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
    Catalog Number:
    m0209l
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    277
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    2 500 units
    Category:
    DNA Polymerases
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    New England Biolabs dna polymerase
    DNA Polymerase I E coli
    DNA Polymerase I E coli 2 500 units
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 99 stars, based on 103 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) Product Images from "Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis"

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    Journal: bioRxiv

    doi: 10.1101/790014

    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.
    Figure Legend Snippet: Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Techniques Used: Selection, Marker, In Vitro, Transformation Assay, Sequencing, Ligation

    Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.
    Figure Legend Snippet: Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Techniques Used: Generated, In Vitro, Sequencing, Variant Assay, Ligation

    28) 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:

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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:

    38) 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

    39) 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

    40) 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

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    Clone Assay:

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    New England Biolabs t7 helicase
    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a <t>T7</t> helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).
    T7 Helicase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs general information bst dna polymerase large fragment
    Verification of UIMA using different <t>DNA</t> polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of <t>Bst</t> DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .
    General Information Bst Dna Polymerase Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs g stearothermophilus bst dna polymerase
    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid <t>(DNA)</t> and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of <t>Bst</t> DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
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    Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Journal: Nature Communications

    Article Title: DNA looping mediates nucleosome transfer

    doi: 10.1038/ncomms13337

    Figure Lengend Snippet: Helicase displacement of a single nucleosome. ( a ) Experimental configuration. A single dsDNA molecule was unwound by a T7 helicase as the two strands of the DNA were held under 12 pN of force by an optical trap, which assisted helicase unwinding but was insufficient to mechanically separate the dsDNA ( Supplementary Fig. 1B ). The nucleosomal DNA template is specified in Supplementary Fig. 2A , Supplementary Table 1 and Methods. ( b ) Representative helicase-unwinding traces on a nucleosomal (black) or naked (grey) template. Helicase unwinding was interrupted by discrete pauses along the DNA template. Dashed lines indicate the dyad locations of the initial positioned nucleosome and the transferred nucleosome. N =49 traces. ( c ) Histogram of nucleosome transfer distance. A transfer distance was obtained from the first transfer event of each trace as indicated by the arrow in Fig. 2b . The histogram was obtained by pooling data from 49 traces. The prediction (not a fit) from the DNA looping model is plotted for comparison. The resulting Pearson test gives a reduced χ 2 of 0.31 with a P value of 0.95 (Methods; Supplementary Fig. 4A ).

    Article Snippet: Replisomes were formed by pre-incubating 1 unit per μl T7 DNA polymerase (NEB) and 1 μM T7 helicase in reaction buffer on ice for 5 min, and then were added to a final concentration of 0.1 unit per μl T7 DNA polymerase (NEB) and 100 nM T7 helicase and incubated at 37 °C for 10 min.

    Techniques:

    Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Journal: Scientific Reports

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

    Techniques: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

    Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .

    Journal: Scientific Reports

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend Snippet: Real-time fluorescence and electrophoresis analysis of UIMA. All reactions shared the same primer (RL) or template (F*R*), and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) The results of real-time fluorescence obtained from the reactions that contained 10 nM template, 1.6 μM primer, and 3.2 U Bst WS DNA polymerase at 63 °C for 180 min. Each test was in triplicate. ( B ) Time course of the UIMA assay. 2.5% agarose gel electrophoresis shows the products of UIMA. The assay time was varied from 30–120 minutes as indicated above each lane. M1 and M2: DNA marker; NEC, NTC and NPC were all incubated for 120 min. Exposure time is 5 s. ( C ) Extension status of template and primer. Template and primer were labeled with the FAM fluorophore. 1: reaction with FAM-labeled primer and template but no Bst ; 2: with FAM-labeled primer, non-labeled template, and Bst ; 3: with FAM-labeled primer and template and Bst ; 4: with FAM-labeled primer and Bst but no template; 5: with non-labeled primer and template, and Bst ; 6: with non-labeled primer, FAM-labeled template, and Bst ; 7: with non-labeled primer and Bst but no template; 8: with non-labeled template and Bst but no primer; 9: with FAM-labeled template and Bst but no primer. All the reactions were incubated at 63 °C for 180 min. Their products were analyzed by 17% denatured polyacrylamide gel electrophoresis (DPAGE). NEC (no-enzyme control): the reaction without Bst 2.0 WS DNA polymerase. NTC (no-template control): control reaction just lacked the template; NPC (no-primer control): control reaction lacked primer RL. Horizontal arrows denoted the 5′-3′ direction of sequences. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S1 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

    Techniques: Fluorescence, Electrophoresis, Incubation, Agarose Gel Electrophoresis, Marker, Labeling, Polyacrylamide Gel Electrophoresis

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Journal: Nature Communications

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    doi: 10.1038/s41467-018-07611-1

    Figure Lengend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Article Snippet: ThermoPol buffer, Taq DNA polymerase, G. stearothermophilus Bst DNA polymerase, LF, and its variants Bst 2.0 DNA polymerase (2.0), and Bst 3.0 DNA polymerase (3.0), DH5α competent cells, and Monarch DNA gel extraction kits were purchased from New England Biolabs (Ipswich, MA).

    Techniques: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis