DNA polymerase. After incubation at 30 °C for 20 min, samples were processed as described in A .

    Techniques Used: Incubation

    , in the presence of 1 ng ( A ) or 1 pg ( B ) of plasmidic DNA as input and 50 nM of φ29 DNA polymerase or the indicated chimerical DNA polymerase. At the left, linear DNA fragments obtained after digesting φ29 DNA with Hind III, used as DNA length markers.
    Figure Legend Snippet: , in the presence of 1 ng ( A ) or 1 pg ( B ) of plasmidic DNA as input and 50 nM of φ29 DNA polymerase or the indicated chimerical DNA polymerase. At the left, linear DNA fragments obtained after digesting φ29 DNA with Hind III, used as DNA length markers.

    Techniques Used:

    29) Product Images from "MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression"

    Article Title: MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002204

    The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.
    Figure Legend Snippet: The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Generated

    Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.
    Figure Legend Snippet: Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Generated, Purification, Incubation

    30) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    31) Product Images from "Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii"

    Article Title: Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii

    Journal: Genes

    doi: 10.3390/genes9110562

    Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.
    Figure Legend Snippet: Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.

    Techniques Used: Polymerase Chain Reaction, Ligation, DNA Sequencing

    32) Product Images from "Secondary structure formation and DNA instability at fragile site FRA16B"

    Article Title: Secondary structure formation and DNA instability at fragile site FRA16B

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1245

    FRA16B DNA synthesis by Klenow fragment of E. coli DNA polymerase I. ( A ) Construction of replication fork templates to examine fork regression. Plasmids containing FRA16B clone 17 fragment (pFRA16B37, Supplementary Figure S1 ) or a G-less cassette (pGLGAP) were constructed, nicked with Nb.BbvCI at a site immediately upstream of the inserts (bold), and incubated with Klenow fragment to generate a single-strand tail by strand displacement. The single-strand tail was converted into a double-strand tail by annealing a complimentary oligonucleotide at the 3′ end of the displaced strand and further incubation with Klenow fragment. To map the location of the double-strand tail, plasmids were linearized so measurements of the two asymmetrical tails could be obtained to determine the amount of fork regression. ( B ) Classification of circular DNA molecules after Klenow fragment reaction by EM. After the second Klenow reaction to create a double-strand tail, reaction mixtures were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. At least 100 molecules from each sample were examined to determine the percentage of molecules with tails, protein-bound, or without a tail. ( C ) Visualization of circular and linear pGLGAP and pFRA16B37 DNAs after synthesis reaction with Klenow fragment. Representative images were montaged and are shown in reverse contrast. ( D ) Location of bound polymerases within FRA16B replication fork templates. The location of the polymerase as a percentage of the total length is plotted as a histogram for all FRA16B molecules that displayed bound polymerase and did not contain a double-stranded tail. The FRA16B fragment is located at 33–50% of the total length from the nearest end. ( E ) Analysis of DNA polymerase pause sites during lagging strand DNA synthesis. Radiolabeled oligomers were annealed to the displaced lagging strand of pGLGAP (lanes 1–3) and pFRA16B37 (lanes 5–7) replication fork templates, and synthesis was carried out with 0.5 U (lanes 1 and 5), 5 U (lanes 2 and 6) or 15 U (lanes 3 and 7) of Klenow fragment. Lane 4 (C) contained the radiolabeled 25-nt oligonucleotide only. The first 12 nt of the newly synthesized complimentary strand are indicated, and the pause sites are boxed. ( F ) Detection of FRA16B replication fork regression. Circular replication fork molecules were linearized with AhdI to produce asymmetrical arms. The location of the double-strand tail was defined as the percentage of total DNA length from the nearest end.
    Figure Legend Snippet: FRA16B DNA synthesis by Klenow fragment of E. coli DNA polymerase I. ( A ) Construction of replication fork templates to examine fork regression. Plasmids containing FRA16B clone 17 fragment (pFRA16B37, Supplementary Figure S1 ) or a G-less cassette (pGLGAP) were constructed, nicked with Nb.BbvCI at a site immediately upstream of the inserts (bold), and incubated with Klenow fragment to generate a single-strand tail by strand displacement. The single-strand tail was converted into a double-strand tail by annealing a complimentary oligonucleotide at the 3′ end of the displaced strand and further incubation with Klenow fragment. To map the location of the double-strand tail, plasmids were linearized so measurements of the two asymmetrical tails could be obtained to determine the amount of fork regression. ( B ) Classification of circular DNA molecules after Klenow fragment reaction by EM. After the second Klenow reaction to create a double-strand tail, reaction mixtures were directly mounted onto carbon-coated copper EM grids and rotary shadowcasted with tungsten. At least 100 molecules from each sample were examined to determine the percentage of molecules with tails, protein-bound, or without a tail. ( C ) Visualization of circular and linear pGLGAP and pFRA16B37 DNAs after synthesis reaction with Klenow fragment. Representative images were montaged and are shown in reverse contrast. ( D ) Location of bound polymerases within FRA16B replication fork templates. The location of the polymerase as a percentage of the total length is plotted as a histogram for all FRA16B molecules that displayed bound polymerase and did not contain a double-stranded tail. The FRA16B fragment is located at 33–50% of the total length from the nearest end. ( E ) Analysis of DNA polymerase pause sites during lagging strand DNA synthesis. Radiolabeled oligomers were annealed to the displaced lagging strand of pGLGAP (lanes 1–3) and pFRA16B37 (lanes 5–7) replication fork templates, and synthesis was carried out with 0.5 U (lanes 1 and 5), 5 U (lanes 2 and 6) or 15 U (lanes 3 and 7) of Klenow fragment. Lane 4 (C) contained the radiolabeled 25-nt oligonucleotide only. The first 12 nt of the newly synthesized complimentary strand are indicated, and the pause sites are boxed. ( F ) Detection of FRA16B replication fork regression. Circular replication fork molecules were linearized with AhdI to produce asymmetrical arms. The location of the double-strand tail was defined as the percentage of total DNA length from the nearest end.

    Techniques Used: DNA Synthesis, Construct, Incubation, Synthesized

    33) Product Images from "Directed evolution of protein enzymes using nonhomologous random recombination"

    Article Title: Directed evolution of protein enzymes using nonhomologous random recombination

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0402202101

    Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry
    Figure Legend Snippet: Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry

    Techniques Used: Ligation

    34) Product Images from "Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog"

    Article Title: Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01726-12

    Binding of HrpX to potential PIP box by EMSA. The interaction of the DNA probe with purified HrpX-MBP is shown. Each lane contains 0.65 μg isotope-labeled probe. Lanes 1 to 3 contain 12.28 μg, 24.56 μg, and 49.12 μg HrpX-MBP, respectively; lane 4 contains MBP (44.48 μg) as the negative control; lane 5 contains the free probe as the positive control; and lanes 6 to 10 contain the same concentration of HrpX-MBP (49.12 μg) and various amounts of unlabeled probe (0.65 μg, 3.23 μg, 13 μg, and 19.5 μg, respectively) as competitors.
    Figure Legend Snippet: Binding of HrpX to potential PIP box by EMSA. The interaction of the DNA probe with purified HrpX-MBP is shown. Each lane contains 0.65 μg isotope-labeled probe. Lanes 1 to 3 contain 12.28 μg, 24.56 μg, and 49.12 μg HrpX-MBP, respectively; lane 4 contains MBP (44.48 μg) as the negative control; lane 5 contains the free probe as the positive control; and lanes 6 to 10 contain the same concentration of HrpX-MBP (49.12 μg) and various amounts of unlabeled probe (0.65 μg, 3.23 μg, 13 μg, and 19.5 μg, respectively) as competitors.

    Techniques Used: Binding Assay, Purification, Labeling, Negative Control, Positive Control, Concentration Assay

    35) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.
    Figure Legend Snippet: Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.

    Techniques Used: Activity Assay, Radioactivity

    Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.
    Figure Legend Snippet: Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay

    36) Product Images from "Biochemical Methods to Characterize RNA Polymerase II Elongation Complexes"

    Article Title: Biochemical Methods to Characterize RNA Polymerase II Elongation Complexes

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2019.01.011

    Site-specific photocrosslinking of ECs. (A). Schematic diagram of the strategy to produce site-specific DNA probes. A single-stranded oligo is used to prime DNA synthesis immediately adjacent to the preferred crosslinking site. A photoreactive nucleotide is incorporated next to a radiolabeled nucleotide by the Klenow fragment of DNA polymerase. The strand is then completed by adding excess nucleotides. (B). A model of the transcription bubble within RNAPII from several crystal structures, including modeling of Spt4/5 (NGN) adjacent to the non-template strand (NTS) (PDB codes, 5C4X, 2EXU, and 3QQC) using PyMol (version 1.7.4 Schrodinger, LLC). Choice of probe sites are indicated. (C). PAGE showing purified labeled templates with probes located at +14, −6, −12, relative to the arrest location designated as +1. (D). EMSA on photoreactive transcription templates in the presence and absence of Spt4/5 (30 nM). (E) Crosslinking and label transfer to Rpb1,2 and Spt5. SDS-PAGE of ECs prepared with probes located at positions +14, −6, −12. As controls, a –UV lane is included for each position, and a mutant version of Spt4/5 (1-418) that does not bind RNAPII is also shown. Both WT and mutant Spt4/5 are at 30 nM and RNAPII is at 5 nM. Black arrows highlight the migration of Rpb1, Rpb2, and Spt5 in the gel.
    Figure Legend Snippet: Site-specific photocrosslinking of ECs. (A). Schematic diagram of the strategy to produce site-specific DNA probes. A single-stranded oligo is used to prime DNA synthesis immediately adjacent to the preferred crosslinking site. A photoreactive nucleotide is incorporated next to a radiolabeled nucleotide by the Klenow fragment of DNA polymerase. The strand is then completed by adding excess nucleotides. (B). A model of the transcription bubble within RNAPII from several crystal structures, including modeling of Spt4/5 (NGN) adjacent to the non-template strand (NTS) (PDB codes, 5C4X, 2EXU, and 3QQC) using PyMol (version 1.7.4 Schrodinger, LLC). Choice of probe sites are indicated. (C). PAGE showing purified labeled templates with probes located at +14, −6, −12, relative to the arrest location designated as +1. (D). EMSA on photoreactive transcription templates in the presence and absence of Spt4/5 (30 nM). (E) Crosslinking and label transfer to Rpb1,2 and Spt5. SDS-PAGE of ECs prepared with probes located at positions +14, −6, −12. As controls, a –UV lane is included for each position, and a mutant version of Spt4/5 (1-418) that does not bind RNAPII is also shown. Both WT and mutant Spt4/5 are at 30 nM and RNAPII is at 5 nM. Black arrows highlight the migration of Rpb1, Rpb2, and Spt5 in the gel.

    Techniques Used: DNA Synthesis, Polyacrylamide Gel Electrophoresis, Purification, Labeling, SDS Page, Mutagenesis, Migration

    37) Product Images from "A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1"

    Article Title: A DNA break inducer activates the anticodon nuclease RloC and the adaptive immunity in Acinetobacter baylyi ADP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt851

    Gka RloC's ACNase activity is sustained by the activating DNA. ( A ) The 5′- 32- P labelled ACNase substrate 7-2'-Om-Glu-ASL. ( B ) Gka RloC's ACNase was activated for 20 min in the presence of ATP and DNA followed by further pre-incubation with DNase buffer (lanes 1–5), or DNase I buffer containing purified oligonucleotides formed by a DNase I digestion of an activating DNA dose (lanes 6–9) or DNase I (lanes 10–13). In lanes 14–17 DNase I was included in the activation mixture. ACNase activity was subsequently assayed as detailed in ‘Materials and Methods’ section. DDR, DNase-I digest residue; After or During, DNase I added after the activation or during the activation, respectively; ASL, [5′- 32 P]7-2'-Om-Glu-ASL; 8mer, labelled cleavage product.
    Figure Legend Snippet: Gka RloC's ACNase activity is sustained by the activating DNA. ( A ) The 5′- 32- P labelled ACNase substrate 7-2'-Om-Glu-ASL. ( B ) Gka RloC's ACNase was activated for 20 min in the presence of ATP and DNA followed by further pre-incubation with DNase buffer (lanes 1–5), or DNase I buffer containing purified oligonucleotides formed by a DNase I digestion of an activating DNA dose (lanes 6–9) or DNase I (lanes 10–13). In lanes 14–17 DNase I was included in the activation mixture. ACNase activity was subsequently assayed as detailed in ‘Materials and Methods’ section. DDR, DNase-I digest residue; After or During, DNase I added after the activation or during the activation, respectively; ASL, [5′- 32 P]7-2'-Om-Glu-ASL; 8mer, labelled cleavage product.

    Techniques Used: Activity Assay, Incubation, Purification, Activation Assay

    38) Product Images from "Mutations in maltose-binding protein that alter affinity and solubility properties"

    Article Title: Mutations in maltose-binding protein that alter affinity and solubility properties

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-010-2696-y

    Sequence of MBP2 and location of mutations. Mutations are indicated in bold under the amino acid sequence. Δ2682 indicates a deletion of a thymine at position 2682 in the DNA sequence, which leads to a frameshift of the subsequent residues in MBP2. Secondary structural elements are indicated above the sequence, arrows for β-sheets and helices for α-helices, according to the PDB file 1ANF (Spurlino et al. 1991 ). The β-sheets are labeled with a letter to indicate the structural element and a number to indicate the strand in that element; e.g., β-sheet “A” is made up of two strands, A1 and A2. Helices are indicated by a number . The numbering of the mutations we obtained ignores the N-terminal methionine present in MBP2, to simplify comparison to the structure and the previous literature. The sequences corresponding to the hinges between the two domains are indicated as bold letters embedded in the sequence
    Figure Legend Snippet: Sequence of MBP2 and location of mutations. Mutations are indicated in bold under the amino acid sequence. Δ2682 indicates a deletion of a thymine at position 2682 in the DNA sequence, which leads to a frameshift of the subsequent residues in MBP2. Secondary structural elements are indicated above the sequence, arrows for β-sheets and helices for α-helices, according to the PDB file 1ANF (Spurlino et al. 1991 ). The β-sheets are labeled with a letter to indicate the structural element and a number to indicate the strand in that element; e.g., β-sheet “A” is made up of two strands, A1 and A2. Helices are indicated by a number . The numbering of the mutations we obtained ignores the N-terminal methionine present in MBP2, to simplify comparison to the structure and the previous literature. The sequences corresponding to the hinges between the two domains are indicated as bold letters embedded in the sequence

    Techniques Used: Sequencing, Labeling

    39) Product Images from "Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri"

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    Journal: Nucleic Acids Research

    doi:

    EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.
    Figure Legend Snippet: EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.

    Techniques Used: Incubation

    EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.
    Figure Legend Snippet: EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.

    Techniques Used: Incubation, Electrophoresis

    DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.
    Figure Legend Snippet: DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.

    Techniques Used: Incubation, Isolation, Agarose Gel Electrophoresis, Staining

    40) Product Images from "Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis"

    Article Title: Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis

    Journal:

    doi: 10.1021/tx9000896

    Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template
    Figure Legend Snippet: Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Techniques Used: Primer Extension Assay

    41) Product Images from "Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification"

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001948

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Figure Legend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Techniques Used: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.
    Figure Legend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Techniques Used: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.
    Figure Legend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Techniques Used: Lamp Assay, Infection, Isolation

    42) Product Images from "A rapid method for assessing the RNA-binding potential of a protein"

    Article Title: A rapid method for assessing the RNA-binding potential of a protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks285

    Schematic of ssRNA Pentaprobe production. pcDNA3.1 vector containing a dsDNA Pentaprobe sequence under the control of a T7 promoter site is linearized and the in vitro transcribed to produce a ssRNA sequence encoding the Pentaprobe sequence. Highlighted is the ApaI restriction site (purple), T7 promoter site (pink), the encoded DNA sequence (blue) and the resulting ssRNA probe sequence (blue).
    Figure Legend Snippet: Schematic of ssRNA Pentaprobe production. pcDNA3.1 vector containing a dsDNA Pentaprobe sequence under the control of a T7 promoter site is linearized and the in vitro transcribed to produce a ssRNA sequence encoding the Pentaprobe sequence. Highlighted is the ApaI restriction site (purple), T7 promoter site (pink), the encoded DNA sequence (blue) and the resulting ssRNA probe sequence (blue).

    Techniques Used: Plasmid Preparation, Sequencing, In Vitro

    43) Product Images from "Transcriptional Activation of the mrkA Promoter of the Klebsiella pneumoniae Type 3 Fimbrial Operon by the c-di-GMP-Dependent MrkH Protein"

    Article Title: Transcriptional Activation of the mrkA Promoter of the Klebsiella pneumoniae Type 3 Fimbrial Operon by the c-di-GMP-Dependent MrkH Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079038

    Analysis of MrkH-8×His binding to the wild-type MrkH and mutant mrkA fragments by EMSA. The two mrkA fragments (wild-type and MrkH box mut-1) spanning from −155 and +116 were each amplified and labeled at the 5′ end with 32 P by PCR, using primer pairs 32 P-mrkA116 and mrkA-155. DNA fragments were each mixed with varying amounts of MrkH-8×His in the presence of 50 µM c-di-GMP. Following incubation at 30°C for 20 min, samples were analyzed on native polyacrylamide gels. F: free DNA. C: protein-DNA complex.
    Figure Legend Snippet: Analysis of MrkH-8×His binding to the wild-type MrkH and mutant mrkA fragments by EMSA. The two mrkA fragments (wild-type and MrkH box mut-1) spanning from −155 and +116 were each amplified and labeled at the 5′ end with 32 P by PCR, using primer pairs 32 P-mrkA116 and mrkA-155. DNA fragments were each mixed with varying amounts of MrkH-8×His in the presence of 50 µM c-di-GMP. Following incubation at 30°C for 20 min, samples were analyzed on native polyacrylamide gels. F: free DNA. C: protein-DNA complex.

    Techniques Used: Binding Assay, Mutagenesis, Amplification, Labeling, Polymerase Chain Reaction, Incubation

    44) Product Images from "Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production"

    Article Title: Assembly PCR oligo maker: a tool for designing oligodeoxynucleotides for constructing long DNA molecules for RNA production

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki380

    The construction of a 191-nt DNA molecule using the oligodeoxynucleotides determined by the Assembly PCR Oligo Maker program. ( a ) The sequence of the 191-nt DNA target to be produced. ( b ) The DNA sequences reported by Assembly PCR Oligo Maker program. Sequences for both steps of the assembly PCR reaction are reported. ( c ) Diagram showing how the four oligodeoxynucleotides anneal to produce the full-length dsDNA product ( d ) Agarose gel showing the results of the first (lane 2) and second (lane 3) PCR steps. The desired 191-nt molecule is visible after the second PCR step. DNA length markers are shown in lane 1.
    Figure Legend Snippet: The construction of a 191-nt DNA molecule using the oligodeoxynucleotides determined by the Assembly PCR Oligo Maker program. ( a ) The sequence of the 191-nt DNA target to be produced. ( b ) The DNA sequences reported by Assembly PCR Oligo Maker program. Sequences for both steps of the assembly PCR reaction are reported. ( c ) Diagram showing how the four oligodeoxynucleotides anneal to produce the full-length dsDNA product ( d ) Agarose gel showing the results of the first (lane 2) and second (lane 3) PCR steps. The desired 191-nt molecule is visible after the second PCR step. DNA length markers are shown in lane 1.

    Techniques Used: Polymerase Cycling Assembly, Sequencing, Produced, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    The assembly PCR method for constructing long DNA molecules. ( a ) In the first PCR step a pool of oligodeoxynucleotides anneal and are ( b ) elongated to produce a full-length DNA molecule. In addition to the full-length product, a host of shorter molecules also results. ( c ) In the second PCR step the desired full-length molecule is selectively amplified from the mixture using primers specific for the desired full-length product.
    Figure Legend Snippet: The assembly PCR method for constructing long DNA molecules. ( a ) In the first PCR step a pool of oligodeoxynucleotides anneal and are ( b ) elongated to produce a full-length DNA molecule. In addition to the full-length product, a host of shorter molecules also results. ( c ) In the second PCR step the desired full-length molecule is selectively amplified from the mixture using primers specific for the desired full-length product.

    Techniques Used: Polymerase Cycling Assembly, Polymerase Chain Reaction, Amplification

    45) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.
    Figure Legend Snippet: Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.

    Techniques Used: Functional Assay, Incubation, Mutagenesis, Variant Assay, Concentration Assay

    Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.
    Figure Legend Snippet: Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

    Techniques Used: Sequencing, Software, Incubation, SDS Page, Autoradiography, Electrophoresis, Expressing, Plasmid Preparation

    Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.
    Figure Legend Snippet: Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Marker

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    46) Product Images from "Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay"

    Article Title: Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01372

    Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.
    Figure Legend Snippet: Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.
    Figure Legend Snippet: LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Positive Control, Negative Control, Marker

    Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Agarose Gel Electrophoresis, Fluorescence, Marker

    Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Polymerase Chain Reaction, Purification, Fluorescence, Agarose Gel Electrophoresis, Marker

    47) Product Images from "PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications"

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    Journal: Scientific Reports

    doi: 10.1038/srep05052

    Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .
    Figure Legend Snippet: Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Techniques Used: Binding Assay, Amplification, Polymerase Chain Reaction

    Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .
    Figure Legend Snippet: Primers that anneal far away from the repetitive DNA perform much better in amplifying the desired product. Taq DNA polymerase (NE Biolabs) was used in the PCR amplification of the indicated region of the pdTALE12 plasmid. PCR conditions are given in the supplementary material .

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation

    PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .
    Figure Legend Snippet: PCR fragments generated upon a typical PCR amplification from the pTAL2 vector with 12.5 TALE DNA-binding repeats. Plasmid map is shown in Supplementary Fig. 3 . Proofreading Pfu polymerase (Bioline) and normal Taq polymerase (NE Biolabs) were used in PCR amplification. PCR conditions are described in the supplementary material .

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Binding Assay

    Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.
    Figure Legend Snippet: Testing the generality of the model to other template DNAs with repetitive sequences. (A). GFP-coding sequences were cloned into the pBasicS1 vector and their integrity were checked by sequencing and restriction enzyme digestions. (B). PCR results obtained with primers 390 and 570. Taq DNA polymerase (NE Biolabs) was used in PCR amplification. The arrow indicates the sequenced artifact product which contained only one copy of the GFP lacking the filler sequence. See the supplementary material for PCR conditions.

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    48) Product Images from "Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity"

    Article Title: Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20120876

    Increased numbers of ribonucleotides in genomic DNA of RNase H2–deficient embryos. Large chromosomal fragments obtained by standard DNA purification were nicked by bacterial RNase HII at ribonucleotide positions, followed by DNA polymerase I–dependent nick translation in the presence of 32 P-dCTP. Labeled DNA was run on a 1% agarose gel (focusing the large chromosomal fragments [several hundred kilobases] in a distinct band) and visualized by autoradiography. Control samples were not digested with RNase HII to detect background single strand breaks. (A) DNA from RNase H2–deficient ( rnh201Δ ) or control yeast (Ctrl) was digested with increasing amounts (1–10 mU/µl) of RNase HII or subjected to nick translation without RNase HII pretreatment. (B) 200 ng DNA from WT (Ctrl) and Rnaseh2b Δ/Δ embryos (obtained by crossing Rnaseh2b FLOX mice with PGK-Cre mice) was digested with 10 mU/µl RNase HII before nick translation. Negative control samples from each embryo were not pretreated with RNase HII. DNA from RNase H2–proficient (WT) and –deficient (Δ) yeast served as controls. (C) Experiment as in B performed with DNA from Rnaseh2c −/− and WT control embryos. All panels in A, B, and C originate from the same exposure of one single gel each.
    Figure Legend Snippet: Increased numbers of ribonucleotides in genomic DNA of RNase H2–deficient embryos. Large chromosomal fragments obtained by standard DNA purification were nicked by bacterial RNase HII at ribonucleotide positions, followed by DNA polymerase I–dependent nick translation in the presence of 32 P-dCTP. Labeled DNA was run on a 1% agarose gel (focusing the large chromosomal fragments [several hundred kilobases] in a distinct band) and visualized by autoradiography. Control samples were not digested with RNase HII to detect background single strand breaks. (A) DNA from RNase H2–deficient ( rnh201Δ ) or control yeast (Ctrl) was digested with increasing amounts (1–10 mU/µl) of RNase HII or subjected to nick translation without RNase HII pretreatment. (B) 200 ng DNA from WT (Ctrl) and Rnaseh2b Δ/Δ embryos (obtained by crossing Rnaseh2b FLOX mice with PGK-Cre mice) was digested with 10 mU/µl RNase HII before nick translation. Negative control samples from each embryo were not pretreated with RNase HII. DNA from RNase H2–proficient (WT) and –deficient (Δ) yeast served as controls. (C) Experiment as in B performed with DNA from Rnaseh2c −/− and WT control embryos. All panels in A, B, and C originate from the same exposure of one single gel each.

    Techniques Used: DNA Purification, Nick Translation, Labeling, Agarose Gel Electrophoresis, Autoradiography, Mouse Assay, Negative Control

    49) Product Images from "Construction of a circular single-stranded DNA template containing a defined lesion"

    Article Title: Construction of a circular single-stranded DNA template containing a defined lesion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.006

    ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.
    Figure Legend Snippet: ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.

    Techniques Used: Sequencing, Binding Assay, Labeling, In Vitro

    50) Product Images from "The Full-length Saccharomyces cerevisiae Sgs1 Protein Is a Vigorous DNA Helicase That Preferentially Unwinds Holliday Junctions *"

    Article Title: The Full-length Saccharomyces cerevisiae Sgs1 Protein Is a Vigorous DNA Helicase That Preferentially Unwinds Holliday Junctions *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.083196

    Sgs1 can unwind long lengths of dsDNA. A , λ phage DNA was digested with HindIII and 32 P-labeled with Klenow fragment of DNA polymerase I at the 3′-ends. This substrate (each fragment at 50 p m molecules) was used for helicase assays at
    Figure Legend Snippet: Sgs1 can unwind long lengths of dsDNA. A , λ phage DNA was digested with HindIII and 32 P-labeled with Klenow fragment of DNA polymerase I at the 3′-ends. This substrate (each fragment at 50 p m molecules) was used for helicase assays at

    Techniques Used: Labeling

    51) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    52) Product Images from "The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1"

    Article Title: The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1

    Journal: Blood

    doi: 10.1182/blood-2008-12-195768

    DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)
    Figure Legend Snippet: DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)

    Techniques Used:

    53) Product Images from "Directed evolution of protein enzymes using nonhomologous random recombination"

    Article Title: Directed evolution of protein enzymes using nonhomologous random recombination

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0402202101

    Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry
    Figure Legend Snippet: Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry

    Techniques Used: Ligation

    54) Product Images from "Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *"

    Article Title: Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.223495

    DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated
    Figure Legend Snippet: DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated

    Techniques Used: In Vitro, Incubation

    55) Product Images from "Investigation of the Streptomyces clavuligerus Cephamycin C Gene Cluster and Its Regulation by the CcaR Protein"

    Article Title: Investigation of the Streptomyces clavuligerus Cephamycin C Gene Cluster and Its Regulation by the CcaR Protein

    Journal: Journal of Bacteriology

    doi:

    Strategy for disruption or deletion of the ccaR gene or deletion of the cob group of genes by gene replacement. The lightly shaded boxes represent the target gene(s), the darker boxes represent other ORFs within the cephamycin cluster, and the clear box represents the antibiotic resistance marker. The antibiotic resistance markers were introduced in both orientations (A and B). (A) Insertion of the apr cassette into the Eco ICRI site of the ccaR gene; (B) deletion of the internal Bam HI/ Nru I fragment containing the ccaR gene and replacement with the tsr marker; (C) deletion of the internal Bam HI/ Eco RI fragment containing the ccaR , orf11 , and blp genes and replacement with the tsr marker; (D) Southern hybridization pattern of Kpn I-digested genomic DNA from the wild type and ccaR :: apr , Δ ccaR :: tsr , and Δ cob :: tsr mutants, using the 1.7- and 3.7-kb Kpn I fragments as hybridization probes. Abbreviations for restriction sites: B, Bam HI; E, Eco RI; Ec, Eco ICRI; K, Kpn I; N, Nru I.
    Figure Legend Snippet: Strategy for disruption or deletion of the ccaR gene or deletion of the cob group of genes by gene replacement. The lightly shaded boxes represent the target gene(s), the darker boxes represent other ORFs within the cephamycin cluster, and the clear box represents the antibiotic resistance marker. The antibiotic resistance markers were introduced in both orientations (A and B). (A) Insertion of the apr cassette into the Eco ICRI site of the ccaR gene; (B) deletion of the internal Bam HI/ Nru I fragment containing the ccaR gene and replacement with the tsr marker; (C) deletion of the internal Bam HI/ Eco RI fragment containing the ccaR , orf11 , and blp genes and replacement with the tsr marker; (D) Southern hybridization pattern of Kpn I-digested genomic DNA from the wild type and ccaR :: apr , Δ ccaR :: tsr , and Δ cob :: tsr mutants, using the 1.7- and 3.7-kb Kpn I fragments as hybridization probes. Abbreviations for restriction sites: B, Bam HI; E, Eco RI; Ec, Eco ICRI; K, Kpn I; N, Nru I.

    Techniques Used: Marker, Hybridization

    The cob group of genes and pSET152 complementation constructs created with these genes. (A) Restriction map of the region of DNA containing the cob genes and unique restriction sites present within this region; (B) diagram of wild-type and mutant constructs created in pSET152 for the complementation experiments using the Δ ccaR :: tsr or Δ cob :: tsr deletion mutant strains. Asterisks represent locations of stop codon-containing TSFs.
    Figure Legend Snippet: The cob group of genes and pSET152 complementation constructs created with these genes. (A) Restriction map of the region of DNA containing the cob genes and unique restriction sites present within this region; (B) diagram of wild-type and mutant constructs created in pSET152 for the complementation experiments using the Δ ccaR :: tsr or Δ cob :: tsr deletion mutant strains. Asterisks represent locations of stop codon-containing TSFs.

    Techniques Used: Construct, Mutagenesis

    56) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Long-range PCR with mixtures of Taq and Neq DNA polymerases. λ DNA fragments of 10 kb (a) and 20 kb (b) were amplified using mixtures of Taq and Neq DNA polymerases at the indicated ratios. Lanes M1 and M3, DNA molecular size markers (1-kb ladder); lane M2, λ/HindIII DNA size markers; lane 1, Taq DNA polymerase; lanes 2 to 11, mixtures of Taq and Neq DNA polymerases in various ratios.
    Figure Legend Snippet: Long-range PCR with mixtures of Taq and Neq DNA polymerases. λ DNA fragments of 10 kb (a) and 20 kb (b) were amplified using mixtures of Taq and Neq DNA polymerases at the indicated ratios. Lanes M1 and M3, DNA molecular size markers (1-kb ladder); lane M2, λ/HindIII DNA size markers; lane 1, Taq DNA polymerase; lanes 2 to 11, mixtures of Taq and Neq DNA polymerases in various ratios.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Extension efficiency of Neq DNA polymerase. The amplification of the λ DNA fragments was performed in a 50-μl reaction mixture containing the optimized PCR buffer for Neq DNA polymerase. Lane M, DNA molecular size markers; lanes 1 to 6, amplified λ DNA fragments of indicated target sizes (kb).
    Figure Legend Snippet: Extension efficiency of Neq DNA polymerase. The amplification of the λ DNA fragments was performed in a 50-μl reaction mixture containing the optimized PCR buffer for Neq DNA polymerase. Lane M, DNA molecular size markers; lanes 1 to 6, amplified λ DNA fragments of indicated target sizes (kb).

    Techniques Used: Amplification, Polymerase Chain Reaction

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.
    Figure Legend Snippet: PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.
    Figure Legend Snippet: Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.

    Techniques Used: Activity Assay, Radioactivity

    Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.
    Figure Legend Snippet: Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay

    Comparison of Neq plus and Taq DNA polymerases, in combination with UDG, in preventing carryover contamination in PCR. To mimic carryover contamination, the 2-kb uracil-DNAs (75 pg) were added to new PCR mixtures that contained 150 pg of the 4-kb target DNAs. The mixtures were preincubated at 25°C for 10 min in the presence (lanes 2 to 7) or absence (lane 1) of 1 U of BMTU 3346 UDG. After heating at 95°C for 5 min, the mixtures were used for normal PCR cycling in the presence of dUTP. PCRs were carried out using Taq DNA polymerase (lanes 2 to 4) and Neq plus DNA polymerase (lanes 1 and 5 to 7). Lane M, DNA molecular size markers; lanes 2 and 5, 20 cycles; lanes 3 and 6, 25 cycles; lanes 1, 4, and 7, 30 cycles.
    Figure Legend Snippet: Comparison of Neq plus and Taq DNA polymerases, in combination with UDG, in preventing carryover contamination in PCR. To mimic carryover contamination, the 2-kb uracil-DNAs (75 pg) were added to new PCR mixtures that contained 150 pg of the 4-kb target DNAs. The mixtures were preincubated at 25°C for 10 min in the presence (lanes 2 to 7) or absence (lane 1) of 1 U of BMTU 3346 UDG. After heating at 95°C for 5 min, the mixtures were used for normal PCR cycling in the presence of dUTP. PCRs were carried out using Taq DNA polymerase (lanes 2 to 4) and Neq plus DNA polymerase (lanes 1 and 5 to 7). Lane M, DNA molecular size markers; lanes 2 and 5, 20 cycles; lanes 3 and 6, 25 cycles; lanes 1, 4, and 7, 30 cycles.

    Techniques Used: Polymerase Chain Reaction

    57) Product Images from "Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces"

    Article Title: Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    Journal: Scientific Reports

    doi: 10.1038/srep12066

    Analysis of P/DNA/dNTP interactions and enzymatic activity of Pol I(KF). A – C Association, dissociation, and elongation under different conditions: association in Mg 2+ -buffer, followed by dissociation in Ca 2+ -buffer (diss. 1), elongation in a 100 μ M dNTP-mix, and dissociation of the polymerase in Mg 2+ -buffer (diss. 2). In A and B, the dissociation phase 2 proceeds after the elongation from the end of the extended oligonucleotide primer in the absence and presence of competing DNA, respectively. C is a control where the polymerase dissociates in the absence of dNTPs but presence of competing DNA in solution. Lines are single exponential fits. D Dissociation rates from dissociation phase 2 as a function of dNTP concentration. The line is a Langmuir fit and yields the dissociation constant for the binding of dNTPs by a polymerase located at the end of dsDNA (without the influence of base-pairing). E : Elongation monitored in real-time by the fluorescence emitted from standing DNA ( F up ) for different dNTP concentrations. F : Elongation rates from E (linear slope) plotted as a function of the dNTP concentration. The line is a Michaelis-Menten fit with K M being the Michaelis constant.
    Figure Legend Snippet: Analysis of P/DNA/dNTP interactions and enzymatic activity of Pol I(KF). A – C Association, dissociation, and elongation under different conditions: association in Mg 2+ -buffer, followed by dissociation in Ca 2+ -buffer (diss. 1), elongation in a 100 μ M dNTP-mix, and dissociation of the polymerase in Mg 2+ -buffer (diss. 2). In A and B, the dissociation phase 2 proceeds after the elongation from the end of the extended oligonucleotide primer in the absence and presence of competing DNA, respectively. C is a control where the polymerase dissociates in the absence of dNTPs but presence of competing DNA in solution. Lines are single exponential fits. D Dissociation rates from dissociation phase 2 as a function of dNTP concentration. The line is a Langmuir fit and yields the dissociation constant for the binding of dNTPs by a polymerase located at the end of dsDNA (without the influence of base-pairing). E : Elongation monitored in real-time by the fluorescence emitted from standing DNA ( F up ) for different dNTP concentrations. F : Elongation rates from E (linear slope) plotted as a function of the dNTP concentration. The line is a Michaelis-Menten fit with K M being the Michaelis constant.

    Techniques Used: Activity Assay, Concentration Assay, Binding Assay, Fluorescence

    58) Product Images from "Efficient purification of DNA fragments using a protein binding membrane"

    Article Title: Efficient purification of DNA fragments using a protein binding membrane

    Journal: Nucleic Acids Research

    doi:

    Retainment of labelled lambda Hin dIII restricted DNA fragments by the membrane cartridge. Lambda Hin dIII DNA fragments are labelled with streptavidin in ( A ), and with fluorescein in ( B ). An aliquot of 1 µg lambda Hin dIII digested DNA fragments were used in each lane. Lane 1, unlabelled lambda Hin dIII fragments; lane 2, unlabelled lambda Hin dIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 3, unlabelled lambda Hin dIII fragments incubated with 5 µg streptavidin and passed through the membrane cartridge; lanes 4 and 5, empty; lane 6, labelled lambda Hin dIII fragments; lane 7, labelled lambda Hin dIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 8, labelled lambda Hin dIII fragments incubated with 5 µg streptavidin (A) or anti-fluorescein antibody (B) and passed through the membrane cartridge.
    Figure Legend Snippet: Retainment of labelled lambda Hin dIII restricted DNA fragments by the membrane cartridge. Lambda Hin dIII DNA fragments are labelled with streptavidin in ( A ), and with fluorescein in ( B ). An aliquot of 1 µg lambda Hin dIII digested DNA fragments were used in each lane. Lane 1, unlabelled lambda Hin dIII fragments; lane 2, unlabelled lambda Hin dIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 3, unlabelled lambda Hin dIII fragments incubated with 5 µg streptavidin and passed through the membrane cartridge; lanes 4 and 5, empty; lane 6, labelled lambda Hin dIII fragments; lane 7, labelled lambda Hin dIII fragments incubated with 5 µg BSA and passed through the membrane cartridge; lane 8, labelled lambda Hin dIII fragments incubated with 5 µg streptavidin (A) or anti-fluorescein antibody (B) and passed through the membrane cartridge.

    Techniques Used: Incubation

    Purification of digested DNA. An aliquot of 200 ng of DNA was used for each lane. ( A ) Lane 1, 100 bp DNA size standard; lane 2, empty; lane 3, PCR product; lane 4, PCR product added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, PCR product restricted by Sfi I endonuclease; lane 7, PCR product restricted by Sfi I endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, PCR product restricted by Not I endonuclease; lane 10, PCR product restricted by Not I endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, PCR product restricted by Not I and Sfi I endonucleases; lane 13, PCR product restricted by Not I and Sfi I endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, 100 bp DNA size standard. ( B ) Lane 1, Lambda Hin dIII size standard; lane 2, empty; lane 3, Asc I digested and biotin labelled plasmid DNA; lane 4, Asc I digested and biotin labelled plasmid DNA added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, Asc I digested and biotin labelled plasmid DNA restricted by Sfi I endonuclease; lane 7, Asc I digested and biotin labelled plasmid DNA restricted by Sfi I endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, Asc I digested and biotin labelled plasmid DNA restricted by Not I endonuclease; lane 10, Asc I digested and biotin labelled plasmid DNA restricted by Not I endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, Asc I digested and biotin labelled plasmid DNA restricted by Not I and Sfi I endonucleases; lane 13, Asc I digested and biotin labelled plasmid DNA restricted by Not I and Sfi I endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, Lambda Hin dIII size standard.
    Figure Legend Snippet: Purification of digested DNA. An aliquot of 200 ng of DNA was used for each lane. ( A ) Lane 1, 100 bp DNA size standard; lane 2, empty; lane 3, PCR product; lane 4, PCR product added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, PCR product restricted by Sfi I endonuclease; lane 7, PCR product restricted by Sfi I endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, PCR product restricted by Not I endonuclease; lane 10, PCR product restricted by Not I endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, PCR product restricted by Not I and Sfi I endonucleases; lane 13, PCR product restricted by Not I and Sfi I endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, 100 bp DNA size standard. ( B ) Lane 1, Lambda Hin dIII size standard; lane 2, empty; lane 3, Asc I digested and biotin labelled plasmid DNA; lane 4, Asc I digested and biotin labelled plasmid DNA added streptavidin and passed through the membrane cartridge; lane 5, empty; lane 6, Asc I digested and biotin labelled plasmid DNA restricted by Sfi I endonuclease; lane 7, Asc I digested and biotin labelled plasmid DNA restricted by Sfi I endonuclease added streptavidin and passed through the membrane cartridge; lane 8, empty; lane 9, Asc I digested and biotin labelled plasmid DNA restricted by Not I endonuclease; lane 10, Asc I digested and biotin labelled plasmid DNA restricted by Not I endonuclease added streptavidin and passed through the membrane cartridge; lane 11, empty; lane 12, Asc I digested and biotin labelled plasmid DNA restricted by Not I and Sfi I endonucleases; lane 13, Asc I digested and biotin labelled plasmid DNA restricted by Not I and Sfi I endonucleases added streptavidin and passed through the membrane cartridge; lane 14, empty; lane 15, Lambda Hin dIII size standard.

    Techniques Used: Purification, Polymerase Chain Reaction, Plasmid Preparation

    59) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.
    Figure Legend Snippet: Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.

    Techniques Used: Mutagenesis, Sequencing, SDS Page

    Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.
    Figure Legend Snippet: Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

    Techniques Used: Sequencing, Software, Incubation, SDS Page, Autoradiography, Electrophoresis, Expressing, Plasmid Preparation

    Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.
    Figure Legend Snippet: Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Marker

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    60) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.
    Figure Legend Snippet: Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.

    Techniques Used: Functional Assay, Incubation, Mutagenesis, Variant Assay, Concentration Assay

    61) Product Images from "Regression of Replication Forks Stalled by Leading-strand Template Damage: II. REGRESSION BY RecA IS INHIBITED BY SSB*"

    Article Title: Regression of Replication Forks Stalled by Leading-strand Template Damage: II. REGRESSION BY RecA IS INHIBITED BY SSB*

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.587907

    SSB stimulates RuvC cleavage of deproteinized stalled forks. A , standard RFR reaction mixtures with deproteinized stalled forks formed in the presence of DNA polymerase I, RNase H, and DNA ligase containing 0.25–2 μ m SSB (increasing in
    Figure Legend Snippet: SSB stimulates RuvC cleavage of deproteinized stalled forks. A , standard RFR reaction mixtures with deproteinized stalled forks formed in the presence of DNA polymerase I, RNase H, and DNA ligase containing 0.25–2 μ m SSB (increasing in

    Techniques Used:

    Formation of a RFR product by RecA in the presence of the replisome is stimulated by sealing the Okazaki fragments. Stalled forks formed in either the absence ( without Ligase ; A) or presence ( with Ligase ; B ) of DNA polymerase I, RNase H, and DNA ligase
    Figure Legend Snippet: Formation of a RFR product by RecA in the presence of the replisome is stimulated by sealing the Okazaki fragments. Stalled forks formed in either the absence ( without Ligase ; A) or presence ( with Ligase ; B ) of DNA polymerase I, RNase H, and DNA ligase

    Techniques Used:

    62) Product Images from "Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes"

    Article Title: Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagen.2009.11.003

    Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.
    Figure Legend Snippet: Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.

    Techniques Used: Binding Assay, Sequencing

    Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.
    Figure Legend Snippet: Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Quantitation Assay, Nucleic Acid Electrophoresis, Amplification, Software

    Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.
    Figure Legend Snippet: Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.

    Techniques Used: In Silico, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

    63) Product Images from "Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †"

    Article Title: Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †

    Journal: Biochemistry

    doi: 10.1021/bi026985+

    Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).
    Figure Legend Snippet: Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).

    Techniques Used: Modification, Labeling

    Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.
    Figure Legend Snippet: Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.

    Techniques Used:

    64) Product Images from "Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins"

    Article Title: Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.00870-17

    Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.
    Figure Legend Snippet: Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.

    Techniques Used: Activation Assay, Binding Assay

    Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.
    Figure Legend Snippet: Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.

    Techniques Used: Binding Assay

    65) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Long-range PCR with mixtures of Taq and Neq DNA polymerases. λ DNA fragments of 10 kb (a) and 20 kb (b) were amplified using mixtures of Taq and Neq DNA polymerases at the indicated ratios. Lanes M1 and M3, DNA molecular size markers (1-kb ladder); lane M2, λ/HindIII DNA size markers; lane 1, Taq DNA polymerase; lanes 2 to 11, mixtures of Taq and Neq DNA polymerases in various ratios.
    Figure Legend Snippet: Long-range PCR with mixtures of Taq and Neq DNA polymerases. λ DNA fragments of 10 kb (a) and 20 kb (b) were amplified using mixtures of Taq and Neq DNA polymerases at the indicated ratios. Lanes M1 and M3, DNA molecular size markers (1-kb ladder); lane M2, λ/HindIII DNA size markers; lane 1, Taq DNA polymerase; lanes 2 to 11, mixtures of Taq and Neq DNA polymerases in various ratios.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Extension efficiency of Neq DNA polymerase. The amplification of the λ DNA fragments was performed in a 50-μl reaction mixture containing the optimized PCR buffer for Neq DNA polymerase. Lane M, DNA molecular size markers; lanes 1 to 6, amplified λ DNA fragments of indicated target sizes (kb).
    Figure Legend Snippet: Extension efficiency of Neq DNA polymerase. The amplification of the λ DNA fragments was performed in a 50-μl reaction mixture containing the optimized PCR buffer for Neq DNA polymerase. Lane M, DNA molecular size markers; lanes 1 to 6, amplified λ DNA fragments of indicated target sizes (kb).

    Techniques Used: Amplification, Polymerase Chain Reaction

    PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.
    Figure Legend Snippet: PCR amplification with Neq DNA polymerase. (a) Effect of pH on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl, 2 mM MgCl 2 , 50 mM KCl, and 0.01% BSA at the indicated pH values. (b) Effect of MgCl 2 on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.01% BSA with the indicated concentrations of MgCl 2 . (c) Effect of KCl on the PCR amplification with Neq DNA polymerase. The amplification of the 1-kb λ DNA fragment was performed in a 50-μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2 , and 0.01% BSA with the indicated concentrations of KCl. Lane M, DNA molecular size markers.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.
    Figure Legend Snippet: Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay

    Comparison of Neq plus and Taq DNA polymerases, in combination with UDG, in preventing carryover contamination in PCR. To mimic carryover contamination, the 2-kb uracil-DNAs (75 pg) were added to new PCR mixtures that contained 150 pg of the 4-kb target DNAs. The mixtures were preincubated at 25°C for 10 min in the presence (lanes 2 to 7) or absence (lane 1) of 1 U of BMTU 3346 UDG. After heating at 95°C for 5 min, the mixtures were used for normal PCR cycling in the presence of dUTP. PCRs were carried out using Taq DNA polymerase (lanes 2 to 4) and Neq plus DNA polymerase (lanes 1 and 5 to 7). Lane M, DNA molecular size markers; lanes 2 and 5, 20 cycles; lanes 3 and 6, 25 cycles; lanes 1, 4, and 7, 30 cycles.
    Figure Legend Snippet: Comparison of Neq plus and Taq DNA polymerases, in combination with UDG, in preventing carryover contamination in PCR. To mimic carryover contamination, the 2-kb uracil-DNAs (75 pg) were added to new PCR mixtures that contained 150 pg of the 4-kb target DNAs. The mixtures were preincubated at 25°C for 10 min in the presence (lanes 2 to 7) or absence (lane 1) of 1 U of BMTU 3346 UDG. After heating at 95°C for 5 min, the mixtures were used for normal PCR cycling in the presence of dUTP. PCRs were carried out using Taq DNA polymerase (lanes 2 to 4) and Neq plus DNA polymerase (lanes 1 and 5 to 7). Lane M, DNA molecular size markers; lanes 2 and 5, 20 cycles; lanes 3 and 6, 25 cycles; lanes 1, 4, and 7, 30 cycles.

    Techniques Used: Polymerase Chain Reaction

    66) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    67) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    68) Product Images from "C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease"

    Article Title: C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease

    Journal: Nature

    doi: 10.1038/nature13124

    R-loops, and not G-quadruplex formation on nascent RNA transcripts, increase abortive transcription within the C9orf72 HRE region in vitro a) RNA transcripts containing many GGGGCC repeats form G-quadruplexes under physiologically relevant KCl concentrations. A colorimetric assay was performed to identify the formation of RNA G-quadruplexes utilizing the enzyme-like peroxidase activity of G-quadruplex•hemin complexes 29 . b) Workflow considerations for the transcriptional assay. The linear plasmid was first annealed ± 100 mM KCl or 100 mM NaCl in 10 mM Tris-HCl, pH 7.4. To prevent salt concentration-dependent effects on the in vitro transcriptional assay, a second adjustment was made to adjust the salts to a final 50 mM concentration in the assay. Reducing the effects on RNA polymerase allowed us to disambiguate the effects of salt on the conformation of DNA versus the nascent RNA. Comparison of DNA annealed in 0 mM KCl and 100 mM NaCl shows similar reduced polymerase processivity, suggesting that possible formation of G-quadruplexes on the nascent RNA transcripts makes a negligible contribution, but does not exclude intrinsic RNA hairpin-induced termination. c) RNase H treatment during transcription reduces the periodic accumulation of abortive transcripts and increases full-length transcripts. Treatment with RNase H, which specifically cleaves RNA•DNA hybrids, during transcription of the C9orf72 repeats causes a shift from truncated transcripts to full-length transcripts, suggestive of the formation of alternative secondary structures, known as R-loops, caused by increased nonduplex DNA during transcription. d) R-loop formation is observed for the plasmid containing the HRE insert, (GGGGCC) 21 , but not for a GFP insert in a control. Treatment with RNase A removes ssRNA but does not affect R-loops. As shown in Figure 2c , the HREs induce the formation of R-loops that cause a decrease in the plasmid mobility but can be relieved with RNase H treatment, which specifically cleaves RNA•DNA hybrids. The addition of RNase A and RNase H has little effect on the mobility of the plasmid containing the GFP insert. Radiolabeling the transcriptional products during in vitro transcription confirms the formation of R-loops, demonstrated by the shift consistent with the supercoiled and relaxed plasmids having altered mobility, which is relieved by treatment with both RNases. The transcripts were bodylabeled by including 20 μCi of α-[ 32 P]UTP (Perkin Elmer) and then performing the in vitro transcription as previously described. e) There is a significant increase in the R-loop-induced plasmid mobility shift for the (GGGGCC) 21 and (GGGGCC) ~70 containing plasmids when compared to the GFP control insert. The shifted supercoiled plasmid bands were quantified by densitometrically measuring each band intensity after treatment with RNase A versus those after treatment with both RNase A and Rnase H (ImageJ, NIH). The overlapping densitometric signal of the supercoiled R-loop smear with the circular plasmid band ( Figure 2b ) prevented accurate quantification and was excluded. Data are means ± s.e.m. n = 3. * P
    Figure Legend Snippet: R-loops, and not G-quadruplex formation on nascent RNA transcripts, increase abortive transcription within the C9orf72 HRE region in vitro a) RNA transcripts containing many GGGGCC repeats form G-quadruplexes under physiologically relevant KCl concentrations. A colorimetric assay was performed to identify the formation of RNA G-quadruplexes utilizing the enzyme-like peroxidase activity of G-quadruplex•hemin complexes 29 . b) Workflow considerations for the transcriptional assay. The linear plasmid was first annealed ± 100 mM KCl or 100 mM NaCl in 10 mM Tris-HCl, pH 7.4. To prevent salt concentration-dependent effects on the in vitro transcriptional assay, a second adjustment was made to adjust the salts to a final 50 mM concentration in the assay. Reducing the effects on RNA polymerase allowed us to disambiguate the effects of salt on the conformation of DNA versus the nascent RNA. Comparison of DNA annealed in 0 mM KCl and 100 mM NaCl shows similar reduced polymerase processivity, suggesting that possible formation of G-quadruplexes on the nascent RNA transcripts makes a negligible contribution, but does not exclude intrinsic RNA hairpin-induced termination. c) RNase H treatment during transcription reduces the periodic accumulation of abortive transcripts and increases full-length transcripts. Treatment with RNase H, which specifically cleaves RNA•DNA hybrids, during transcription of the C9orf72 repeats causes a shift from truncated transcripts to full-length transcripts, suggestive of the formation of alternative secondary structures, known as R-loops, caused by increased nonduplex DNA during transcription. d) R-loop formation is observed for the plasmid containing the HRE insert, (GGGGCC) 21 , but not for a GFP insert in a control. Treatment with RNase A removes ssRNA but does not affect R-loops. As shown in Figure 2c , the HREs induce the formation of R-loops that cause a decrease in the plasmid mobility but can be relieved with RNase H treatment, which specifically cleaves RNA•DNA hybrids. The addition of RNase A and RNase H has little effect on the mobility of the plasmid containing the GFP insert. Radiolabeling the transcriptional products during in vitro transcription confirms the formation of R-loops, demonstrated by the shift consistent with the supercoiled and relaxed plasmids having altered mobility, which is relieved by treatment with both RNases. The transcripts were bodylabeled by including 20 μCi of α-[ 32 P]UTP (Perkin Elmer) and then performing the in vitro transcription as previously described. e) There is a significant increase in the R-loop-induced plasmid mobility shift for the (GGGGCC) 21 and (GGGGCC) ~70 containing plasmids when compared to the GFP control insert. The shifted supercoiled plasmid bands were quantified by densitometrically measuring each band intensity after treatment with RNase A versus those after treatment with both RNase A and Rnase H (ImageJ, NIH). The overlapping densitometric signal of the supercoiled R-loop smear with the circular plasmid band ( Figure 2b ) prevented accurate quantification and was excluded. Data are means ± s.e.m. n = 3. * P

    Techniques Used: Abortive Initiation Assay, In Vitro, Colorimetric Assay, Activity Assay, Transcription Factor Assay, Plasmid Preparation, Concentration Assay, Radioactivity, Mobility Shift

    69) Product Images from "New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase"

    Article Title: New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37513-7

    Processivity assay of ϕ29 DNA polymerase mutant Y101A. The assay was carried out as described in Materials and Methods by using a 5′ labelled sp1/sp1c + 18 (15/33 mer) depicted at the top of the figure as substrate, in the presence of the indicated concentrations of wild-type or mutant ϕ29 DNA polymerases. As a control of non-processive elongation, the Klenow DNA polymerase (units) was used. Asterisk indicates the 5′- 32 P-labelled end of the primer strand. c: control DNA.
    Figure Legend Snippet: Processivity assay of ϕ29 DNA polymerase mutant Y101A. The assay was carried out as described in Materials and Methods by using a 5′ labelled sp1/sp1c + 18 (15/33 mer) depicted at the top of the figure as substrate, in the presence of the indicated concentrations of wild-type or mutant ϕ29 DNA polymerases. As a control of non-processive elongation, the Klenow DNA polymerase (units) was used. Asterisk indicates the 5′- 32 P-labelled end of the primer strand. c: control DNA.

    Techniques Used: Mutagenesis

    70) Product Images from "DNA Polymerase I Is Essential for Growth of Methylobacterium dichloromethanicum DM4 with Dichloromethane"

    Article Title: DNA Polymerase I Is Essential for Growth of Methylobacterium dichloromethanicum DM4 with Dichloromethane

    Journal: Journal of Bacteriology

    doi:

    DNA polymerase I activity of wild-type M. dichloromethanicum DM4 and of the polA mutant. Shown is an autoradiogram of cell extract protein (100 μg) of wild-type strain DM4 (lane 1), mutant DM4-1445 (lane 2), and complemented mutant DM4-1445(pME8112) (lane 3), separated by SDS-PAGE in a gel containing nicked DNA, after incubation with dideoxynucleotides and α- 32 P-labeled dCTP (see Materials and Methods). Lane M, prestained marker from the scanned gel; lanes 4 and 5, E. coli DNA polymerase I and Klenow fragment, respectively (0.02 U [∼0.1 ng] each).
    Figure Legend Snippet: DNA polymerase I activity of wild-type M. dichloromethanicum DM4 and of the polA mutant. Shown is an autoradiogram of cell extract protein (100 μg) of wild-type strain DM4 (lane 1), mutant DM4-1445 (lane 2), and complemented mutant DM4-1445(pME8112) (lane 3), separated by SDS-PAGE in a gel containing nicked DNA, after incubation with dideoxynucleotides and α- 32 P-labeled dCTP (see Materials and Methods). Lane M, prestained marker from the scanned gel; lanes 4 and 5, E. coli DNA polymerase I and Klenow fragment, respectively (0.02 U [∼0.1 ng] each).

    Techniques Used: Activity Assay, Mutagenesis, SDS Page, Incubation, Labeling, Marker

    71) Product Images from "Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells"

    Article Title: Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-015-0205-1

    Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products
    Figure Legend Snippet: Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products

    Techniques Used: Flow Cytometry, Modification, Produced, Amplification, Polymerase Chain Reaction

    72) Product Images from "Transposable Element 'roo' Attaches to Nuclear Matrix of the Drosophila melanogaster"

    Article Title: Transposable Element 'roo' Attaches to Nuclear Matrix of the Drosophila melanogaster

    Journal: Journal of Insect Science

    doi: 10.1673/031.013.11101

    Analysis of roo MAR sequence. A: Genome view of distribution of roo MAR sequence in Drosophila melanogaster . B: Analysis of roo MAR with MAR-WIZ program. The regions with matrix association potential are shown as peaks in the graph. The matrix potential is shown on the Y-axis, and DNA in base pairs is shown on the X-axis. Sequences corresponding to the peaks are given below. Sequences relevant for MAR association are underlined. C: Sequence alignment of the roo MAR with the matrix-associated region of the gypsy transposable element using ClustalW program. On the gypsy sequence, topoisomerase II cleavage sites are marked with brackets and labelled 1–7. Sequences following ATC rule and an A-box are underlined. High quality figures are available online.
    Figure Legend Snippet: Analysis of roo MAR sequence. A: Genome view of distribution of roo MAR sequence in Drosophila melanogaster . B: Analysis of roo MAR with MAR-WIZ program. The regions with matrix association potential are shown as peaks in the graph. The matrix potential is shown on the Y-axis, and DNA in base pairs is shown on the X-axis. Sequences corresponding to the peaks are given below. Sequences relevant for MAR association are underlined. C: Sequence alignment of the roo MAR with the matrix-associated region of the gypsy transposable element using ClustalW program. On the gypsy sequence, topoisomerase II cleavage sites are marked with brackets and labelled 1–7. Sequences following ATC rule and an A-box are underlined. High quality figures are available online.

    Techniques Used: Sequencing

    A: Sequence of MAR18 ( roo transposon) clone found in MAR of Drosophila melanogaster . B: Southern blot analysis of PCR amplified roo LTR and control regions. Left panel shows the resolution of PCR amplicons on a 1.2% agarose gel. roo LTR (lane 1), Wnt4 control (lane 2), Arc control (lane 3), Wnt6 control (lane 4), 100 bp ladder (lane M). The right panel shows Southern hybridization of the gel with 32P-labelled MAR DNA. High quality figures are available online.
    Figure Legend Snippet: A: Sequence of MAR18 ( roo transposon) clone found in MAR of Drosophila melanogaster . B: Southern blot analysis of PCR amplified roo LTR and control regions. Left panel shows the resolution of PCR amplicons on a 1.2% agarose gel. roo LTR (lane 1), Wnt4 control (lane 2), Arc control (lane 3), Wnt6 control (lane 4), 100 bp ladder (lane M). The right panel shows Southern hybridization of the gel with 32P-labelled MAR DNA. High quality figures are available online.

    Techniques Used: Sequencing, Southern Blot, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Hybridization

    A: Flow chart of steps used for the isolation of MAR DNA from Drosophila melanogaster embryos. B: Ethidium bromide stained 1% agarose gel showing size distribution of MAR DNA from D. melanogaster embryos. Genomic DNA (lane 1); MAR DNA (lane 2); Isolated MAR DNA digested with DNase I (lane 3); 100 bp DNA marker (Lane M). High quality figures are available online.
    Figure Legend Snippet: A: Flow chart of steps used for the isolation of MAR DNA from Drosophila melanogaster embryos. B: Ethidium bromide stained 1% agarose gel showing size distribution of MAR DNA from D. melanogaster embryos. Genomic DNA (lane 1); MAR DNA (lane 2); Isolated MAR DNA digested with DNase I (lane 3); 100 bp DNA marker (Lane M). High quality figures are available online.

    Techniques Used: Flow Cytometry, Isolation, Staining, Agarose Gel Electrophoresis, Marker

    73) Product Images from "Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2"

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.013946-0

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Figure Legend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Techniques Used: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    74) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.
    Figure Legend Snippet: Functional characterization of mutants in the Y194 priming residue. ( A ) Template-independent TP-deoxyadenylation products of wild type (lane 1) and increasing concentrations of Y194F and Y194A TP mutants. Reactions were carried out triggered with 1 mM MnCl 2 and incubated for 30 min. ( B ) Comparative analysis of wild type and Y194A and Y194F mutant TPs interaction with the DNA polymerase. The reactions were triggered with 1 mM MnCl 2 in the presence of the indicated TP variant and, after 2.5 min, the competitor YFPTP fusion protein was added and the samples were incubated again for 2.5 min. See Materials and Methods for details. The effect of the TP variants concentration on the relative YFPTP deoxyadenylation, from three independent experiments (mean and standard error), is shown in panel C.

    Techniques Used: Functional Assay, Incubation, Mutagenesis, Variant Assay, Concentration Assay

    Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.
    Figure Legend Snippet: Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

    Techniques Used: Sequencing, Software, Incubation, SDS Page, Autoradiography, Electrophoresis, Expressing, Plasmid Preparation

    75) Product Images from "Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes"

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks316

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.
    Figure Legend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Techniques Used: Purification, Polymerase Chain Reaction, Construct

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    Amplification:

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    Synthesized:

    Article Title: LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells
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    Autoradiography:

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    Construct:

    Article Title: LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells
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    Real-time Polymerase Chain Reaction:

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    Microarray:

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    Incubation:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
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    Article Title: Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays
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    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
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    Article Title: Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon
    Article Snippet: .. Second-strand synthesis was for 2 h at 16°C in a total reaction volume of 50 μl containing first-strand reaction products, second-strand buffer (Invitrogen), 250 μM deoxynucleoside triphosphates, 0.06 U of DNA ligase (Ambion) per μl, 0.26 U of DNA polymerase I (New England Biolabs) per μl, and 0.012 U of RNase H (Ambion) per μl followed by the addition of 3.3 U of T4 DNA polymerase (3 U per μl; New England Biolabs) and a further 15 min of incubation at 16°C. .. Second-strand reaction products were purified by phenol-chloroform-isoamyl alcohol extraction in Phaselock microcentrifuge tubes (Eppendorf) according to the manufacturer's instructions and ethanol precipitated.

    Luciferase:

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    Modification:

    Article Title: Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon
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    Western Blot:

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    Derivative Assay:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs). .. Following ligation of library adapters , cDNA fragments were treated with uracil-DNA glycosylase (New England Biolabs) to remove uracils introduced during second-strand synthesis so that library fragments produced in the subsequent amplification step were all derived from first-strand cDNA.

    Hybridization:

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro
    Article Snippet: The 3′ end was labeled with the Klenow fragment of DNA polymerase I (New England Biolabs) and [α-32 P]dCTP. .. Hybridization was done at 56°C, and S1 digestion was performed as previously described ( , ).

    Article Title: Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays
    Article Snippet: Two micrograms of total genomic DNA of each of the different strains was used as a template for direct incorporation of [33 P]dATP (Amersham Pharmacia, Freiburg, Germany) by a randomly primed polymerization reaction using 0.75 μg of random hexamer primers (New England Biolabs, Frankfurt [Main], Germany) and 10 U of Klenow fragment of DNA polymerase I (New England Biolabs) according to the manufacturers' recommendations. .. Prior to hybridization, the DNA macroarrays were rinsed in 2× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2 PO4 , and 1 mM EDTA [pH 7.7]) solution and subsequently prehybridized for 3 h at 65°C in 5 ml of hybridization solution (5× SSPE, 2% sodium dodecyl sulfate, 1× Denhardt's solution, 100 μg of sheared denatured herring sperm DNA/ml).

    Article Title: PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ †PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ † ‡
    Article Snippet: The second strand was generated by incubating the first-strand reaction product with Second Strand Reaction Buffer (Invitrogen), DNA ligase (10 units), DNA polymerase I (40 units), and RNase H (2 units) for 2 h at 16°C; 20 units of T4 DNA polymerase was then added, and this step was followed by incubation at 16°C for another 5 min (New England BioLabs). .. At this point the dscDNA was ready for fragmentation and labeling for microarray hybridization.

    Ligation:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs). .. Following ligation of library adapters , cDNA fragments were treated with uracil-DNA glycosylase (New England Biolabs) to remove uracils introduced during second-strand synthesis so that library fragments produced in the subsequent amplification step were all derived from first-strand cDNA.

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp). ..

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). .. Libraries were prepared from a 150–200-bp size-selected fraction following adapter ligation and agarose gel separation.

    Article Title: RNA-Seq analysis to capture the transcriptome landscape of a single cell
    Article Snippet: .. Ac-BSA (Sigma, cat. no. B8894) Acidic Tyrode’s solution (Sigma, cat. no. T1788) 1× PBS (pH 7.2) (Gibco, cat. no. 14249-95) 10× PCR Buffer II and 25 mM MgCl2 (Applied Biosystems, cat. no. 4379878) Nonidet P-40 SP (Roche, cat. no. 11332473001) SuperScript III Reverse Transcriptase with 0.1 M DTT (Invitrogen, cat. no. 18080-044 or 18080-085) RNase Inhibitor (Cloned) (40 U μl−1 ) (Applied Biosystems, cat. no. AM2682) SUPERase-In™ (20 U μl−1 ) (Applied Biosystems, cat. no. AM2694) T4 gene 32 Protein (Roche, cat. no. 972983) Exonuclease I (New England Biolabs, cat. no. M0293S) Terminal Transferase (TdT) (Invitrogen, cat. no. 10533-065 or 10533-073) 100 mM dATP (Promega, cat. no. U1201) Nuclease-free Water, 1 L (Applied Biosystems, cat. no. AM9932) RNase H (Invitrogen, cat. no. 18021-014 or 18021-071) TaKaRa Ex Taq™ HS (Includes: 10× ExTaq Buffer (mg2+ plus) and dNTP mixture) (Takara Bio Inc, cat. no. RR006A or RR006B) QIAquick PCR Purification Kit (250) (Qiagen, cat. no. 28106) QIAquick Gel Extraction Kit (250) (Qiagen, cat. no. 28706) RNeasy Mini Kit (50) (Qiagen, cat. no. 74104) PCR tubes, 0.5 ml (Eppendorf, cat. no. 951010057) PCR tubes, 0.2 ml thin-wall (MLS) Filtered pipettor tips (MLS) SYBR Green PCR Mastermix (Applied Biosystems, cat. no. 4334973) GeneAmp® dNTP Blend (100 mM) (Applied Biosystems, cat. no. N8080261) 3 M sodium acetate (pH 5.5) (Applied Biosystems/Ambion, cat. no. AM9740) 1 M Tris, pH 8.0 (100 ml) (Applied Biosystems/Ambion, AM9855G) Nuclease-free Water (1 L) (Applied Biosystems/Ambion, cat. no. AM9932) 10× NEBuffer2 (New England BioLabs® Inc., B7002S) Ethanol (Sigma-Aldrich®, cat. no. E7023) Ethylene glycol (American Bioanalytical, cat. no. AB00455-01000) Covaris microTUBE with AFA fiber and Snap-Cap with pre-slit Teflon/silicone/Teflon septa (Covaris™ Inc., cat. no. 520045) End-It™ DNA End-Repair Kit (Epicentre®, cat. no. ER0720) DNA Polymerase I (E. coli), (10U μl−1 ) (New England BioLabs® Inc., cat. no. M0209L) Quick Ligation™ kit (New England Biolabs, cat. no. M2200L) SYBR GreenER® qPCR SuperMix Universal (Invitrogen™ Corporation, cat. no. 11762-100) OR SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) Agilent DNA 1000 Kit (Agilent Technologies, cat. no. 5067-1504) Agencourt® AMPure® 60 ml kit (Agencourt, cat. no. 000130) AmpliTaq® DNA Polymerase, LD with Buffer I (Applied Biosystems, cat. no. N8080157) Cloned Pfu polymerase (2.5 U μl−1 ) (Stratagene, cat. no. 600153) Invitrogen Platinum® PCR SuperMix (Invitrogen™ Corporation, cat. no. 11306-016) Quant-iT™ dsDNA HS Assay Kit, 100 assays (Invitrogen™ Corporation, cat. no. ) SOLiD™ Fragment Library Oligos Kit (Applied Biosystems, cat. no. 4401151) .. Brown-flaming micropipette puller (Sutter Instrument Co., Model P-80) Covaris™ S2 System, (for system materials summary, see “Covaris™ S2 System Materials Summary,” SOLiD ™ System 2.0 Site Preparation Guide .)

    Footprinting:

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: Paragraph title: EMSA and DNase I footprinting analysis of the pIJ101 kilB promoter. ... Relevant S. lividans TK23 cultures used to obtain KorB-containing cell extracts were grown as indicated above until culture densities reached 0.5 to 0.7 OD600 units, whereupon they were harvested, and cell extracts containing active KorB protein were prepared as described earlier ( ) in S30 cell lysis buffer, except that subsequent to lysis cell extracts were centrifuged twice for 30 min at 30,000 × g . kilB promoter binding by KorB proteins was performed as described by Tai and Cohen , with the following modifications: a 0.1-kb Eco RI fragment from pGSP311 containing the Fsp I- Bst EII portion of the kilB promoter region was labeled on both ends by using [α-32 P]dATP (6,000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of DNA polymerase I (New England BioLabs), and the labeled fragment was then purified from excess radionuclides by using an S300 column (Amersham Biosciences).

    Nick Translation:

    Article Title: Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression
    Article Snippet: Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen). .. Then, nick translation was carried out with DNA polymerase I (New England Biolabs) and dNTPs for 8 min at 8°C.

    Infection:

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: Total RNA isolated from five M. brunnea f.sp. multigermtubi -infected and control leaves was pooled by treatment, respectively. .. The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).

    Generated:

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro
    Article Snippet: The S1 probe used to determine luciferase RNA levels was generated by linearization of the Luc-SL vector with Dra II, which cuts in the luciferase coding region. .. The 3′ end was labeled with the Klenow fragment of DNA polymerase I (New England Biolabs) and [α-32 P]dCTP.

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). .. Libraries were sequenced on Illumina HiSeq 4000.

    Article Title: PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ †PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ † ‡
    Article Snippet: .. The second strand was generated by incubating the first-strand reaction product with Second Strand Reaction Buffer (Invitrogen), DNA ligase (10 units), DNA polymerase I (40 units), and RNase H (2 units) for 2 h at 16°C; 20 units of T4 DNA polymerase was then added, and this step was followed by incubation at 16°C for another 5 min (New England BioLabs). .. The dscDNA was cleaned of RNA by adding RNAse III (Shortcut; New England BioLabs) and incubating at 37°C for 10 min, and then the sample was cleaned by phenol-chloroform extraction.

    Article Title: Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon
    Article Snippet: Target RNA was generated in a T7 polymerase-based linear amplification reaction based on a modified version of a published protocol ( ). .. Second-strand synthesis was for 2 h at 16°C in a total reaction volume of 50 μl containing first-strand reaction products, second-strand buffer (Invitrogen), 250 μM deoxynucleoside triphosphates, 0.06 U of DNA ligase (Ambion) per μl, 0.26 U of DNA polymerase I (New England Biolabs) per μl, and 0.012 U of RNase H (Ambion) per μl followed by the addition of 3.3 U of T4 DNA polymerase (3 U per μl; New England Biolabs) and a further 15 min of incubation at 16°C.

    Polymerase Chain Reaction:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs). .. Library amplification was performed with eight cycles of PCR using the KAPA Bio HiFi polymerase and barcoded primers.

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas). .. Libraries were amplified with 9–11 PCR cycles and sequenced (50 bp paired end) on a HiSeq1000 platform (Illumina).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: The PCR amplification step was performed for 12 cycles. .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp).

    Article Title: RNA-Seq analysis to capture the transcriptome landscape of a single cell
    Article Snippet: .. Ac-BSA (Sigma, cat. no. B8894) Acidic Tyrode’s solution (Sigma, cat. no. T1788) 1× PBS (pH 7.2) (Gibco, cat. no. 14249-95) 10× PCR Buffer II and 25 mM MgCl2 (Applied Biosystems, cat. no. 4379878) Nonidet P-40 SP (Roche, cat. no. 11332473001) SuperScript III Reverse Transcriptase with 0.1 M DTT (Invitrogen, cat. no. 18080-044 or 18080-085) RNase Inhibitor (Cloned) (40 U μl−1 ) (Applied Biosystems, cat. no. AM2682) SUPERase-In™ (20 U μl−1 ) (Applied Biosystems, cat. no. AM2694) T4 gene 32 Protein (Roche, cat. no. 972983) Exonuclease I (New England Biolabs, cat. no. M0293S) Terminal Transferase (TdT) (Invitrogen, cat. no. 10533-065 or 10533-073) 100 mM dATP (Promega, cat. no. U1201) Nuclease-free Water, 1 L (Applied Biosystems, cat. no. AM9932) RNase H (Invitrogen, cat. no. 18021-014 or 18021-071) TaKaRa Ex Taq™ HS (Includes: 10× ExTaq Buffer (mg2+ plus) and dNTP mixture) (Takara Bio Inc, cat. no. RR006A or RR006B) QIAquick PCR Purification Kit (250) (Qiagen, cat. no. 28106) QIAquick Gel Extraction Kit (250) (Qiagen, cat. no. 28706) RNeasy Mini Kit (50) (Qiagen, cat. no. 74104) PCR tubes, 0.5 ml (Eppendorf, cat. no. 951010057) PCR tubes, 0.2 ml thin-wall (MLS) Filtered pipettor tips (MLS) SYBR Green PCR Mastermix (Applied Biosystems, cat. no. 4334973) GeneAmp® dNTP Blend (100 mM) (Applied Biosystems, cat. no. N8080261) 3 M sodium acetate (pH 5.5) (Applied Biosystems/Ambion, cat. no. AM9740) 1 M Tris, pH 8.0 (100 ml) (Applied Biosystems/Ambion, AM9855G) Nuclease-free Water (1 L) (Applied Biosystems/Ambion, cat. no. AM9932) 10× NEBuffer2 (New England BioLabs® Inc., B7002S) Ethanol (Sigma-Aldrich®, cat. no. E7023) Ethylene glycol (American Bioanalytical, cat. no. AB00455-01000) Covaris microTUBE with AFA fiber and Snap-Cap with pre-slit Teflon/silicone/Teflon septa (Covaris™ Inc., cat. no. 520045) End-It™ DNA End-Repair Kit (Epicentre®, cat. no. ER0720) DNA Polymerase I (E. coli), (10U μl−1 ) (New England BioLabs® Inc., cat. no. M0209L) Quick Ligation™ kit (New England Biolabs, cat. no. M2200L) SYBR GreenER® qPCR SuperMix Universal (Invitrogen™ Corporation, cat. no. 11762-100) OR SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) Agilent DNA 1000 Kit (Agilent Technologies, cat. no. 5067-1504) Agencourt® AMPure® 60 ml kit (Agencourt, cat. no. 000130) AmpliTaq® DNA Polymerase, LD with Buffer I (Applied Biosystems, cat. no. N8080157) Cloned Pfu polymerase (2.5 U μl−1 ) (Stratagene, cat. no. 600153) Invitrogen Platinum® PCR SuperMix (Invitrogen™ Corporation, cat. no. 11306-016) Quant-iT™ dsDNA HS Assay Kit, 100 assays (Invitrogen™ Corporation, cat. no. ) SOLiD™ Fragment Library Oligos Kit (Applied Biosystems, cat. no. 4401151) .. Brown-flaming micropipette puller (Sutter Instrument Co., Model P-80) Covaris™ S2 System, (for system materials summary, see “Covaris™ S2 System Materials Summary,” SOLiD ™ System 2.0 Site Preparation Guide .)

    Binding Assay:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
    Article Snippet: Ten picomoles of synthetic double-stranded DNA oligonucleotide with a 5′ overhang was labeled with [γ-32 P]dCTP (3,000 Ci/mmol; PerkinElmer, Waltham, MA) using the Klenow fragment of DNA polymerase I (New England BioLabs). .. For the electrophoretic mobility shift assays (EMSA), 2 μl of a reticulocyte lysate containing TCF4 was mixed with 0.4 pmol of radiolabeled probes in binding buffer [20-μl final volume, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 7% glycerol, 0.05% Nonidet P-40, 0.1 mg/ml poly(dI-dC), 70 mM KCl, 50 mM dithiothreitol, 1 mM MgCl2 ].

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: .. Relevant S. lividans TK23 cultures used to obtain KorB-containing cell extracts were grown as indicated above until culture densities reached 0.5 to 0.7 OD600 units, whereupon they were harvested, and cell extracts containing active KorB protein were prepared as described earlier ( ) in S30 cell lysis buffer, except that subsequent to lysis cell extracts were centrifuged twice for 30 min at 30,000 × g . kilB promoter binding by KorB proteins was performed as described by Tai and Cohen , with the following modifications: a 0.1-kb Eco RI fragment from pGSP311 containing the Fsp I- Bst EII portion of the kilB promoter region was labeled on both ends by using [α-32 P]dATP (6,000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of DNA polymerase I (New England BioLabs), and the labeled fragment was then purified from excess radionuclides by using an S300 column (Amersham Biosciences). .. In a total volume of 20 μl, approximately 17 μg of each relevant S. lividans TK23 cell extract was added to 30,000 counts of labeled fragment per min that had been preincubated in binding buffer , and following further incubation for 10 min at room temperature, these samples were electrophoresed on a 5% nondenaturing polyacrylamide gel in Tris-glycine buffer as described earlier ( ).

    RNA Sequencing Assay:

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Paragraph title: Nuclear strand-specific RNA-Seq ... Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas).

    Article Title: LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells
    Article Snippet: Paragraph title: RNA sequencing ... Second strand was synthesized with deoxyuridine triphosphate to generate strand asymmetry using DNA polymerase I [M0209L, New England Biolabs (NEB)] and the Escherichia coli Ligase (L6090L, Enzymatics).

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: Paragraph title: RNA-seq ... Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

    Magnetic Beads:

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: RNA-seq Total RNA was isolated from cells and purified using poly-dT oligo-attached magnetic beads. .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

    Article Title: Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression
    Article Snippet: Total RNA was incubated for 5 min at 65°C, followed by incubation with the coated magnetic beads for 10 min at 45°C. .. Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen).

    Mutagenesis:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
    Article Snippet: Ten picomoles of synthetic double-stranded DNA oligonucleotide with a 5′ overhang was labeled with [γ-32 P]dCTP (3,000 Ci/mmol; PerkinElmer, Waltham, MA) using the Klenow fragment of DNA polymerase I (New England BioLabs). .. For confirming the specificity of the DNA-TCF4 complex, a 50-fold excess of unlabeled double-stranded wild-type or mutant oligonucleotide or 2 μg of anti-MYC (Developmental Studies Hybridoma Bank) or anti-HA (Sigma-Aldrich) mouse antibody was added into the reaction mixture 15 min prior to the addition of the labeled probe.

    Isolation:

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Nuclear RNA was isolated using TRIsure (Bioline), treated with DNaseI (Roche) and re-purified using an RNeasy Mini Kit (Qiagen). .. Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas).

    Article Title: LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells
    Article Snippet: RNA sequencing Total RNA from ESCs, EBs, 293T cells, MBs, and MTs was isolated with RNeasy (QIAGEN) and reverse transcribed using SuperScript III and random hexamers (Life Technologies) to synthesize the first strand. .. Second strand was synthesized with deoxyuridine triphosphate to generate strand asymmetry using DNA polymerase I [M0209L, New England Biolabs (NEB)] and the Escherichia coli Ligase (L6090L, Enzymatics).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: Messenger RNA (mRNA) was isolated with the Dynabeads mRNA Purification Kit (Invitrogen), followed by shearing with RNA fragmentation reagent (Ambion) at 72 °C. .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp).

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: RNA-seq Total RNA was isolated from cells and purified using poly-dT oligo-attached magnetic beads. .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: Total RNA isolated from five M. brunnea f.sp. multigermtubi -infected and control leaves was pooled by treatment, respectively. .. The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).

    Article Title: Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression
    Article Snippet: Total RNA was isolated using Tri-reagent (Ambion) and DNase-treated (Ambion). .. Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen).

    Size-exclusion Chromatography:

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: 250 ng nuclear RNA was fragmented using a Covaris E220 instrument at standard RNA settings for 60 sec. Fragmented RNA was precipitated and first strand synthesis was carried out using SuperScript III (Invitrogen) with 4 µg of actinomycin D (Sigma). .. Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas).

    Labeling:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
    Article Snippet: .. Ten picomoles of synthetic double-stranded DNA oligonucleotide with a 5′ overhang was labeled with [γ-32 P]dCTP (3,000 Ci/mmol; PerkinElmer, Waltham, MA) using the Klenow fragment of DNA polymerase I (New England BioLabs). .. For the electrophoretic mobility shift assays (EMSA), 2 μl of a reticulocyte lysate containing TCF4 was mixed with 0.4 pmol of radiolabeled probes in binding buffer [20-μl final volume, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 7% glycerol, 0.05% Nonidet P-40, 0.1 mg/ml poly(dI-dC), 70 mM KCl, 50 mM dithiothreitol, 1 mM MgCl2 ].

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro
    Article Snippet: .. The 3′ end was labeled with the Klenow fragment of DNA polymerase I (New England Biolabs) and [α-32 P]dCTP. .. Hybridization was done at 56°C, and S1 digestion was performed as previously described ( , ).

    Article Title: PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ †PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip ▿ † ‡
    Article Snippet: The second strand was generated by incubating the first-strand reaction product with Second Strand Reaction Buffer (Invitrogen), DNA ligase (10 units), DNA polymerase I (40 units), and RNase H (2 units) for 2 h at 16°C; 20 units of T4 DNA polymerase was then added, and this step was followed by incubation at 16°C for another 5 min (New England BioLabs). .. At this point the dscDNA was ready for fragmentation and labeling for microarray hybridization.

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: .. Relevant S. lividans TK23 cultures used to obtain KorB-containing cell extracts were grown as indicated above until culture densities reached 0.5 to 0.7 OD600 units, whereupon they were harvested, and cell extracts containing active KorB protein were prepared as described earlier ( ) in S30 cell lysis buffer, except that subsequent to lysis cell extracts were centrifuged twice for 30 min at 30,000 × g . kilB promoter binding by KorB proteins was performed as described by Tai and Cohen , with the following modifications: a 0.1-kb Eco RI fragment from pGSP311 containing the Fsp I- Bst EII portion of the kilB promoter region was labeled on both ends by using [α-32 P]dATP (6,000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of DNA polymerase I (New England BioLabs), and the labeled fragment was then purified from excess radionuclides by using an S300 column (Amersham Biosciences). .. In a total volume of 20 μl, approximately 17 μg of each relevant S. lividans TK23 cell extract was added to 30,000 counts of labeled fragment per min that had been preincubated in binding buffer , and following further incubation for 10 min at room temperature, these samples were electrophoresed on a 5% nondenaturing polyacrylamide gel in Tris-glycine buffer as described earlier ( ).

    Purification:

    Article Title: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome
    Article Snippet: Nucleotides were removed with mini quick spin DNA columns (Roche) and second strand synthesis was performed using E.coli DNA ligase (NEB), DNA Polymerase I (NEB) and RNase H (Fermentas), replacing dTTP with dUTP (Fermentas). .. Following purification on QIAquick columns (Qiagen), TruSeq Illumina adapters were ligated with T4 DNA Ligase (Enzymatics).

    Article Title: UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿ †
    Article Snippet: All DNA-modifying enzymes used (restriction endonucleases, DNA polymerase I (Pol I) Klenow fragment, T4 DNA ligase) were purchased from New England Biolabs and used according to the manufacturer's recommendations. .. The reaction was then run on a 1% agarose gel, and the linear fragment was extracted and purified using the QIAEX II kit (Qiagen).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: Messenger RNA (mRNA) was isolated with the Dynabeads mRNA Purification Kit (Invitrogen), followed by shearing with RNA fragmentation reagent (Ambion) at 72 °C. .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp).

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: RNA-seq Total RNA was isolated from cells and purified using poly-dT oligo-attached magnetic beads. .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB).

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: .. Relevant S. lividans TK23 cultures used to obtain KorB-containing cell extracts were grown as indicated above until culture densities reached 0.5 to 0.7 OD600 units, whereupon they were harvested, and cell extracts containing active KorB protein were prepared as described earlier ( ) in S30 cell lysis buffer, except that subsequent to lysis cell extracts were centrifuged twice for 30 min at 30,000 × g . kilB promoter binding by KorB proteins was performed as described by Tai and Cohen , with the following modifications: a 0.1-kb Eco RI fragment from pGSP311 containing the Fsp I- Bst EII portion of the kilB promoter region was labeled on both ends by using [α-32 P]dATP (6,000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of DNA polymerase I (New England BioLabs), and the labeled fragment was then purified from excess radionuclides by using an S300 column (Amersham Biosciences). .. In a total volume of 20 μl, approximately 17 μg of each relevant S. lividans TK23 cell extract was added to 30,000 counts of labeled fragment per min that had been preincubated in binding buffer , and following further incubation for 10 min at room temperature, these samples were electrophoresed on a 5% nondenaturing polyacrylamide gel in Tris-glycine buffer as described earlier ( ).

    Article Title: RNA-Seq analysis to capture the transcriptome landscape of a single cell
    Article Snippet: .. Ac-BSA (Sigma, cat. no. B8894) Acidic Tyrode’s solution (Sigma, cat. no. T1788) 1× PBS (pH 7.2) (Gibco, cat. no. 14249-95) 10× PCR Buffer II and 25 mM MgCl2 (Applied Biosystems, cat. no. 4379878) Nonidet P-40 SP (Roche, cat. no. 11332473001) SuperScript III Reverse Transcriptase with 0.1 M DTT (Invitrogen, cat. no. 18080-044 or 18080-085) RNase Inhibitor (Cloned) (40 U μl−1 ) (Applied Biosystems, cat. no. AM2682) SUPERase-In™ (20 U μl−1 ) (Applied Biosystems, cat. no. AM2694) T4 gene 32 Protein (Roche, cat. no. 972983) Exonuclease I (New England Biolabs, cat. no. M0293S) Terminal Transferase (TdT) (Invitrogen, cat. no. 10533-065 or 10533-073) 100 mM dATP (Promega, cat. no. U1201) Nuclease-free Water, 1 L (Applied Biosystems, cat. no. AM9932) RNase H (Invitrogen, cat. no. 18021-014 or 18021-071) TaKaRa Ex Taq™ HS (Includes: 10× ExTaq Buffer (mg2+ plus) and dNTP mixture) (Takara Bio Inc, cat. no. RR006A or RR006B) QIAquick PCR Purification Kit (250) (Qiagen, cat. no. 28106) QIAquick Gel Extraction Kit (250) (Qiagen, cat. no. 28706) RNeasy Mini Kit (50) (Qiagen, cat. no. 74104) PCR tubes, 0.5 ml (Eppendorf, cat. no. 951010057) PCR tubes, 0.2 ml thin-wall (MLS) Filtered pipettor tips (MLS) SYBR Green PCR Mastermix (Applied Biosystems, cat. no. 4334973) GeneAmp® dNTP Blend (100 mM) (Applied Biosystems, cat. no. N8080261) 3 M sodium acetate (pH 5.5) (Applied Biosystems/Ambion, cat. no. AM9740) 1 M Tris, pH 8.0 (100 ml) (Applied Biosystems/Ambion, AM9855G) Nuclease-free Water (1 L) (Applied Biosystems/Ambion, cat. no. AM9932) 10× NEBuffer2 (New England BioLabs® Inc., B7002S) Ethanol (Sigma-Aldrich®, cat. no. E7023) Ethylene glycol (American Bioanalytical, cat. no. AB00455-01000) Covaris microTUBE with AFA fiber and Snap-Cap with pre-slit Teflon/silicone/Teflon septa (Covaris™ Inc., cat. no. 520045) End-It™ DNA End-Repair Kit (Epicentre®, cat. no. ER0720) DNA Polymerase I (E. coli), (10U μl−1 ) (New England BioLabs® Inc., cat. no. M0209L) Quick Ligation™ kit (New England Biolabs, cat. no. M2200L) SYBR GreenER® qPCR SuperMix Universal (Invitrogen™ Corporation, cat. no. 11762-100) OR SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) Agilent DNA 1000 Kit (Agilent Technologies, cat. no. 5067-1504) Agencourt® AMPure® 60 ml kit (Agencourt, cat. no. 000130) AmpliTaq® DNA Polymerase, LD with Buffer I (Applied Biosystems, cat. no. N8080157) Cloned Pfu polymerase (2.5 U μl−1 ) (Stratagene, cat. no. 600153) Invitrogen Platinum® PCR SuperMix (Invitrogen™ Corporation, cat. no. 11306-016) Quant-iT™ dsDNA HS Assay Kit, 100 assays (Invitrogen™ Corporation, cat. no. ) SOLiD™ Fragment Library Oligos Kit (Applied Biosystems, cat. no. 4401151) .. Brown-flaming micropipette puller (Sutter Instrument Co., Model P-80) Covaris™ S2 System, (for system materials summary, see “Covaris™ S2 System Materials Summary,” SOLiD ™ System 2.0 Site Preparation Guide .)

    Article Title: Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon
    Article Snippet: Second-strand synthesis was for 2 h at 16°C in a total reaction volume of 50 μl containing first-strand reaction products, second-strand buffer (Invitrogen), 250 μM deoxynucleoside triphosphates, 0.06 U of DNA ligase (Ambion) per μl, 0.26 U of DNA polymerase I (New England Biolabs) per μl, and 0.012 U of RNase H (Ambion) per μl followed by the addition of 3.3 U of T4 DNA polymerase (3 U per μl; New England Biolabs) and a further 15 min of incubation at 16°C. .. Second-strand reaction products were purified by phenol-chloroform-isoamyl alcohol extraction in Phaselock microcentrifuge tubes (Eppendorf) according to the manufacturer's instructions and ethanol precipitated.

    Sequencing:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Paragraph title: RNA Extraction and Illumina Sequencing ... Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: Paragraph title: RNA Extraction and Transcriptome Sequencing ... Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp).

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: Paragraph title: DGE library construction and sequencing ... The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs).

    Article Title: Ubiquitously transcribed genes use alternative polyadenylation to achieve tissue-specific expression
    Article Snippet: An oligo(dT) primer containing a VN-anker, a uridine, the sequence of the sequencing adapter, and biotin (/5BiosG/CAGACGTGTGCTCTTCCGATCTTTTTTTTrUTTTTTTTTVN) was attached to streptavidin-coated magnetic beads (Invitrogen, M280). .. Second strand synthesis was carried out using second strand synthesis buffer (Invitrogen), dNTPs, DNA polymerase I (New England Biolabs), Escherichia coli ligase (New England Biolabs), and RNase H (Invitrogen).

    Electrophoretic Mobility Shift Assay:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
    Article Snippet: Ten picomoles of synthetic double-stranded DNA oligonucleotide with a 5′ overhang was labeled with [γ-32 P]dCTP (3,000 Ci/mmol; PerkinElmer, Waltham, MA) using the Klenow fragment of DNA polymerase I (New England BioLabs). .. For the electrophoretic mobility shift assays (EMSA), 2 μl of a reticulocyte lysate containing TCF4 was mixed with 0.4 pmol of radiolabeled probes in binding buffer [20-μl final volume, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.5), 7% glycerol, 0.05% Nonidet P-40, 0.1 mg/ml poly(dI-dC), 70 mM KCl, 50 mM dithiothreitol, 1 mM MgCl2 ].

    Lysis:

    Article Title: Common and Distinguishing Regulatory and Expression Characteristics of the Highly Related KorB Proteins of Streptomycete Plasmids pIJ101 and pSB24.2
    Article Snippet: .. Relevant S. lividans TK23 cultures used to obtain KorB-containing cell extracts were grown as indicated above until culture densities reached 0.5 to 0.7 OD600 units, whereupon they were harvested, and cell extracts containing active KorB protein were prepared as described earlier ( ) in S30 cell lysis buffer, except that subsequent to lysis cell extracts were centrifuged twice for 30 min at 30,000 × g . kilB promoter binding by KorB proteins was performed as described by Tai and Cohen , with the following modifications: a 0.1-kb Eco RI fragment from pGSP311 containing the Fsp I- Bst EII portion of the kilB promoter region was labeled on both ends by using [α-32 P]dATP (6,000 Ci/mmol; Amersham Biosciences) and the Klenow fragment of DNA polymerase I (New England BioLabs), and the labeled fragment was then purified from excess radionuclides by using an S300 column (Amersham Biosciences). .. In a total volume of 20 μl, approximately 17 μg of each relevant S. lividans TK23 cell extract was added to 30,000 counts of labeled fragment per min that had been preincubated in binding buffer , and following further incubation for 10 min at room temperature, these samples were electrophoresed on a 5% nondenaturing polyacrylamide gel in Tris-glycine buffer as described earlier ( ).

    cDNA Library Assay:

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). .. Briefly, one individual single-end cDNA library was constructed for each sample.

    Chromatin Immunoprecipitation:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Each RNA sample was run on an Agilent 2100 Bioanalyzer with an RNA Nano 6000 Chip to verify RNA quality and estimate quantity. .. Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs).

    Plasmid Preparation:

    Article Title: UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿ †
    Article Snippet: Paragraph title: Plasmid constructions and transfer of alleles to the chromosome. ... All DNA-modifying enzymes used (restriction endonucleases, DNA polymerase I (Pol I) Klenow fragment, T4 DNA ligase) were purchased from New England Biolabs and used according to the manufacturer's recommendations.

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro
    Article Snippet: The S1 probe used to determine luciferase RNA levels was generated by linearization of the Luc-SL vector with Dra II, which cuts in the luciferase coding region. .. The 3′ end was labeled with the Klenow fragment of DNA polymerase I (New England Biolabs) and [α-32 P]dCTP.

    SYBR Green Assay:

    Article Title: RNA-Seq analysis to capture the transcriptome landscape of a single cell
    Article Snippet: .. Ac-BSA (Sigma, cat. no. B8894) Acidic Tyrode’s solution (Sigma, cat. no. T1788) 1× PBS (pH 7.2) (Gibco, cat. no. 14249-95) 10× PCR Buffer II and 25 mM MgCl2 (Applied Biosystems, cat. no. 4379878) Nonidet P-40 SP (Roche, cat. no. 11332473001) SuperScript III Reverse Transcriptase with 0.1 M DTT (Invitrogen, cat. no. 18080-044 or 18080-085) RNase Inhibitor (Cloned) (40 U μl−1 ) (Applied Biosystems, cat. no. AM2682) SUPERase-In™ (20 U μl−1 ) (Applied Biosystems, cat. no. AM2694) T4 gene 32 Protein (Roche, cat. no. 972983) Exonuclease I (New England Biolabs, cat. no. M0293S) Terminal Transferase (TdT) (Invitrogen, cat. no. 10533-065 or 10533-073) 100 mM dATP (Promega, cat. no. U1201) Nuclease-free Water, 1 L (Applied Biosystems, cat. no. AM9932) RNase H (Invitrogen, cat. no. 18021-014 or 18021-071) TaKaRa Ex Taq™ HS (Includes: 10× ExTaq Buffer (mg2+ plus) and dNTP mixture) (Takara Bio Inc, cat. no. RR006A or RR006B) QIAquick PCR Purification Kit (250) (Qiagen, cat. no. 28106) QIAquick Gel Extraction Kit (250) (Qiagen, cat. no. 28706) RNeasy Mini Kit (50) (Qiagen, cat. no. 74104) PCR tubes, 0.5 ml (Eppendorf, cat. no. 951010057) PCR tubes, 0.2 ml thin-wall (MLS) Filtered pipettor tips (MLS) SYBR Green PCR Mastermix (Applied Biosystems, cat. no. 4334973) GeneAmp® dNTP Blend (100 mM) (Applied Biosystems, cat. no. N8080261) 3 M sodium acetate (pH 5.5) (Applied Biosystems/Ambion, cat. no. AM9740) 1 M Tris, pH 8.0 (100 ml) (Applied Biosystems/Ambion, AM9855G) Nuclease-free Water (1 L) (Applied Biosystems/Ambion, cat. no. AM9932) 10× NEBuffer2 (New England BioLabs® Inc., B7002S) Ethanol (Sigma-Aldrich®, cat. no. E7023) Ethylene glycol (American Bioanalytical, cat. no. AB00455-01000) Covaris microTUBE with AFA fiber and Snap-Cap with pre-slit Teflon/silicone/Teflon septa (Covaris™ Inc., cat. no. 520045) End-It™ DNA End-Repair Kit (Epicentre®, cat. no. ER0720) DNA Polymerase I (E. coli), (10U μl−1 ) (New England BioLabs® Inc., cat. no. M0209L) Quick Ligation™ kit (New England Biolabs, cat. no. M2200L) SYBR GreenER® qPCR SuperMix Universal (Invitrogen™ Corporation, cat. no. 11762-100) OR SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) Agilent DNA 1000 Kit (Agilent Technologies, cat. no. 5067-1504) Agencourt® AMPure® 60 ml kit (Agencourt, cat. no. 000130) AmpliTaq® DNA Polymerase, LD with Buffer I (Applied Biosystems, cat. no. N8080157) Cloned Pfu polymerase (2.5 U μl−1 ) (Stratagene, cat. no. 600153) Invitrogen Platinum® PCR SuperMix (Invitrogen™ Corporation, cat. no. 11306-016) Quant-iT™ dsDNA HS Assay Kit, 100 assays (Invitrogen™ Corporation, cat. no. ) SOLiD™ Fragment Library Oligos Kit (Applied Biosystems, cat. no. 4401151) .. Brown-flaming micropipette puller (Sutter Instrument Co., Model P-80) Covaris™ S2 System, (for system materials summary, see “Covaris™ S2 System Materials Summary,” SOLiD ™ System 2.0 Site Preparation Guide .)

    RNA Extraction:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Paragraph title: RNA Extraction and Illumina Sequencing ... Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: Paragraph title: RNA Extraction and Transcriptome Sequencing ... Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp).

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro
    Article Snippet: Paragraph title: RNA extraction and analysis. ... The 3′ end was labeled with the Klenow fragment of DNA polymerase I (New England Biolabs) and [α-32 P]dCTP.

    Selection:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Polyadenylated RNA transcripts were enriched from total RNA with two rounds of selection on Dynabeads Oligo(dT)25 (Invitrogen). .. Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp). ..

    Agarose Gel Electrophoresis:

    Article Title: UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿UvrD303, a Hyperhelicase Mutant That Antagonizes RecA-Dependent SOS Expression by a Mechanism That Depends on Its C Terminus ▿ †
    Article Snippet: All DNA-modifying enzymes used (restriction endonucleases, DNA polymerase I (Pol I) Klenow fragment, T4 DNA ligase) were purchased from New England Biolabs and used according to the manufacturer's recommendations. .. The reaction was then run on a 1% agarose gel, and the linear fragment was extracted and purified using the QIAEX II kit (Qiagen).

    Article Title: New Zealand Tree and Giant Wētā (Orthoptera) Transcriptomics Reveal Divergent Selection Patterns in Metabolic Loci
    Article Snippet: .. Second-strand synthesis was carried out with RNase H (Invitrogen) and DNA polymerase I (New England Biolabs), followed by end-repair, adaptor ligation, and agarose gel selection (250 ± 20 bp). ..

    Article Title: Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants
    Article Snippet: The second-strand cDNA was synthesized using RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). .. Libraries were prepared from a 150–200-bp size-selected fraction following adapter ligation and agarose gel separation.

    In Vitro:

    Article Title: Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis
    Article Snippet: Paragraph title: In vitro protein translation and EMSA. ... Ten picomoles of synthetic double-stranded DNA oligonucleotide with a 5′ overhang was labeled with [γ-32 P]dCTP (3,000 Ci/mmol; PerkinElmer, Waltham, MA) using the Klenow fragment of DNA polymerase I (New England BioLabs).

    Article Title: Phospholipid Scramblase 1 Potentiates the Antiviral Activity of Interferon
    Article Snippet: Second-strand synthesis was for 2 h at 16°C in a total reaction volume of 50 μl containing first-strand reaction products, second-strand buffer (Invitrogen), 250 μM deoxynucleoside triphosphates, 0.06 U of DNA ligase (Ambion) per μl, 0.26 U of DNA polymerase I (New England Biolabs) per μl, and 0.012 U of RNase H (Ambion) per μl followed by the addition of 3.3 U of T4 DNA polymerase (3 U per μl; New England Biolabs) and a further 15 min of incubation at 16°C. .. In vitro transcription was performed by using the T7 megascript kit (Ambion) according to a modified protocol in which purified cDNA was combined with 1 μl (each) of 10× ATP, GTP, CTP, and UTP and 1 μl of T7 enzyme mix in a 10-μl reaction volume and incubated for 9 h at 37°C.

    Random Hexamer Labeling:

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). .. Libraries were sequenced on Illumina HiSeq 4000.

    Article Title: Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays
    Article Snippet: .. Two micrograms of total genomic DNA of each of the different strains was used as a template for direct incorporation of [33 P]dATP (Amersham Pharmacia, Freiburg, Germany) by a randomly primed polymerization reaction using 0.75 μg of random hexamer primers (New England Biolabs, Frankfurt [Main], Germany) and 10 U of Klenow fragment of DNA polymerase I (New England Biolabs) according to the manufacturers' recommendations. .. Unincorporated nucleotides were removed with Microspin S 200 HR spin columns (Amersham Pharmacia).

    Produced:

    Article Title: Parallel Histories of Horizontal Gene Transfer Facilitated Extreme Reduction of Endosymbiont Genomes in Sap-Feeding Insects
    Article Snippet: Strand specificity was achieved by replacing dTTP with dUTP in the nucleotide pool for second-strand cDNA synthesis, which was performed with DNA polymerase I (New England Biolabs). .. Following ligation of library adapters , cDNA fragments were treated with uracil-DNA glycosylase (New England Biolabs) to remove uracils introduced during second-strand synthesis so that library fragments produced in the subsequent amplification step were all derived from first-strand cDNA.

    Article Title: A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass
    Article Snippet: .. Fragmentation was generated, first strand cDNAs were synthesized with random hexamer primer, and second strand cDNAs were synthesized using DNA Polymerase I and RNase H. The cDNAs were ligated with adaptors and produced libraries by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). .. Libraries were sequenced on Illumina HiSeq 4000.

    Gel Extraction:

    Article Title: RNA-Seq analysis to capture the transcriptome landscape of a single cell
    Article Snippet: .. Ac-BSA (Sigma, cat. no. B8894) Acidic Tyrode’s solution (Sigma, cat. no. T1788) 1× PBS (pH 7.2) (Gibco, cat. no. 14249-95) 10× PCR Buffer II and 25 mM MgCl2 (Applied Biosystems, cat. no. 4379878) Nonidet P-40 SP (Roche, cat. no. 11332473001) SuperScript III Reverse Transcriptase with 0.1 M DTT (Invitrogen, cat. no. 18080-044 or 18080-085) RNase Inhibitor (Cloned) (40 U μl−1 ) (Applied Biosystems, cat. no. AM2682) SUPERase-In™ (20 U μl−1 ) (Applied Biosystems, cat. no. AM2694) T4 gene 32 Protein (Roche, cat. no. 972983) Exonuclease I (New England Biolabs, cat. no. M0293S) Terminal Transferase (TdT) (Invitrogen, cat. no. 10533-065 or 10533-073) 100 mM dATP (Promega, cat. no. U1201) Nuclease-free Water, 1 L (Applied Biosystems, cat. no. AM9932) RNase H (Invitrogen, cat. no. 18021-014 or 18021-071) TaKaRa Ex Taq™ HS (Includes: 10× ExTaq Buffer (mg2+ plus) and dNTP mixture) (Takara Bio Inc, cat. no. RR006A or RR006B) QIAquick PCR Purification Kit (250) (Qiagen, cat. no. 28106) QIAquick Gel Extraction Kit (250) (Qiagen, cat. no. 28706) RNeasy Mini Kit (50) (Qiagen, cat. no. 74104) PCR tubes, 0.5 ml (Eppendorf, cat. no. 951010057) PCR tubes, 0.2 ml thin-wall (MLS) Filtered pipettor tips (MLS) SYBR Green PCR Mastermix (Applied Biosystems, cat. no. 4334973) GeneAmp® dNTP Blend (100 mM) (Applied Biosystems, cat. no. N8080261) 3 M sodium acetate (pH 5.5) (Applied Biosystems/Ambion, cat. no. AM9740) 1 M Tris, pH 8.0 (100 ml) (Applied Biosystems/Ambion, AM9855G) Nuclease-free Water (1 L) (Applied Biosystems/Ambion, cat. no. AM9932) 10× NEBuffer2 (New England BioLabs® Inc., B7002S) Ethanol (Sigma-Aldrich®, cat. no. E7023) Ethylene glycol (American Bioanalytical, cat. no. AB00455-01000) Covaris microTUBE with AFA fiber and Snap-Cap with pre-slit Teflon/silicone/Teflon septa (Covaris™ Inc., cat. no. 520045) End-It™ DNA End-Repair Kit (Epicentre®, cat. no. ER0720) DNA Polymerase I (E. coli), (10U μl−1 ) (New England BioLabs® Inc., cat. no. M0209L) Quick Ligation™ kit (New England Biolabs, cat. no. M2200L) SYBR GreenER® qPCR SuperMix Universal (Invitrogen™ Corporation, cat. no. 11762-100) OR SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) Agilent DNA 1000 Kit (Agilent Technologies, cat. no. 5067-1504) Agencourt® AMPure® 60 ml kit (Agencourt, cat. no. 000130) AmpliTaq® DNA Polymerase, LD with Buffer I (Applied Biosystems, cat. no. N8080157) Cloned Pfu polymerase (2.5 U μl−1 ) (Stratagene, cat. no. 600153) Invitrogen Platinum® PCR SuperMix (Invitrogen™ Corporation, cat. no. 11306-016) Quant-iT™ dsDNA HS Assay Kit, 100 assays (Invitrogen™ Corporation, cat. no. ) SOLiD™ Fragment Library Oligos Kit (Applied Biosystems, cat. no. 4401151) .. Brown-flaming micropipette puller (Sutter Instrument Co., Model P-80) Covaris™ S2 System, (for system materials summary, see “Covaris™ S2 System Materials Summary,” SOLiD ™ System 2.0 Site Preparation Guide .)

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    New England Biolabs dna polymerase
    End protection by <t>DNA</t> ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a <t>non-ligatable</t> 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    doi:

    Figure Lengend Snippet: End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Article Snippet: To generate a non-ligatable substrate, a single nucleotide (dTTP) was incorporated using DNA polymerase I large Klenow fragment (New England Biolabs).

    Techniques: Incubation, Labeling, Recombinant, Agarose Gel Electrophoresis, Autoradiography

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Journal: Nature biotechnology

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    doi: 10.1038/nbt736

    Figure Lengend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Article Snippet: Restriction endonucleases, T4 DNA ligase, Vent DNA polymerase, T4 polynucleotide kinase, and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA).

    Techniques: Ligation, Binding Assay

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Journal: Applied and Environmental Microbiology

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    doi: 10.1128/AEM.00624-08

    Figure Lengend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Article Snippet: Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans .

    Techniques: Sequencing, Produced, Software, Binding Assay

    Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.

    Journal: Journal of Bacteriology

    Article Title: Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69

    doi:

    Figure Lengend Snippet: Diagrammatic representation of λZAPII genomic library screening for RB69 DNA fragments (A) and partial restriction maps of the gene 46-43 regions of T4 and RB69 (B). Endonucleases Dra I and Ssp ). The solid horizontal bars designated PBS3K1, SP101, and SPR45-5 (A) represent 32 P-labeled riboprobes that were used to identify recombinant plasmids carrying the DNA fragments PBY16, PBS3, and LY6, respectively (see Materials and Methods). The SP101 probe corresponds to an internal Ssp I fragment of RB69 gene 43 , PBS3K1 corresponds to a Kpn I deletion of PBS3, and SPR45-5 corresponds to a 3′-terminal gene 45 segment that was generated from purified RB69 phage DNA by PCR amplification. ▿ in panel A denotes a terminal deletion for the respective gene. Restriction site abbreviations in panel B: H, Hin dIII; Sa, Sal I; Sc, Sac I; P, Pst I.

    Article Snippet: The T4 genomic region that specifies DNA polymerase and the DNA polymerase accessory proteins (T4 gene 46-43 cluster [Fig. ]) is known to include three types of genetic elements: structural genes for essential replication proteins (gp46, gp45, gp44/62, and gp43), structural genes for seemingly nonessential regulatory proteins (RpbA and RegA), and untranslated intercistronic nucleotide sequences that harbor signals for transcriptional and posttranscriptional regulation of the structural genes ( ).

    Techniques: Library Screening, Labeling, Recombinant, Generated, Purification, Polymerase Chain Reaction, Amplification

    Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).

    Journal: Journal of Bacteriology

    Article Title: Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69

    doi:

    Figure Lengend Snippet: Complementation between phage-encoded and plasmid-encoded T4 and RB69 DNA polymerase accessory proteins. The abilities of polymerase accessory proteins to be functionally exchanged between T4 and RB69 were examined by burst size measurements (Materials and Methods) and qualitative spot tests. (A) Results of plasmid-phage complementation tests involving gene 45 and genes 44 and 62 together; (B and C) results of similar tests involving genes 44 and 62 separately. In the “Spot test” blocks, each pair of spots represents growth responses (cell lysis) from 5 μl of two phage concentrations, ∼10 4 and ∼10 7 /ml, respectively. Numbers shown in parentheses in the “Relative burst size” blocks are the actual bursts corresponding to the 1.0 reference values for the pairs of infections compared. Note that although the T4 and RB69 counterparts of gp45 and the gp44/62 complex can exchange effectively, with some preferences by the gene functions to support replication of the phage from which they originated (values in panel A), the gp44(RB69)/gp62(T4) combination is largely inactive for phage replication (C). Also note that plasmid-expressed wild-type (wt) RB69 gene 44 is inhibitory to replication of wild-type T4 (T4 wt phage on pRB69g44-bearing host [B]).

    Article Snippet: The T4 genomic region that specifies DNA polymerase and the DNA polymerase accessory proteins (T4 gene 46-43 cluster [Fig. ]) is known to include three types of genetic elements: structural genes for essential replication proteins (gp46, gp45, gp44/62, and gp43), structural genes for seemingly nonessential regulatory proteins (RpbA and RegA), and untranslated intercistronic nucleotide sequences that harbor signals for transcriptional and posttranscriptional regulation of the structural genes ( ).

    Techniques: Plasmid Preparation, Lysis

    Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.

    Journal: Journal of Bacteriology

    Article Title: Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69

    doi:

    Figure Lengend Snippet: Nucleotide sequences of the T4 and RB69 gene 44-62 junctures (A) and expression of cloned gene 62 from RB69 and T4 (B). (A) Open reading frames for genes 44 and 62 , which are separated by one base pair at the g 44-62 ). No such structure can be predicted for RB69. (B) Results of plasmid-mediated expression of comparable genomic segments encompassing the gene 45-62 intervals from T4 mutant 44amN82 and RB69 mutant 44am51 . The desired DNA segments were PCR amplified from the respective phage mutants as well as wild-type strains and cloned in λpLN vector, and their plasmid-mediated expression was subsequently analyzed in E. coli CAJ70 as described in Materials and Methods. The short arrows mark the positions of gp45, gp44, and gp62 bands on the SDS-PAGE autoradiogram (10% gel) from the experiment; dots mark the positions of gp44 amber fragments. Note that expression of gene 62 (from either T4 or RB69) is lower when DNA from gene 44 amber mutants is used than with DNA carrying the wild-type gene 44 alleles.

    Article Snippet: The T4 genomic region that specifies DNA polymerase and the DNA polymerase accessory proteins (T4 gene 46-43 cluster [Fig. ]) is known to include three types of genetic elements: structural genes for essential replication proteins (gp46, gp45, gp44/62, and gp43), structural genes for seemingly nonessential regulatory proteins (RpbA and RegA), and untranslated intercistronic nucleotide sequences that harbor signals for transcriptional and posttranscriptional regulation of the structural genes ( ).

    Techniques: Expressing, Clone Assay, Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Amplification, SDS Page