dntps  (New England Biolabs)


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    Structured Review

    New England Biolabs dntps
    Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except <t>ddNTPs</t> substituted for <t>dNTPs,</t> and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.
    Dntps, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining"

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn164

    Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.
    Figure Legend Snippet: Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.

    Techniques Used: Labeling, Non-Homologous End Joining, Ligation, Polyacrylamide Gel Electrophoresis

    2) Product Images from "A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase"

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879122

    Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) Representative quantification of dTTP using the published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31 to 0.08 pmol) are not shown. ( B ) Representative quantification of dTTP using Q5 DNA polymerase, long oligonucleotide template and EvaGreen detection chemistry. ( C - D ) dTTP signal generated by the fluorometric methods from mouse liver extracts with and without 0.5 pmol dTTP spike-in calibrant. The final extract volume was diluted to 80 µl per 40 mg of initial tissue weight. ( E - F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of the non-linear curve fit.
    Figure Legend Snippet: Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) Representative quantification of dTTP using the published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31 to 0.08 pmol) are not shown. ( B ) Representative quantification of dTTP using Q5 DNA polymerase, long oligonucleotide template and EvaGreen detection chemistry. ( C - D ) dTTP signal generated by the fluorometric methods from mouse liver extracts with and without 0.5 pmol dTTP spike-in calibrant. The final extract volume was diluted to 80 µl per 40 mg of initial tissue weight. ( E - F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of the non-linear curve fit.

    Techniques Used: Hydrolysis Assay, Generated, Fluorescence

    3) Product Images from "Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase"

    Article Title: Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm416

    ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.
    Figure Legend Snippet: ( a ) Template-primer strand duplex used in the running start experiment showing the numbering scheme. ( b ) Typical running start primer extension experiments catalyzed by BF as a function of incubation time (min). Extension of the primer strand, a 22-mer with the terminal 3′-G base opposite the template base C labeled ‘–3’ on the template strand. The position of the [BP]G* is labeled ‘0’ (25-mer). The ‘–1’ position denotes a 24-mer. The lane marked ‘M’ contains 22-mer, 24-mer, 25-mer and 43-mer primer strands (the 43-mer corresponds to the length of a fully extended primer strand). Reactions were initiated with 4.5 nM of the primer/template duplex (1:1.5 ratio), 20 nM of BF, and 200 μM of dNTPs at 37°C. ( c ) Typical single step 2′-deoxynucleotide triphosphate (dNTP) insertion catalyzed by BF. The primer strand was 24 nucleotides long with the terminal 3′-C positioned opposite the template base labeled ‘–1’ which corresponds to the darkest bands in this figure. Incorporation of each individual dNTP opposite the [BP]G* adduct results in the lighter bands just above the darkest bands, where A = dATP, C = dCTP, G = dGTP, T = dTTP, and Ctr = ‘–1’ and ‘0’ position marker. Reactions were initiated at 2 mM of each dNTP, 15 nM of [DNA], and 2 nM of BF at 37°C for 30 min.

    Techniques Used: Incubation, Labeling, Marker

    4) Product Images from "Chemical transcription roadblocking for nascent RNA display"

    Article Title: Chemical transcription roadblocking for nascent RNA display

    Journal: bioRxiv

    doi: 10.1101/2019.12.26.888743

    Additional DNA template quality analyses (A) Non-denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (B) Non-denaturing PAGE of DNA templates in which the initial PCR amplification was split to perform translesion synthesis with either standard dNTPs or a thermostable dNTP mixture in which dATP and dCTP were substituted with 2-amino-dATP and 5-propynyl-dCTP. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (C) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification that was performed after several freeze-thaw cycles that occurred over the course of data collection. The size marker is the Low Range ssRNA Ladder (New England Biolabs). (D) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification and a 5’ biotin-TEG modification. The size marker is the Low Range ssRNA Ladder (New England Biolabs).
    Figure Legend Snippet: Additional DNA template quality analyses (A) Non-denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (B) Non-denaturing PAGE of DNA templates in which the initial PCR amplification was split to perform translesion synthesis with either standard dNTPs or a thermostable dNTP mixture in which dATP and dCTP were substituted with 2-amino-dATP and 5-propynyl-dCTP. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (C) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification that was performed after several freeze-thaw cycles that occurred over the course of data collection. The size marker is the Low Range ssRNA Ladder (New England Biolabs). (D) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification and a 5’ biotin-TEG modification. The size marker is the Low Range ssRNA Ladder (New England Biolabs).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Marker, Polymerase Chain Reaction, Amplification, Translesion Synthesis

    5) Product Images from "Chemical transcription roadblocking for nascent RNA display"

    Article Title: Chemical transcription roadblocking for nascent RNA display

    Journal: bioRxiv

    doi: 10.1101/2019.12.26.888743

    Additional DNA template quality analyses (A) Non-denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (B) Non-denaturing PAGE of DNA templates in which the initial PCR amplification was split to perform translesion synthesis with either standard dNTPs or a thermostable dNTP mixture in which dATP and dCTP were substituted with 2-amino-dATP and 5-propynyl-dCTP. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (C) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification that was performed after several freeze-thaw cycles that occurred over the course of data collection. The size marker is the Low Range ssRNA Ladder (New England Biolabs). (D) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification and a 5’ biotin-TEG modification. The size marker is the Low Range ssRNA Ladder (New England Biolabs).
    Figure Legend Snippet: Additional DNA template quality analyses (A) Non-denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (B) Non-denaturing PAGE of DNA templates in which the initial PCR amplification was split to perform translesion synthesis with either standard dNTPs or a thermostable dNTP mixture in which dATP and dCTP were substituted with 2-amino-dATP and 5-propynyl-dCTP. The size marker is the Quick-Load 100 bp DNA Ladder (New England Biolabs). (C) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification that was performed after several freeze-thaw cycles that occurred over the course of data collection. The size marker is the Low Range ssRNA Ladder (New England Biolabs). (D) Denaturing PAGE quality analysis of DNA template with an internal desthiobiotin-TEG modification and a 5’ biotin-TEG modification. The size marker is the Low Range ssRNA Ladder (New England Biolabs).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Marker, Polymerase Chain Reaction, Amplification, Translesion Synthesis

    6) Product Images from "Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids"

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr858

    Replication of P/T DNA with a single 6-MI at the template coding position. ( A ) Sequences used; 6-MI is designated as ‘g’, the coding base is underlined. ( B ) Primer usage as a function of time. The 61 nM unmodified (left) or 6-MI-modified concentrations (right). P/T constructs were incubated with 0.05 nM KF in the presence of 35 μM concentrations of the four canonical dNTPs at 37°C for various times: 0.25 min (lane 2); 0.5 min (lane 3); 0.75 min (lane 4); 1 min (lane 5); 1.5 min (lane 6); 2 min (lane 7); 4 min (lane 8); and primer alone (lane 1). ( C ) Single nucleotide incorporation across from G or a single 6-MI coding base in the template DNA. The 61 nM concentrations of unmodified (circles) or 6-MI modified (squares) DNA constructs were incubated with increasing concentrations of KF for 10 min at 37°C in the presence of 350 μM of dCTP, dTTP, dATP or dGTP, as indicated. Replication was measured using protocol (ii). Primer utilization was calculated as {1- [the intensity of the gel band of the primer strand]/[the intensity of the gel bands corresponding to the primer plus longer products]} .
    Figure Legend Snippet: Replication of P/T DNA with a single 6-MI at the template coding position. ( A ) Sequences used; 6-MI is designated as ‘g’, the coding base is underlined. ( B ) Primer usage as a function of time. The 61 nM unmodified (left) or 6-MI-modified concentrations (right). P/T constructs were incubated with 0.05 nM KF in the presence of 35 μM concentrations of the four canonical dNTPs at 37°C for various times: 0.25 min (lane 2); 0.5 min (lane 3); 0.75 min (lane 4); 1 min (lane 5); 1.5 min (lane 6); 2 min (lane 7); 4 min (lane 8); and primer alone (lane 1). ( C ) Single nucleotide incorporation across from G or a single 6-MI coding base in the template DNA. The 61 nM concentrations of unmodified (circles) or 6-MI modified (squares) DNA constructs were incubated with increasing concentrations of KF for 10 min at 37°C in the presence of 350 μM of dCTP, dTTP, dATP or dGTP, as indicated. Replication was measured using protocol (ii). Primer utilization was calculated as {1- [the intensity of the gel band of the primer strand]/[the intensity of the gel bands corresponding to the primer plus longer products]} .

    Techniques Used: Modification, Construct, Incubation

    7) Product Images from "A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2"

    Article Title: A highly divergent archaeo-eukaryotic primase from the Thermococcus nautilus plasmid, pTN2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1385

    Terminal transferase activity of PolpTN2 ( A ) and PolpTN2Δ 311–923 ( B ). 5′ 32 P-labelled substrates were incubated with PolpTN2 or PolpTN2Δ 311–923 and non-labelled dNTPs in the presence of 10 mM of Mg 2+ as described in Materials and Methods, followed by denaturing gel electrophoresis and autoradiography. A radiolabelled DNA ladder is included to the left and in the middle of the gel. The ladder also serves as a negative control as it consists of the templates used in the experiment. Templates consisted of 1 : a single-stranded 52-mer (ss TT forward, see Supplementary Table S1 ) and 2 : a double-stranded 52-mer (ss TT forward annealed to its complementary oligonucleotide ss TT reverse). Asterisks indicate the position of the 32 P label.
    Figure Legend Snippet: Terminal transferase activity of PolpTN2 ( A ) and PolpTN2Δ 311–923 ( B ). 5′ 32 P-labelled substrates were incubated with PolpTN2 or PolpTN2Δ 311–923 and non-labelled dNTPs in the presence of 10 mM of Mg 2+ as described in Materials and Methods, followed by denaturing gel electrophoresis and autoradiography. A radiolabelled DNA ladder is included to the left and in the middle of the gel. The ladder also serves as a negative control as it consists of the templates used in the experiment. Templates consisted of 1 : a single-stranded 52-mer (ss TT forward, see Supplementary Table S1 ) and 2 : a double-stranded 52-mer (ss TT forward annealed to its complementary oligonucleotide ss TT reverse). Asterisks indicate the position of the 32 P label.

    Techniques Used: Activity Assay, Incubation, Nucleic Acid Electrophoresis, Autoradiography, Negative Control

    8) Product Images from "Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations"

    Article Title: Molecular Mechanisms of Tenofovir Resistance Conferred by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing a Diserine Insertion after Residue 69 and Multiple Thymidine Analog-Associated Mutations

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.3.992-1003.2004

    ATP-dependent excision of TFV chain-terminated primers by wild-type and mutant RTs. (A) A 5′ end-labeled primer-template terminated with TFV was incubated with wild-type (WT), FS-SSS, or FS RT enzymes, followed by the addition of 3.2 mM ATP at 0.1, 5, 15, 30, and 45 min (indicated from left to right) at 37°C. The RT enzymes were heat inactivated, followed by the addition of dNTPs and the Klenow fragment to extend the unblocked primers to yield 50-nt full-length products (extended primer). The reaction products were separated through 8 M urea-16% polyacrylamide sequencing gels and exposed to PhosphorImager screens. (B) The percentages of rescued primers over time were determined for the wild type (WT), FS-SSS, and FS and are means ± standard deviations from three experiments.
    Figure Legend Snippet: ATP-dependent excision of TFV chain-terminated primers by wild-type and mutant RTs. (A) A 5′ end-labeled primer-template terminated with TFV was incubated with wild-type (WT), FS-SSS, or FS RT enzymes, followed by the addition of 3.2 mM ATP at 0.1, 5, 15, 30, and 45 min (indicated from left to right) at 37°C. The RT enzymes were heat inactivated, followed by the addition of dNTPs and the Klenow fragment to extend the unblocked primers to yield 50-nt full-length products (extended primer). The reaction products were separated through 8 M urea-16% polyacrylamide sequencing gels and exposed to PhosphorImager screens. (B) The percentages of rescued primers over time were determined for the wild type (WT), FS-SSS, and FS and are means ± standard deviations from three experiments.

    Techniques Used: Mutagenesis, Labeling, Incubation, Sequencing

    9) Product Images from "Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids"

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr858

    Replication of P/T DNA with a single 6-MI at the template coding position. ( A ) Sequences used; 6-MI is designated as ‘g’, the coding base is underlined. ( B ) Primer usage as a function of time. The 61 nM unmodified (left) or 6-MI-modified concentrations (right). P/T constructs were incubated with 0.05 nM KF in the presence of 35 μM concentrations of the four canonical dNTPs at 37°C for various times: 0.25 min (lane 2); 0.5 min (lane 3); 0.75 min (lane 4); 1 min (lane 5); 1.5 min (lane 6); 2 min (lane 7); 4 min (lane 8); and primer alone (lane 1). ( C ) Single nucleotide incorporation across from G or a single 6-MI coding base in the template DNA. The 61 nM concentrations of unmodified (circles) or 6-MI modified (squares) DNA constructs were incubated with increasing concentrations of KF for 10 min at 37°C in the presence of 350 μM of dCTP, dTTP, dATP or dGTP, as indicated. Replication was measured using protocol (ii). Primer utilization was calculated as {1- [the intensity of the gel band of the primer strand]/[the intensity of the gel bands corresponding to the primer plus longer products]} .
    Figure Legend Snippet: Replication of P/T DNA with a single 6-MI at the template coding position. ( A ) Sequences used; 6-MI is designated as ‘g’, the coding base is underlined. ( B ) Primer usage as a function of time. The 61 nM unmodified (left) or 6-MI-modified concentrations (right). P/T constructs were incubated with 0.05 nM KF in the presence of 35 μM concentrations of the four canonical dNTPs at 37°C for various times: 0.25 min (lane 2); 0.5 min (lane 3); 0.75 min (lane 4); 1 min (lane 5); 1.5 min (lane 6); 2 min (lane 7); 4 min (lane 8); and primer alone (lane 1). ( C ) Single nucleotide incorporation across from G or a single 6-MI coding base in the template DNA. The 61 nM concentrations of unmodified (circles) or 6-MI modified (squares) DNA constructs were incubated with increasing concentrations of KF for 10 min at 37°C in the presence of 350 μM of dCTP, dTTP, dATP or dGTP, as indicated. Replication was measured using protocol (ii). Primer utilization was calculated as {1- [the intensity of the gel band of the primer strand]/[the intensity of the gel bands corresponding to the primer plus longer products]} .

    Techniques Used: Modification, Construct, Incubation

    10) Product Images from "A transcription and translation-coupled DNA replication system using rolling-circle replication"

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    Journal: Scientific Reports

    doi: 10.1038/srep10404

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    Figure Legend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.
    Figure Legend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Techniques Used: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    11) Product Images from "A multistep damage recognition mechanism for global genomic nucleotide excision repair"

    Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair

    Journal: Genes & Development

    doi: 10.1101/gad.866301

    Binding of XPC–HR23B is not sufficient for the NER incision, but damage is also required. ( A ) The indicated substrates were internally labeled with 32 P and used for the dual incision assay involving the XP-C whole cell extract and various amounts of XPC–HR23B. The DNA samples were subjected to denaturing PAGE followed by autoradiography. (M) 32 P-labeled 25-bp ladder. ( B ) A map of Hae III-cutting sites in the DNA substrates, where the size of each fragment (in base pairs) is indicated. ( C ) The nonlabeled closed circular DNA substrates indicated were incubated in the cell-free NER reactions including the XP-C whole cell extract, various amounts of XPC–HR23B, and aphidicolin. The purified DNA samples were subjected to gap-filling DNA synthesis with T4 DNA polymerase and radiolabeled dNTPs, digestion with Hae III, and nondenaturing PAGE followed by autoradiography.
    Figure Legend Snippet: Binding of XPC–HR23B is not sufficient for the NER incision, but damage is also required. ( A ) The indicated substrates were internally labeled with 32 P and used for the dual incision assay involving the XP-C whole cell extract and various amounts of XPC–HR23B. The DNA samples were subjected to denaturing PAGE followed by autoradiography. (M) 32 P-labeled 25-bp ladder. ( B ) A map of Hae III-cutting sites in the DNA substrates, where the size of each fragment (in base pairs) is indicated. ( C ) The nonlabeled closed circular DNA substrates indicated were incubated in the cell-free NER reactions including the XP-C whole cell extract, various amounts of XPC–HR23B, and aphidicolin. The purified DNA samples were subjected to gap-filling DNA synthesis with T4 DNA polymerase and radiolabeled dNTPs, digestion with Hae III, and nondenaturing PAGE followed by autoradiography.

    Techniques Used: Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Incubation, Purification, DNA Synthesis

    Related Articles

    Amplification:

    Article Title: Chemical transcription roadblocking for nascent RNA display
    Article Snippet: .. Briefly, five 100 μl reactions containing 81.5 μl of water, 10 μl Thermo Pol Buffer (New England Biolabs, Ipswich, MA), 2 μl of 10 mM dNTPs (New England Biolabs), 2.5 μl of 10 μM oligonucleotide A (unmodified forward primer; ), 2.5 μl of 10 μM oligonucleotide C (unmodified reverse primer; ) or oligonucleotide F (5’ biotinylated reverse primer, ), 1 μl of Vent Exo- DNA polymerase (New England Biolabs), and 0.5 μl of 0.1 nM oligonucleotide G (template oligonucleotide, ) were amplified for 30 PCR cycles. .. Following amplification, 100 μl reactions were combined into two 250 μl pools and precipitated by adding 25 μl of 3M sodium acetate (NaOAc) pH 5.5 and 750 μl of cold 100% ethanol (EtOH), chilling at −80C for 15 minutes, and centrifugation at 20,000 x g for 15 minutes.

    Ligation:

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining
    Article Snippet: .. Ligation was initiated by addition of dNTPs or individual ddNTPs to 25 μM (or 25 μM each), MgCl2 to 5 mM and 200 ng supercoiled plasmid DNA (Litmus38; New England Biolabs, Ipswich, MA, USA). ..

    Purification:

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids
    Article Snippet: .. When present, the concentration of betaine used as a small molecule solvent additive was 5.5 M. DNA replication studies were performed using two protocols. (i) KF exo- DNA polymerase was purified as previously described ( ) and polymerase activity was measured in buffer containing 20 mM HEPES (pH 7.9), 100 mM sodium acetate, 10 mM Mg(OAc)2 and 1 mM DTT, using 3 µM concentrations of P/T DNA and 500 µM dNTPs, and incubated at 25°C for time periods up to 1 h. (ii) KF DNAP was purchased from New England BioLabs and assays were performed at 37°C for the time periods indicated in reaction mixtures containing 10 mM Tris–HCl (pH 7.9), 10 mM MgCl2 , 50 mM NaCl, 1 mM DTT and quantities of DNA template, KF DNAP and dNTPs specified for each experiment. .. In both cases primer strands were labeled at the 5′-terminus with γ−P32 -ATP and DNA products were separated on 12–15% sequencing gels in 7 M urea and then visualized and quantified using the Molecular Dynamics PhosphorImager with ImageQuant software.

    Concentration Assay:

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids
    Article Snippet: .. When present, the concentration of betaine used as a small molecule solvent additive was 5.5 M. DNA replication studies were performed using two protocols. (i) KF exo- DNA polymerase was purified as previously described ( ) and polymerase activity was measured in buffer containing 20 mM HEPES (pH 7.9), 100 mM sodium acetate, 10 mM Mg(OAc)2 and 1 mM DTT, using 3 µM concentrations of P/T DNA and 500 µM dNTPs, and incubated at 25°C for time periods up to 1 h. (ii) KF DNAP was purchased from New England BioLabs and assays were performed at 37°C for the time periods indicated in reaction mixtures containing 10 mM Tris–HCl (pH 7.9), 10 mM MgCl2 , 50 mM NaCl, 1 mM DTT and quantities of DNA template, KF DNAP and dNTPs specified for each experiment. .. In both cases primer strands were labeled at the 5′-terminus with γ−P32 -ATP and DNA products were separated on 12–15% sequencing gels in 7 M urea and then visualized and quantified using the Molecular Dynamics PhosphorImager with ImageQuant software.

    Incubation:

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids
    Article Snippet: .. When present, the concentration of betaine used as a small molecule solvent additive was 5.5 M. DNA replication studies were performed using two protocols. (i) KF exo- DNA polymerase was purified as previously described ( ) and polymerase activity was measured in buffer containing 20 mM HEPES (pH 7.9), 100 mM sodium acetate, 10 mM Mg(OAc)2 and 1 mM DTT, using 3 µM concentrations of P/T DNA and 500 µM dNTPs, and incubated at 25°C for time periods up to 1 h. (ii) KF DNAP was purchased from New England BioLabs and assays were performed at 37°C for the time periods indicated in reaction mixtures containing 10 mM Tris–HCl (pH 7.9), 10 mM MgCl2 , 50 mM NaCl, 1 mM DTT and quantities of DNA template, KF DNAP and dNTPs specified for each experiment. .. In both cases primer strands were labeled at the 5′-terminus with γ−P32 -ATP and DNA products were separated on 12–15% sequencing gels in 7 M urea and then visualized and quantified using the Molecular Dynamics PhosphorImager with ImageQuant software.

    Activity Assay:

    Article Title: Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids
    Article Snippet: .. When present, the concentration of betaine used as a small molecule solvent additive was 5.5 M. DNA replication studies were performed using two protocols. (i) KF exo- DNA polymerase was purified as previously described ( ) and polymerase activity was measured in buffer containing 20 mM HEPES (pH 7.9), 100 mM sodium acetate, 10 mM Mg(OAc)2 and 1 mM DTT, using 3 µM concentrations of P/T DNA and 500 µM dNTPs, and incubated at 25°C for time periods up to 1 h. (ii) KF DNAP was purchased from New England BioLabs and assays were performed at 37°C for the time periods indicated in reaction mixtures containing 10 mM Tris–HCl (pH 7.9), 10 mM MgCl2 , 50 mM NaCl, 1 mM DTT and quantities of DNA template, KF DNAP and dNTPs specified for each experiment. .. In both cases primer strands were labeled at the 5′-terminus with γ−P32 -ATP and DNA products were separated on 12–15% sequencing gels in 7 M urea and then visualized and quantified using the Molecular Dynamics PhosphorImager with ImageQuant software.

    Translesion Synthesis:

    Article Title: Chemical transcription roadblocking for nascent RNA display
    Article Snippet: .. Translesion synthesis reactions were prepared by combining the eluted DNA with 100 μl of Thermo Pol Buffer (New England Biolabs), 20 μl of 10 mM dNTPs (New England Biolabs), 10 μl of Sulfolobus DNA polymerase IV (New England Biolabs) and nuclease-free water to 1 ml. .. The 1 ml master mix was split into 100 μl aliquots and incubated at 55C for 1 hour.

    Polymerase Chain Reaction:

    Article Title: Chemical transcription roadblocking for nascent RNA display
    Article Snippet: .. Briefly, five 100 μl reactions containing 81.5 μl of water, 10 μl Thermo Pol Buffer (New England Biolabs, Ipswich, MA), 2 μl of 10 mM dNTPs (New England Biolabs), 2.5 μl of 10 μM oligonucleotide A (unmodified forward primer; ), 2.5 μl of 10 μM oligonucleotide C (unmodified reverse primer; ) or oligonucleotide F (5’ biotinylated reverse primer, ), 1 μl of Vent Exo- DNA polymerase (New England Biolabs), and 0.5 μl of 0.1 nM oligonucleotide G (template oligonucleotide, ) were amplified for 30 PCR cycles. .. Following amplification, 100 μl reactions were combined into two 250 μl pools and precipitated by adding 25 μl of 3M sodium acetate (NaOAc) pH 5.5 and 750 μl of cold 100% ethanol (EtOH), chilling at −80C for 15 minutes, and centrifugation at 20,000 x g for 15 minutes.

    Plasmid Preparation:

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining
    Article Snippet: .. Ligation was initiated by addition of dNTPs or individual ddNTPs to 25 μM (or 25 μM each), MgCl2 to 5 mM and 200 ng supercoiled plasmid DNA (Litmus38; New England Biolabs, Ipswich, MA, USA). ..

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    New England Biolabs dntps
    Relative quantity of circularized MIP products. The y-axis shows the ‘percentage of unique reads’, with unique reads defined as those having distinct barcodes and arm sequence combinations. Since the number of unique reads increases as total number of reads increases, in order to normalize it, we randomly selected 2 million reads for each condition, counted the number of unique reads and calculated the percentage of unique reads. As the amount of gDNA or hybridization time increased, more unique reads were detected, indicating that more circularized product was obtained. A 1:500 gDNA:probe ratio and <t>10x</t> <t>dNTPs</t> were used for all conditions.
    Dntps, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Relative quantity of circularized MIP products. The y-axis shows the ‘percentage of unique reads’, with unique reads defined as those having distinct barcodes and arm sequence combinations. Since the number of unique reads increases as total number of reads increases, in order to normalize it, we randomly selected 2 million reads for each condition, counted the number of unique reads and calculated the percentage of unique reads. As the amount of gDNA or hybridization time increased, more unique reads were detected, indicating that more circularized product was obtained. A 1:500 gDNA:probe ratio and 10x dNTPs were used for all conditions.

    Journal: Nucleic Acids Research

    Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes

    doi: 10.1093/nar/gku1188

    Figure Lengend Snippet: Relative quantity of circularized MIP products. The y-axis shows the ‘percentage of unique reads’, with unique reads defined as those having distinct barcodes and arm sequence combinations. Since the number of unique reads increases as total number of reads increases, in order to normalize it, we randomly selected 2 million reads for each condition, counted the number of unique reads and calculated the percentage of unique reads. As the amount of gDNA or hybridization time increased, more unique reads were detected, indicating that more circularized product was obtained. A 1:500 gDNA:probe ratio and 10x dNTPs were used for all conditions.

    Article Snippet: Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C.

    Techniques: Sequencing, Hybridization

    Comparison of capture efficiencies under different conditions. The effects of varying ( A ) the gDNA:probe ratio, and ( B ) the amount of dNTPs are shown. Band intensity (∼200 bp, red arrows) is proportional to the amount of captured product on each of the capture parameters, because the number of PCR cycles was held constant at 26 cycles. The amount of captured products was saturated at 1:500 gDNA:probe ratio and 10x dNTPs. ( C ) and ( D ) Captured products were detected around 200 bp for all conditions, and only products in these bands were separated and used for further analysis.

    Journal: Nucleic Acids Research

    Article Title: microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes

    doi: 10.1093/nar/gku1188

    Figure Lengend Snippet: Comparison of capture efficiencies under different conditions. The effects of varying ( A ) the gDNA:probe ratio, and ( B ) the amount of dNTPs are shown. Band intensity (∼200 bp, red arrows) is proportional to the amount of captured product on each of the capture parameters, because the number of PCR cycles was held constant at 26 cycles. The amount of captured products was saturated at 1:500 gDNA:probe ratio and 10x dNTPs. ( C ) and ( D ) Captured products were detected around 200 bp for all conditions, and only products in these bands were separated and used for further analysis.

    Article Snippet: Then, 2 U of AmpliTaq® DNA polymerase (Life Technologies), 4 U of Ampligase DNA ligase (Epicentre® ), 10x dNTPs (NEB® Inc), 0.2 μl of Ampligase buffer (Epicentre® ) were added and the mixtures were incubated for 24 h at 60°C.

    Techniques: Polymerase Chain Reaction

    Translesion DNA synthesis by human DNA polymerases κ , η , ι , or Rev1 with unmodified or LdG-containing DNA substrate. Reaction conditions are described in the Materials and Methods section. (A) Primer extension reactions in the presence of all four dNTPs at their physiological concentrations (i.e., 10 μ M for dGTP and 40 μ . Changes in catalytic efficiency relative to a native base pair were calculated from ( k cat / K m,dCTP ) unmodified /( k cat / K m,dCTP ) LdG and indicated as x-fold decrease.

    Journal: Chemical research in toxicology

    Article Title: Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ

    doi: 10.1021/acs.chemrestox.7b00227

    Figure Lengend Snippet: Translesion DNA synthesis by human DNA polymerases κ , η , ι , or Rev1 with unmodified or LdG-containing DNA substrate. Reaction conditions are described in the Materials and Methods section. (A) Primer extension reactions in the presence of all four dNTPs at their physiological concentrations (i.e., 10 μ M for dGTP and 40 μ . Changes in catalytic efficiency relative to a native base pair were calculated from ( k cat / K m,dCTP ) unmodified /( k cat / K m,dCTP ) LdG and indicated as x-fold decrease.

    Article Snippet: Unlabeled dNTPs, T4 polynucleotide kinase, and uracil DNA glycosylase (UDG) were from New England Biolabs (Ipswich, MA).

    Techniques: DNA Synthesis

    Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) Representative quantification of dTTP using the published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31 to 0.08 pmol) are not shown. ( B ) Representative quantification of dTTP using Q5 DNA polymerase, long oligonucleotide template and EvaGreen detection chemistry. ( C - D ) dTTP signal generated by the fluorometric methods from mouse liver extracts with and without 0.5 pmol dTTP spike-in calibrant. The final extract volume was diluted to 80 µl per 40 mg of initial tissue weight. ( E - F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of the non-linear curve fit.

    Journal: bioRxiv

    Article Title: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

    doi: 10.1101/2019.12.17.879122

    Figure Lengend Snippet: Long synthetic DNA oligonucleotides, an inhibitor-resistant high-fidelity DNA polymerase and EvaGreen detection chemistry allow quantification of dNTPs from mouse liver extracts. ( A ) Representative quantification of dTTP using the published fluorometric probe-hydrolysis-based assay. For clarity, curves from three lowest standard samples (0.31 to 0.08 pmol) are not shown. ( B ) Representative quantification of dTTP using Q5 DNA polymerase, long oligonucleotide template and EvaGreen detection chemistry. ( C - D ) dTTP signal generated by the fluorometric methods from mouse liver extracts with and without 0.5 pmol dTTP spike-in calibrant. The final extract volume was diluted to 80 µl per 40 mg of initial tissue weight. ( E - F ) Standard curves generated from the end-point baseline-corrected fluorescence values. The lowest y-axis value shows the background signal. Gray lines present the 95% confidence interval of the non-linear curve fit.

    Article Snippet: Q5 DNA polymerase and EvaGreen-based assay for dNTPs The following reaction concentrations were found to be optimal: 1x Q5 reaction buffer: 0.25 µM primer and 0.2 µM template, 25 µM non-limiting dNTPs, 1x EvaGreen (Biotum) and 20 U/ml Q5 DNA polymerase (New England Biolabs).

    Techniques: Hydrolysis Assay, Generated, Fluorescence

    Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.

    Journal: Nucleic Acids Research

    Article Title: End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

    doi: 10.1093/nar/gkn164

    Figure Lengend Snippet: Factors required for joining of noncomplementary ends. ( A ) A diagram of the standard 280 bp substrate labeled internally with 32 P (asterisk), and possessing 3′ TT overhangs. Arrows indicate direction of synthesis by pol μ after alignment of ends by core NHEJ factors Ku and XL. ( B ) All reactions used 5 nM DNA substrate as illustrated in (A), and products analyzed after 5 min reactions. 25 nM Ku and 50 nM XL were added as indicated (+). Polymerase μ or λ was added at 25 nM or 250 nM (10×). S, substrate; P, concatamer ligation products. ( C ) Reactions performed as in B except ddNTPs substituted for dNTPs, and synthesis at one end analyzed by denaturing PAGE as described in methods. S, substrate; P, +1 synthesis product.

    Article Snippet: Ligation was initiated by addition of dNTPs or individual ddNTPs to 25 μM (or 25 μM each), MgCl2 to 5 mM and 200 ng supercoiled plasmid DNA (Litmus38; New England Biolabs, Ipswich, MA, USA).

    Techniques: Labeling, Non-Homologous End Joining, Ligation, Polyacrylamide Gel Electrophoresis