ts k1 dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs ts k1 dna polymerase
    Phylogenetic tree showing the evolutionary relationship between Ts <t>K1</t> DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Ts K1 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1"

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.1149

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Figure Legend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Techniques Used:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)
    Figure Legend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Techniques Used: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)
    Figure Legend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Techniques Used: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Standard Deviation

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn575

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

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    3) 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

    4) 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

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.587907

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

    Techniques Used:

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

    Techniques Used:

    6) 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

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

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

    Journal: Journal of Bacteriology

    doi:

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

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

    8) Product Images from "Producing molecular biology reagents without purification"

    Article Title: Producing molecular biology reagents without purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0252507

    Activity of Bst-LF evaporated cellular reagents. Bst-LF DNA polymerase cellular reagents evaporated at the indicated temperature with (GF800) or without (None) solid support were tested after 2 months of preparation by performing LAMP-OSD reactions with or without human gapd plasmid DNA templates. LAMP-OSD reactions performed using commercially obtained pure Bst 2.0 enzyme or with lyophilized Bst-LF cellular reagents (Lyo CR) served as controls. Amplification curves observed in real-time by measuring increase in fluorescence of OSD probes are depicted. Data shown are representative of three biological replicates.
    Figure Legend Snippet: Activity of Bst-LF evaporated cellular reagents. Bst-LF DNA polymerase cellular reagents evaporated at the indicated temperature with (GF800) or without (None) solid support were tested after 2 months of preparation by performing LAMP-OSD reactions with or without human gapd plasmid DNA templates. LAMP-OSD reactions performed using commercially obtained pure Bst 2.0 enzyme or with lyophilized Bst-LF cellular reagents (Lyo CR) served as controls. Amplification curves observed in real-time by measuring increase in fluorescence of OSD probes are depicted. Data shown are representative of three biological replicates.

    Techniques Used: Activity Assay, Plasmid Preparation, Amplification, Fluorescence

    9) Product Images from "Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats"

    Article Title: Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn025

    RCA of pHC or pHC 120–130 plasmid DNA. ( A ) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). ( B ) Restriction endonuclease digest of plasmid pHC 120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC 120–130 . Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).
    Figure Legend Snippet: RCA of pHC or pHC 120–130 plasmid DNA. ( A ) Restriction endonuclease digest of plasmid pHC generated a linear 1796 bp fragment with XhoI (lane 2), or 771 bp and 1025 bp fragments with HindIII (lane 3). RCA of plasmid pHC is shown in lanes 4–11. No RCA product was observed in the absence of template (lane 4) or ϕ29 DNA polymerase (lane 5). RCA products from duplicate reactions containing template and ϕ29 DNA polymerase migrated as high molecular weight DNA (lanes 6 and 7), slower than the largest marker DNA (10 kb). Restriction endonuclease digest of duplicate amplification products using XhoI (lanes 8 and 10) or HindIII (lanes 9 and 11) generated the same pattern as plasmid pHC template. Lanes 1 and 12 contain 1 kb DNA ladder (NEB). ( B ) Restriction endonuclease digest of plasmid pHC 120–130 generated a linear 2194–2224 bp fragment with XhoI (lane 2), 1169–1199 bp and 1025 bp fragments with HindIII (lane 3) and 379–409 bp and 1815 bp with Acc65I and BsrGI (lane 4). Acc65I and BsrGI cleave on either side of the repeat tract. RCA products were observed from duplicate reactions containing both template and ϕ29 polymerase (lanes 7 and 8) but not in the absence of template (lane 5) or ϕ29 polymerase (lane 6). Restriction endonuclease digest of duplicate RCA products using XhoI (lanes 9 and 12), HindIII (lanes 10 and 13) or Acc65I-BsrGI (lanes 11 and 14) generated the same pattern as plasmid pHC 120–130 . Fragments containing repeats are marked with arrows. Lanes 1 and 15 contain 1 kb DNA ladder (NEB).

    Techniques Used: Plasmid Preparation, Generated, Molecular Weight, Marker, Amplification

    10) 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

    11) Product Images from "Dynamic assembly of primers on nucleic acid templates"

    Article Title: Dynamic assembly of primers on nucleic acid templates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl625

    Mismatched templates using 9°N. PAGE gel (20%) of primer extension assays with mismatched DNA 38mer templates using 9°N. Final concentrations: dNTPs (100 μM each, for A, T, G, C, with 33 nM [α- 32 P]dCTP) template (30 pmol, 3 μM), 3′-terminal 6mer fragment carrying a 5′-amino group (20 pmol, 2 μM) and 5′-terminal 8mer carrying a 3′-CH 2 CHO unit (20 pmol, 2 μM). Reaction mixtures were brought to 70°C for (30 s), and dNTPs were added. The mixtures were incubated for 2 min at the appropriate temperature. Reactions were terminated with quench buffer (5 μl, formamide EDTA, dyes, dilution factor of 33%). An aliquot (1 μl) was loaded on a polyacrylamide gel (20%, urea 7 M) and resolved. Single nucleotide mismatches for the 3′-terminal fragment inhibits extension of primers to full-length product [19mer (N + 13) and 27mer (N + 21)] ( A ). Depending on the location of the mismatches in the 5′-fragment, decreased amounts of full-length product ( B ) are observed.
    Figure Legend Snippet: Mismatched templates using 9°N. PAGE gel (20%) of primer extension assays with mismatched DNA 38mer templates using 9°N. Final concentrations: dNTPs (100 μM each, for A, T, G, C, with 33 nM [α- 32 P]dCTP) template (30 pmol, 3 μM), 3′-terminal 6mer fragment carrying a 5′-amino group (20 pmol, 2 μM) and 5′-terminal 8mer carrying a 3′-CH 2 CHO unit (20 pmol, 2 μM). Reaction mixtures were brought to 70°C for (30 s), and dNTPs were added. The mixtures were incubated for 2 min at the appropriate temperature. Reactions were terminated with quench buffer (5 μl, formamide EDTA, dyes, dilution factor of 33%). An aliquot (1 μl) was loaded on a polyacrylamide gel (20%, urea 7 M) and resolved. Single nucleotide mismatches for the 3′-terminal fragment inhibits extension of primers to full-length product [19mer (N + 13) and 27mer (N + 21)] ( A ). Depending on the location of the mismatches in the 5′-fragment, decreased amounts of full-length product ( B ) are observed.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation

    12) 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:

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn575

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

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

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

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37513-7

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

    Techniques Used: Mutagenesis

    15) 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

    16) Product Images from "Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1"

    Article Title: Rapid induction of Alternative Lengthening of Telomeres by depletion of the histone chaperone ASF1

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2754

    ASF1 knockdown generates ECTR ( a ) Telomere Restriction Fragment (TRF) analysis of telomeric DNA. Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. ( b, c ) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking φ29 or DNA. ( d ) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value
    Figure Legend Snippet: ASF1 knockdown generates ECTR ( a ) Telomere Restriction Fragment (TRF) analysis of telomeric DNA. Left: Digested DNA (10µg) from independent prolonged ASF1 knockdowns (days 17 and 14 post transfection) in HeLa LT was resolved on a 0.7% TBE agarose gel. DNA from siControl transfected cells, Saos-2 and U2OS cells was resolved in parallel. Right: Lines traces of siControl (black) and siASF1 (red) gel lanes. ( b, c ) C-circle assays in siControl and siASF1 transfected IMR90-hTERT and HeLa LT. The ALT positive U2OS control is on the left. Negative controls are reactions lacking φ29 or DNA. ( d ) Quantification of C-circles in siRNA and drug treated HeLa LT (light grey bar) and U2OS (dark grey bar). In both (c) and (d), C-circle levels are calculated relative to those of U2OS cells (dark grey bar, far left of graph). Data represent means ±SDs of at least 3 experiments. *** indicates p-value

    Techniques Used: Transfection, Agarose Gel Electrophoresis

    17) Product Images from "Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1"

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.1149

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Figure Legend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Techniques Used:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)
    Figure Legend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Techniques Used: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)
    Figure Legend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Techniques Used: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Standard Deviation

    18) 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

    19) 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

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

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

    Journal: Nucleic Acids Research

    doi:

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

    Techniques Used: Incubation

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

    Techniques Used: Incubation, Electrophoresis

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

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

    21) Product Images from "Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches"

    Article Title: Expression, Purification, and Refolding of Chikungunya Virus Full-Length Envelope E2 Protein along with B-Cell and T-Cell Epitope Analyses Using Immuno-Informatics Approaches

    Journal: ACS Omega

    doi: 10.1021/acsomega.1c05975

    PCR amplification of CHIKV E2-FL and truncated fragments used in the study. (A) PCR amplification of the CHIKV E2 full-length gene (1269 bp). (B) PCR amplification of truncated E2 fragments, E2-ΔC (1095 bp), and E2-ΔNC (993 bp). Vector: linearized pQE-30 Xa vector. Marker: 1 Kb DNA ladder; last three marker bands are indicated on the right.
    Figure Legend Snippet: PCR amplification of CHIKV E2-FL and truncated fragments used in the study. (A) PCR amplification of the CHIKV E2 full-length gene (1269 bp). (B) PCR amplification of truncated E2 fragments, E2-ΔC (1095 bp), and E2-ΔNC (993 bp). Vector: linearized pQE-30 Xa vector. Marker: 1 Kb DNA ladder; last three marker bands are indicated on the right.

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

    22) Product Images from "Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis"

    Article Title: Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64541-z

    Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.
    Figure Legend Snippet: Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.

    Techniques Used:

    23) Product Images from "Telomerase Inhibitor Imetelstat (GRN163L) Limits the Lifespan of Human Pancreatic Cancer Cells"

    Article Title: Telomerase Inhibitor Imetelstat (GRN163L) Limits the Lifespan of Human Pancreatic Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085155

    Effects of chronic GRN163L on telomere maintenance. Cells were treated every 2–3 days with no drug (CTR), mismatch oligo (MIS) or GRN163L (GRN). A–B) Southern blot analysis of the telomeres. At the indicated population doubling (PD), genomic DNA samples were collected and subsequently processed for telomere size measurements, as described in Figure 1A . C–D) Telomere size measurements. Median telomere sizes are shown for the drug- and control-treated CAPAN1 and CD18 cells. E) Detection of ALT by quantitative PCR. Samples tested included CAPAN1 and CD18 cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN), all of which harvested at the end of the growth curves presented in Figure 3 . Also included were stocks of CAPAN1, CD18 and VA13 cells. Samples were tested in triplicate with (+) and without (−) the Φ29 DNA polymerase. A representative dot blot is shown.
    Figure Legend Snippet: Effects of chronic GRN163L on telomere maintenance. Cells were treated every 2–3 days with no drug (CTR), mismatch oligo (MIS) or GRN163L (GRN). A–B) Southern blot analysis of the telomeres. At the indicated population doubling (PD), genomic DNA samples were collected and subsequently processed for telomere size measurements, as described in Figure 1A . C–D) Telomere size measurements. Median telomere sizes are shown for the drug- and control-treated CAPAN1 and CD18 cells. E) Detection of ALT by quantitative PCR. Samples tested included CAPAN1 and CD18 cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN), all of which harvested at the end of the growth curves presented in Figure 3 . Also included were stocks of CAPAN1, CD18 and VA13 cells. Samples were tested in triplicate with (+) and without (−) the Φ29 DNA polymerase. A representative dot blot is shown.

    Techniques Used: Southern Blot, Real-time Polymerase Chain Reaction, Dot Blot

    24) Product Images from "RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae"

    Article Title: RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn980

    Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.
    Figure Legend Snippet: Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.

    Techniques Used: Titration, Labeling, Marker, Generated, Activity Assay, Quantitation Assay, Binding Assay

    Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.
    Figure Legend Snippet: Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.

    Techniques Used: Sequencing, End Labeling, Labeling, Incubation, Marker, Electrophoresis, Activity Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1245

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

    Techniques Used: DNA Synthesis, Construct, Incubation, Synthesized

    26) Product Images from "Benchmarking the Effectiveness and Accuracy of Multiple Mitochondrial DNA Variant Callers: Practical Implications for Clinical Application"

    Article Title: Benchmarking the Effectiveness and Accuracy of Multiple Mitochondrial DNA Variant Callers: Practical Implications for Clinical Application

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2022.692257

    Accuracy comparison of mtDNA variant callers at heteroplasmy threshold of 1%, across different Taq polymerases and DNA extraction protocols using a synthetic benchmark dataset. (A) F1 scores of four variant callers using Clontech Taq polymerase and total DNA extraction. (B) F1 scores of four variant callers using Herk Taq polymerase and total DNA extraction. (C) F1 scores of four variant callers using NEB Taq polymerase and total DNA extraction. (D) F1 scores of four variant callers using Clontech Taq polymerase and PCR products. (E) F1 scores of four variant callers using Herk Taq polymerase and PCR products. (F) F1 scores of four variant callers using NEB Taq polymerase and PCR products.
    Figure Legend Snippet: Accuracy comparison of mtDNA variant callers at heteroplasmy threshold of 1%, across different Taq polymerases and DNA extraction protocols using a synthetic benchmark dataset. (A) F1 scores of four variant callers using Clontech Taq polymerase and total DNA extraction. (B) F1 scores of four variant callers using Herk Taq polymerase and total DNA extraction. (C) F1 scores of four variant callers using NEB Taq polymerase and total DNA extraction. (D) F1 scores of four variant callers using Clontech Taq polymerase and PCR products. (E) F1 scores of four variant callers using Herk Taq polymerase and PCR products. (F) F1 scores of four variant callers using NEB Taq polymerase and PCR products.

    Techniques Used: Variant Assay, DNA Extraction, Polymerase Chain Reaction

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

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

    Journal: Scientific Reports

    doi: 10.1038/srep05052

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

    Techniques Used: Binding Assay, Amplification, Polymerase Chain Reaction

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

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation

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

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

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

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

    28) Product Images from "The Complete Genome Sequence of Hyperthermophile Dictyoglomus turgidum DSM 6724™ Reveals a Specialized Carbohydrate Fermentor"

    Article Title: The Complete Genome Sequence of Hyperthermophile Dictyoglomus turgidum DSM 6724™ Reveals a Specialized Carbohydrate Fermentor

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01979

    Multiple sequence alignment of all five Dictyoglomus (4 from turgidum and one from thermophilum ) nucleotidyltransferase domains of DNA polymerase beta family protein sequences using MAFFT software (Katoh et al., 2002 ) . From top to bottom: Dtur_0104, 114AA; Dtur_0317, 121AA, Dtur_0545, 135AA, Dtur_1295, 99AA, Dicth_0227, 114AA. Dtur_1295 is located in the prophage region of D. turdigum .
    Figure Legend Snippet: Multiple sequence alignment of all five Dictyoglomus (4 from turgidum and one from thermophilum ) nucleotidyltransferase domains of DNA polymerase beta family protein sequences using MAFFT software (Katoh et al., 2002 ) . From top to bottom: Dtur_0104, 114AA; Dtur_0317, 121AA, Dtur_0545, 135AA, Dtur_1295, 99AA, Dicth_0227, 114AA. Dtur_1295 is located in the prophage region of D. turdigum .

    Techniques Used: Sequencing, Software

    29) 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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks316

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

    Techniques Used: Purification, Polymerase Chain Reaction, Construct

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

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

    Journal:

    doi: 10.1021/tx9000896

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

    Techniques Used: Primer Extension Assay

    32) 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

    33) Product Images from "Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1"

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.1149

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Figure Legend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Techniques Used:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)
    Figure Legend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Techniques Used: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)
    Figure Legend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Techniques Used: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Standard Deviation

    34) Product Images from "Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1"

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.1149

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Figure Legend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Techniques Used:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)
    Figure Legend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Techniques Used: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)
    Figure Legend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Techniques Used: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values
    Figure Legend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Techniques Used: Standard Deviation

    35) Product Images from "Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation"

    Article Title: Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

    Journal: eLife

    doi: 10.7554/eLife.04988

    Pol α requires CMG for priming activity during unwinding of forked DNA. ( A ) Scheme of assays comparing Pol α activity using either CMG helicase or the strand displacing ϕ29 polymerase. ( B ) Autoradiograph of DNA products using either 32 P-dCTP (leading) or 32 P-dGTP (lagging). Use of a DNA-primed leading strand fork (PF) or an unprimed fork (UF) is indicated in the figure. Pol α was present at 10 nM, and reactions were for 20 min. Lanes 1 and 2 represent control reactions of ϕ29 polymerase alone. DOI: http://dx.doi.org/10.7554/eLife.04988.006
    Figure Legend Snippet: Pol α requires CMG for priming activity during unwinding of forked DNA. ( A ) Scheme of assays comparing Pol α activity using either CMG helicase or the strand displacing ϕ29 polymerase. ( B ) Autoradiograph of DNA products using either 32 P-dCTP (leading) or 32 P-dGTP (lagging). Use of a DNA-primed leading strand fork (PF) or an unprimed fork (UF) is indicated in the figure. Pol α was present at 10 nM, and reactions were for 20 min. Lanes 1 and 2 represent control reactions of ϕ29 polymerase alone. DOI: http://dx.doi.org/10.7554/eLife.04988.006

    Techniques Used: Activity Assay, Autoradiography

    Okazaki Fragments are produced along the entire DNA. ( A ) Restriction enzyme map of the 3.2 kb substrate for Psi I and Ear I. ( B ) Lagging strand reactions were performed as detailed in ‘Materials and methods’ using an unprimed forked DNA, CMG, RPA, and either Pol α (lanes 4–6) or Pol α and Pol ε (lanes 7–9), then were either untreated (lanes 4, 7), treated with Psi I (lanes 5, 8), or treated with Ear I (lanes 6, 9). A control leading strand reaction using only ϕ29 Pol is shown in lanes 10–12. Pol α without CMG (lanes 1–3) and ϕ29 alone (lanes 13–15) gave no lagging strand products. The (*) mark incomplete digestion products. The reaction products were analyzed on a native 2% agarose gel. DOI: http://dx.doi.org/10.7554/eLife.04988.007
    Figure Legend Snippet: Okazaki Fragments are produced along the entire DNA. ( A ) Restriction enzyme map of the 3.2 kb substrate for Psi I and Ear I. ( B ) Lagging strand reactions were performed as detailed in ‘Materials and methods’ using an unprimed forked DNA, CMG, RPA, and either Pol α (lanes 4–6) or Pol α and Pol ε (lanes 7–9), then were either untreated (lanes 4, 7), treated with Psi I (lanes 5, 8), or treated with Ear I (lanes 6, 9). A control leading strand reaction using only ϕ29 Pol is shown in lanes 10–12. Pol α without CMG (lanes 1–3) and ϕ29 alone (lanes 13–15) gave no lagging strand products. The (*) mark incomplete digestion products. The reaction products were analyzed on a native 2% agarose gel. DOI: http://dx.doi.org/10.7554/eLife.04988.007

    Techniques Used: Produced, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis

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

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

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.006

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

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

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

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

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

    doi: 10.1093/dnares/dsv010

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

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

    38) 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

    39) 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

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    New England Biolabs ts k1 dna polymerase
    Phylogenetic tree showing the evolutionary relationship between Ts <t>K1</t> DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Ts K1 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase
    Schematic diagram of the inverted-nested two-step <t>PCR</t> (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 <t>DNA</t> 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.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Standard Deviation

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkn575

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

    Article Snippet: Primer extension with Klenow fragment of DNA polymerase Primer extension experiments using large fragment (Klenow) of DNA polymerase I (New England Biolabs) were performed at 25°C using the HIV-1 tat reverse primer (5′-AATACTATGGTCCACACAACTATTGCT-3′) that was unmodified or contained a single OXP modification.

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw673

    Figure Lengend 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.

    Article Snippet: In order to analyze whether, besides template-independent deoxyadenylation, Bam35 TP could serve as primer for viral DNA replication in vitro , we incubated the Bam35 TP-DNA with the TP and the DNA polymerase in the presence of dNTPs using magnesium as cofactor, obtaining a band of the expected size of the Bam35 genome (about 15 kB, Figure , lanes 4–5).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw673

    Figure Lengend 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.

    Article Snippet: In order to analyze whether, besides template-independent deoxyadenylation, Bam35 TP could serve as primer for viral DNA replication in vitro , we incubated the Bam35 TP-DNA with the TP and the DNA polymerase in the presence of dNTPs using magnesium as cofactor, obtaining a band of the expected size of the Bam35 genome (about 15 kB, Figure , lanes 4–5).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw673

    Figure Lengend 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.

    Article Snippet: In order to analyze whether, besides template-independent deoxyadenylation, Bam35 TP could serve as primer for viral DNA replication in vitro , we incubated the Bam35 TP-DNA with the TP and the DNA polymerase in the presence of dNTPs using magnesium as cofactor, obtaining a band of the expected size of the Bam35 genome (about 15 kB, Figure , lanes 4–5).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw673

    Figure Lengend 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.

    Article Snippet: In order to analyze whether, besides template-independent deoxyadenylation, Bam35 TP could serve as primer for viral DNA replication in vitro , we incubated the Bam35 TP-DNA with the TP and the DNA polymerase in the presence of dNTPs using magnesium as cofactor, obtaining a band of the expected size of the Bam35 genome (about 15 kB, Figure , lanes 4–5).

    Techniques: Incubation, Sequencing, Autoradiography

    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.

    Journal: Genes

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

    doi: 10.3390/genes9110562

    Figure Lengend 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.

    Article Snippet: PCR (50 μL) reactions were mixed on ice with dimethyl sulfoxide (DMSO), buffer, deoxynucleotide triphosphate mix, primers , template (spooled genomic DNA or PCR product), and DNA polymerase according to the supplier (New England Biolabs).

    Techniques: Polymerase Chain Reaction, Ligation, DNA Sequencing