dna modifying enzymes  (New England Biolabs)


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    Structured Review

    New England Biolabs dna modifying enzymes
    Genome-wide base composition bias curves in Illumina reads from PCR-free human <t>DNA</t> libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or <t>NEBNext</t> Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Dna Modifying Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    2) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    3) Product Images from "Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells"

    Article Title: Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl703

    Effects of induced expression of TFAM-stop and TFAM-MycHis and of RNAi knockdown of TFAM expression. Mitochondrial proteins, DNA and RNA were analysed from Flp-In™ T-Rex™ -293 cells stably transfected with the TFAM-stop (a–c) or TFAM-MycHis construct (d–f), induced over the times indicated or from HEK293T cells (g–i) following transfection with siRNAs Si2 and Si4 over the times indicated. In each case, error bars indicate means ± SEs from at least three independent experiments. a.u., arbitrary units. Measurements of mtDNA levels (a, d and g) are arbitrarily normalized to the mean values for uninduced or untreated cells. For cells under TFAM induction, the measurements were made by two independent methods, Southern blotting and Q-PCR and the values plotted for each time point are the means of measurements by the two methods, shown in Supplementary Figures 1 and 2. ( b , e and h ) show TFAM protein levels normalized to the mtDNA levels shown in ( a , d and g ), then normalized against the level in uninduced or untreated cells. ( c , f and i ) show ND3 mRNA levels normalized first against the 5S rRNA loading control, then against the mtDNA levels shown in (a, d and g), then finally against the level in uninduced or untreated cells. Samples of the raw data and compiled data for TFAM protein, mtDNA and RNA levels on which this figure is based, are shown in Supplementary Figures 1–3.
    Figure Legend Snippet: Effects of induced expression of TFAM-stop and TFAM-MycHis and of RNAi knockdown of TFAM expression. Mitochondrial proteins, DNA and RNA were analysed from Flp-In™ T-Rex™ -293 cells stably transfected with the TFAM-stop (a–c) or TFAM-MycHis construct (d–f), induced over the times indicated or from HEK293T cells (g–i) following transfection with siRNAs Si2 and Si4 over the times indicated. In each case, error bars indicate means ± SEs from at least three independent experiments. a.u., arbitrary units. Measurements of mtDNA levels (a, d and g) are arbitrarily normalized to the mean values for uninduced or untreated cells. For cells under TFAM induction, the measurements were made by two independent methods, Southern blotting and Q-PCR and the values plotted for each time point are the means of measurements by the two methods, shown in Supplementary Figures 1 and 2. ( b , e and h ) show TFAM protein levels normalized to the mtDNA levels shown in ( a , d and g ), then normalized against the level in uninduced or untreated cells. ( c , f and i ) show ND3 mRNA levels normalized first against the 5S rRNA loading control, then against the mtDNA levels shown in (a, d and g), then finally against the level in uninduced or untreated cells. Samples of the raw data and compiled data for TFAM protein, mtDNA and RNA levels on which this figure is based, are shown in Supplementary Figures 1–3.

    Techniques Used: Expressing, Stable Transfection, Transfection, Construct, Southern Blot, Polymerase Chain Reaction

    Effects of TFAM overexpression and knockdown on mtDNA topology. One-dimensional agarose gel blots, hybridized with O H probe. ( a ) mtDNA from uninduced cells and from cells induced to overexpress TFAM-stop for 48 h, fractionated on a 0.4% agarose gel run in TBE. Only the high molecular weight portion of the gel is shown. Samples were either untreated (U) or treated with T7 gp3 endonuclease (gp3), topoisomerase I (tI), topoisomerase IV (tIV) or topoisomerase IV plus T7 gp3 endonuclease. Identity of the main topoisomers was inferred from enzymatic sensitivity and confirmed by other treatments (data not shown). DNA from cells treated with TFAM-specific siRNAs (RNAi) for 24 h was run on a separate gel. ( b ) MtDNA from TFAM-induced, uninduced and siRNA-treated cells, fractionated on 0.4% agarose gels run in TBE. Only the low molecular weight portion of each gel is shown. First 4 lanes of upper panel are equally exposed, whereas the right-most two lanes are ∼3-fold overloaded, to reveal the presence of 7S DNA in induced cells. Samples were either heated for 2 min at 95°C (+) or left unheated, as indicated.
    Figure Legend Snippet: Effects of TFAM overexpression and knockdown on mtDNA topology. One-dimensional agarose gel blots, hybridized with O H probe. ( a ) mtDNA from uninduced cells and from cells induced to overexpress TFAM-stop for 48 h, fractionated on a 0.4% agarose gel run in TBE. Only the high molecular weight portion of the gel is shown. Samples were either untreated (U) or treated with T7 gp3 endonuclease (gp3), topoisomerase I (tI), topoisomerase IV (tIV) or topoisomerase IV plus T7 gp3 endonuclease. Identity of the main topoisomers was inferred from enzymatic sensitivity and confirmed by other treatments (data not shown). DNA from cells treated with TFAM-specific siRNAs (RNAi) for 24 h was run on a separate gel. ( b ) MtDNA from TFAM-induced, uninduced and siRNA-treated cells, fractionated on 0.4% agarose gels run in TBE. Only the low molecular weight portion of each gel is shown. First 4 lanes of upper panel are equally exposed, whereas the right-most two lanes are ∼3-fold overloaded, to reveal the presence of 7S DNA in induced cells. Samples were either heated for 2 min at 95°C (+) or left unheated, as indicated.

    Techniques Used: Over Expression, Agarose Gel Electrophoresis, Molecular Weight

    4) Product Images from "Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA"

    Article Title: Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA

    Journal: Nucleic Acids Research

    doi:

    Substrate properties of modified dNTP derivatives in primer extension assays. The incorporation of each modified dNTP derivative was evaluated in the homopolymer template assay. A 10 pmol concentration of the DNA substrate (homopolymer template annealed to a 5′ digoxigenin- labeled primer), 1 U of DNA pol (Vent R exo – or wild-type Taq ) and 50 µM of each dNTP derivative to be tested were incubated in a DNA pol assay as outlined in Materials and Methods. The primer extension products were resolved on a 7 M urea–12% (v/v) polyacrylamide gel and identified after detection of the 5′ digoxigenin label on the primer. ( A ) Evaluation of dUTP derivatives. Lanes 1–10, Vent R exo – DNA pol; lanes 11–19, Taq DNA pol. Lane 1, primer alone (negative control); lanes 2 and 11, dTTP (5 µM); lanes 3 and 12, dTTP (50 µM) (positive control); lanes 4 and 13, biotin-dUTP; lanes 5 and 14, RhG-dUTP; lanes 6 and 15, Atto655-dUTP; lanes 7 and 16, GB1-dUTP; lanes 8 and 17, GB3-dUTP; lanes 9 and 18, Evo30-dUTP; and lanes 10 and 19, Alexa 488-dUTP. Arrowheads indicate the positions of the unextended primer as well as the primer elongated by 18 dTs. The arrow indicates the shift in product size due to the incorporation of labeled nucleotides.
    Figure Legend Snippet: Substrate properties of modified dNTP derivatives in primer extension assays. The incorporation of each modified dNTP derivative was evaluated in the homopolymer template assay. A 10 pmol concentration of the DNA substrate (homopolymer template annealed to a 5′ digoxigenin- labeled primer), 1 U of DNA pol (Vent R exo – or wild-type Taq ) and 50 µM of each dNTP derivative to be tested were incubated in a DNA pol assay as outlined in Materials and Methods. The primer extension products were resolved on a 7 M urea–12% (v/v) polyacrylamide gel and identified after detection of the 5′ digoxigenin label on the primer. ( A ) Evaluation of dUTP derivatives. Lanes 1–10, Vent R exo – DNA pol; lanes 11–19, Taq DNA pol. Lane 1, primer alone (negative control); lanes 2 and 11, dTTP (5 µM); lanes 3 and 12, dTTP (50 µM) (positive control); lanes 4 and 13, biotin-dUTP; lanes 5 and 14, RhG-dUTP; lanes 6 and 15, Atto655-dUTP; lanes 7 and 16, GB1-dUTP; lanes 8 and 17, GB3-dUTP; lanes 9 and 18, Evo30-dUTP; and lanes 10 and 19, Alexa 488-dUTP. Arrowheads indicate the positions of the unextended primer as well as the primer elongated by 18 dTs. The arrow indicates the shift in product size due to the incorporation of labeled nucleotides.

    Techniques Used: Modification, Concentration Assay, Labeling, Incubation, Negative Control, Positive Control

    Incorporation of modified dNTPs into natural DNA. A 1 pmol concentration of a natural DNA substrate (5′ biotin-labeled 300 bp PCR product annealed to a 5′ biotin-labeled primer), 2 U of DNA pol (Vent R exo – ) and 0.2 mM of each of the four dNTPs were used to test modified dNTP derivatives in the primer extension assay as outlined in Materials and Methods. One of the four natural dNTPs was gradually (in steps of 20%) substituted by its modified derivative until it was completely (100%) replaced. After the reaction, the products were separated on a 7 M urea–6% (v/v) polyacrylamide gel followed by transfer onto a nylon membrane and the DNA visualized after detecting for the 5′ biotin label, which is on both the natural DNA template and the extended primer. The levels of replacement of a given natural dNTP by its modified derivative in the dNTP mixture are indicated as a percentage. The level of the 300 bp natural DNA template delineates the baseline position with natural dNTP exclusively (indicated by an arrowhead). Lanes 1–6, GB1-dUTP; lanes 7–12, GB2-dUTP; lanes 13–18, GB3-dUTP; lanes 25–29, biotin-dUTP. In lanes 19–24, double dNTP substitutions at 50 and 100% are shown. Lanes 19 and 20, RhG-dCTP and GB1-dUTP; lanes 21 and 22, RhG-dCTP and GB2-dUTP; lanes 23 and 24, RhG-dCTP and GB3-dUTP. For each modified dNTP derivative, gradual substitution leads to a corresponding shift of modified product DNA. Arrows indicate the final product of the 100% substitutions (lanes 6, 12, 18 and 29) and the 50% as well as the 100% substitutions of the double dNTP substitutions (lanes 19–24).
    Figure Legend Snippet: Incorporation of modified dNTPs into natural DNA. A 1 pmol concentration of a natural DNA substrate (5′ biotin-labeled 300 bp PCR product annealed to a 5′ biotin-labeled primer), 2 U of DNA pol (Vent R exo – ) and 0.2 mM of each of the four dNTPs were used to test modified dNTP derivatives in the primer extension assay as outlined in Materials and Methods. One of the four natural dNTPs was gradually (in steps of 20%) substituted by its modified derivative until it was completely (100%) replaced. After the reaction, the products were separated on a 7 M urea–6% (v/v) polyacrylamide gel followed by transfer onto a nylon membrane and the DNA visualized after detecting for the 5′ biotin label, which is on both the natural DNA template and the extended primer. The levels of replacement of a given natural dNTP by its modified derivative in the dNTP mixture are indicated as a percentage. The level of the 300 bp natural DNA template delineates the baseline position with natural dNTP exclusively (indicated by an arrowhead). Lanes 1–6, GB1-dUTP; lanes 7–12, GB2-dUTP; lanes 13–18, GB3-dUTP; lanes 25–29, biotin-dUTP. In lanes 19–24, double dNTP substitutions at 50 and 100% are shown. Lanes 19 and 20, RhG-dCTP and GB1-dUTP; lanes 21 and 22, RhG-dCTP and GB2-dUTP; lanes 23 and 24, RhG-dCTP and GB3-dUTP. For each modified dNTP derivative, gradual substitution leads to a corresponding shift of modified product DNA. Arrows indicate the final product of the 100% substitutions (lanes 6, 12, 18 and 29) and the 50% as well as the 100% substitutions of the double dNTP substitutions (lanes 19–24).

    Techniques Used: Modification, Concentration Assay, Labeling, Polymerase Chain Reaction, Primer Extension Assay

    Complete substitution of all four natural dNTPs by biotinylated derivatives. The DNA pol reactions were performed as outlined in Materials and Methods. In (A)–(C), 1 pmol of natural DNA substrate (5′ biotin-labeled 300 bp pUC19 PCR product), 2.5 U of Vent R exo – DNA pol and 0.2 mM of each of the four dNTPs were used in a primer extension assay. After the reaction, the DNA synthesis products were separated on a 1.5% agarose gel followed by ethidium bromide staining. ( A ) A single natural dNTP at a time was replaced by the corresponding biotin-dNTP derivative. Lane 1, control reaction with only natural dNTPs; lane 2, dATP replaced by biotin-dATP; lane 3, dCTP replaced by biotin-dCTP; lane 4, dGTP replaced by biotin-dGTP; lane 5, dUTP replaced by biotin-dUTP. ( B ) Stepwise replacement of all four dNTPs simultaneously by the corresponding biotin-dNTPs. Lane 1, control reaction with natural dNTPs; lanes 2–6, substitution levels of 20, 40, 60, 80 and 100%, respectively. ( C ) Complete replacement of one natural dNTP after another until all the four natural dNTPs were replaced by the corresponding biotin-dNTPs. Lane 1, control reaction with natural dNTPs; lane 2, dATP replaced by biotin-dATP; lane 3, dATP and dCTP replaced by biotin-dATP and biotin-dCTP; lane 4, dATP, dCTP and dGTP replaced by biotin-dATP, biotin-dCTP and biotin-dGTP; lane 5, all natural dNTPs replaced by biotin-dATP, biotin-dCTP, biotin-dGTP and biotin-dUTP. Note that the 100% substituted final 300 bp products are present in (C) lane 5 and (B) lane 6, and they migrate at the same position. ( D ) Experiments were carried out as described in (A) using linearized pUC19 DNA (lanes 1–8 and 14–17) or the 300 bp pUC19 PCR product (lanes 9–13) as a template. To facilitate newly synthesized DNA identification, in all cases one of the primers used had a 5′ biotin label. For PCR amplification (with one or two primers), 0.1 pmol of Hin dIII-linearized pUC19 template was used to introduce the biotinylated dNTP derivatives (lanes 1–8). In the primer extension reactions, 1 pmol of 5′ biotin-labeled 300 bp pUC19 PCR product or linearized pUC19 DNA was used (lanes 9–17). Lane 9 represents a control reaction where only natural dNTPs are included. The order of substitution in all reaction blocks was: biotin-dATP (lanes 1, 5, 10 and 14), biotin-dATP and biotin-dCTP (lanes 2, 6, 11 and 15), biotin-dATP, biotin-dCTP and biotin-dGTP (lanes 3, 7, 12 and 16) and all four natural dNTPs replaced (lanes 4, 8, 13 and 17). Arrowheads indicate the position of the 300 bp natural DNA template.
    Figure Legend Snippet: Complete substitution of all four natural dNTPs by biotinylated derivatives. The DNA pol reactions were performed as outlined in Materials and Methods. In (A)–(C), 1 pmol of natural DNA substrate (5′ biotin-labeled 300 bp pUC19 PCR product), 2.5 U of Vent R exo – DNA pol and 0.2 mM of each of the four dNTPs were used in a primer extension assay. After the reaction, the DNA synthesis products were separated on a 1.5% agarose gel followed by ethidium bromide staining. ( A ) A single natural dNTP at a time was replaced by the corresponding biotin-dNTP derivative. Lane 1, control reaction with only natural dNTPs; lane 2, dATP replaced by biotin-dATP; lane 3, dCTP replaced by biotin-dCTP; lane 4, dGTP replaced by biotin-dGTP; lane 5, dUTP replaced by biotin-dUTP. ( B ) Stepwise replacement of all four dNTPs simultaneously by the corresponding biotin-dNTPs. Lane 1, control reaction with natural dNTPs; lanes 2–6, substitution levels of 20, 40, 60, 80 and 100%, respectively. ( C ) Complete replacement of one natural dNTP after another until all the four natural dNTPs were replaced by the corresponding biotin-dNTPs. Lane 1, control reaction with natural dNTPs; lane 2, dATP replaced by biotin-dATP; lane 3, dATP and dCTP replaced by biotin-dATP and biotin-dCTP; lane 4, dATP, dCTP and dGTP replaced by biotin-dATP, biotin-dCTP and biotin-dGTP; lane 5, all natural dNTPs replaced by biotin-dATP, biotin-dCTP, biotin-dGTP and biotin-dUTP. Note that the 100% substituted final 300 bp products are present in (C) lane 5 and (B) lane 6, and they migrate at the same position. ( D ) Experiments were carried out as described in (A) using linearized pUC19 DNA (lanes 1–8 and 14–17) or the 300 bp pUC19 PCR product (lanes 9–13) as a template. To facilitate newly synthesized DNA identification, in all cases one of the primers used had a 5′ biotin label. For PCR amplification (with one or two primers), 0.1 pmol of Hin dIII-linearized pUC19 template was used to introduce the biotinylated dNTP derivatives (lanes 1–8). In the primer extension reactions, 1 pmol of 5′ biotin-labeled 300 bp pUC19 PCR product or linearized pUC19 DNA was used (lanes 9–17). Lane 9 represents a control reaction where only natural dNTPs are included. The order of substitution in all reaction blocks was: biotin-dATP (lanes 1, 5, 10 and 14), biotin-dATP and biotin-dCTP (lanes 2, 6, 11 and 15), biotin-dATP, biotin-dCTP and biotin-dGTP (lanes 3, 7, 12 and 16) and all four natural dNTPs replaced (lanes 4, 8, 13 and 17). Arrowheads indicate the position of the 300 bp natural DNA template.

    Techniques Used: Labeling, Polymerase Chain Reaction, Primer Extension Assay, DNA Synthesis, Agarose Gel Electrophoresis, Staining, Synthesized, Amplification, Introduce

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    Amplification:

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    Synthesized:

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    Construct:

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    Adsorption:

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    Incubation:

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    Activity Assay:

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    Cell Culture:

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    Expressing:

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    Article Snippet: Paragraph title: Expression plasmids and gene assembly ... Restriction- and DNA modifying enzymes and Vent-DNA polymerase (New England Biolabs, Beverly, MA) were used as recommended by the manufacturers.

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs. .. Afterward cyaA was deleted using Red® /ET® recombination technology and the kanamycin cassette was removed using the 709-FLPe/amp expression vector in accordance to the Quick and Easy E. coli Gene Deletion Kit (Gene Bridges, Germany).

    Infection:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Small aliquots of infected cell culture supernatants were collected at various times postinfection, clarified, and stored at −80°C for virus titration at a later time. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    other:

    Article Title: Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells
    Article Snippet: Enzymatic treatment of DNA MtDNA samples were treated with the following DNA-modifying enzymes under conditions recommended by the manufacturers: T7 gp3 endonuclease (New England Biolabs), topoisomerase I (New England Biolabs) and topoisomerase IV (John Innes Enterprises).

    Article Title: Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation
    Article Snippet: Restriction and DNA-modifying enzymes and calf intestine phosphatase (CIP) were obtained from New England Biolabs (Beverly, Mass.) or from Eurogentec (Seraing, Belgium).

    Article Title: Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA
    Article Snippet: Restriction endonucleases, DNA-modifying enzymes and VentR exo– DNA pol were obtained from New England Biolabs.

    DNA Labeling:

    Article Title: Deletion mutations in N-terminal ?1 helix render heat labile enterotoxin B subunit susceptible to degradation
    Article Snippet: All restriction enzymes, DNA-modifying enzymes, and DNA markers were purchased from New England Biolabs (Ipswich, MA) and used as recommended by the suppliers. .. The ECL-Western blotting kit and multiprime DNA labeling kit were procured from Amersham (Piscataway, NJ).

    Polymerase Chain Reaction:

    Article Title: Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris
    Article Snippet: Restriction- and DNA modifying enzymes and Vent-DNA polymerase (New England Biolabs, Beverly, MA) were used as recommended by the manufacturers. .. Gene assembly was conducted by introduction of suitable restriction sites using modifying PCR primers.

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
    Article Snippet: Materials All DNA modifying enzymes and NEBNext Ultra II Library Prep Kit were provided by New England Biolabs (NEB) (Ipswich, MA). .. Illumina TruSeq DNA PCR-Free LT Library Preparation Kit was obtained from Illumina (San Diego, CA) and Kapa Hyper Prep Kit was obtained from Kapa Biosystems (Wilmington, MA).

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
    Article Snippet: All DNA modifying enzymes and NEBNext Ultra II Library Prep Kit were provided by New England Biolabs (NEB) (Ipswich, MA). .. Illumina TruSeq DNA PCR-Free LT Library Preparation Kit was obtained from Illumina (San Diego, CA) and Kapa Hyper Prep Kit was obtained from Kapa Biosystems (Wilmington, MA).

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: .. All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs. .. Escherichia coli strain KV1 for bacterial two-hybrid analysis was constructed as follows: The luxCDABE operon from Photorhabdus luminescens was amplified from pBAD/HisA-Lux ( ) and integrated into E. coli LF1 as essentially described previously by .

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA). .. SuperScript II was obtained from Invitrogen (Carlsbad, CA).

    Binding Assay:

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs. .. To keep the ability of blue/white screening, a synthetic ribosomal binding site predicted by RBS calculator ( ; ) was introduced upstream of the lacZ start site.

    Nucleic Acid Purification:

    Article Title: Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)
    Article Snippet: DNA modifying enzymes, deoxyribonucleotides and DNA ladders were purchased from New England Biolabs (Hitchin, UK), Thermo Fisher Scientific (Loughborough, UK) and Agilent Technologies (Cheadle, UK). .. Nucleic acid purification kits were purchased from Qiagen (Manchester, UK), Machery-Nagel (Düren, Germany) and Omega Bio-tek (Norcross, USA).

    Plaque Assay:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Virus quantitation was performed by plaque assay on Vero cells. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Mutagenesis:

    Article Title: Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double-stranded DNA cleavage activity whereas Asp218 is not
    Article Snippet: DNA-modifying enzymes, restriction endonucleases, IMPACT-T7 cloning system, and chitin resin were procured from New England Biolabs (UK). .. Mutagenesis primers were obtained from Sigma-Genosys.

    Isolation:

    Article Title: Deletion mutations in N-terminal ?1 helix render heat labile enterotoxin B subunit susceptible to degradation
    Article Snippet: All restriction enzymes, DNA-modifying enzymes, and DNA markers were purchased from New England Biolabs (Ipswich, MA) and used as recommended by the suppliers. .. The TRIzol-RNA isolation kit and E. coli S30 cell extract were obtained from Invitrogen (Carlsbad, CA) and Promega (Madison, WI), respectively.

    Article Title: Mutant TEM ?-Lactamase Producing Resistance to Ceftazidime, Ampicillins, and ?-Lactamase Inhibitors
    Article Snippet: Restriction endonucleases, DNA-modifying enzymes, T4 DNA polymerase, and Taq DNA polymerase were purchased from New England Biolabs or BRL; Pfu Turbo DNA polymerase was purchased from Stratagene; and DNaseI was purchased from Sigma. .. Plasmid DNA was isolated with a QIAprep Spin Miniprep kit (Qiagen Inc.).

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: Plasmid DNA was isolated using the Hi Yield® Plasmid Mini Kit from Süd Laborbedarf. .. All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs.

    Purification:

    Article Title: Evolutionary Link between the Mycobacterial Plasmid pAL5000 Replication Protein RepB and the Extracytoplasmic Function Family of ? Factors
    Article Snippet: Other chemicals for protein expression, purification, and analysis, of the highest grade of purity, were obtained from SRL Laboratories, India. .. Restriction enzymes and DNA-modifying enzymes such as polynucleotide kinase were purchased from New England BioLabs (NEB).

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: DNA fragments were purified from agarose gels using the Hi Yield® Gel/PCR DNA fragment extraction kit from Süd Laborbedarf. .. All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs.

    Sequencing:

    Article Title: Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris
    Article Snippet: Restriction- and DNA modifying enzymes and Vent-DNA polymerase (New England Biolabs, Beverly, MA) were used as recommended by the manufacturers. .. All PCR-derived fragments were sequence verified after cloning.

    Gel Extraction:

    Article Title: Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double-stranded DNA cleavage activity whereas Asp218 is not
    Article Snippet: DNA-modifying enzymes, restriction endonucleases, IMPACT-T7 cloning system, and chitin resin were procured from New England Biolabs (UK). .. DNA gel extraction kit was purchased from QIAGEN.

    IA:

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
    Article Snippet: Materials All DNA modifying enzymes and NEBNext Ultra II Library Prep Kit were provided by New England Biolabs (NEB) (Ipswich, MA). .. DNA oligos were synthesized by Integrated DNA Technologies (Coralville, IA) and dissolved in nuclease-free water prior to use (Supplementary Table ).

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
    Article Snippet: All DNA modifying enzymes and NEBNext Ultra II Library Prep Kit were provided by New England Biolabs (NEB) (Ipswich, MA). .. DNA oligos were synthesized by Integrated DNA Technologies (Coralville, IA) and dissolved in nuclease-free water prior to use (Supplementary Table ).

    Titration:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Small aliquots of infected cell culture supernatants were collected at various times postinfection, clarified, and stored at −80°C for virus titration at a later time. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Plasmid Preparation:

    Article Title: The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity *
    Article Snippet: DNA-modifying enzymes, PNGaseF and EndoH, were from New England Biolabs (Frankfurt am Main, Germany). .. Vector plasmid pEGFP-N1 (encoding the enhanced variant of GFP) and the monoclonal anti-GFP antibody were from Clontech.

    Article Title: Mutant TEM ?-Lactamase Producing Resistance to Ceftazidime, Ampicillins, and ?-Lactamase Inhibitors
    Article Snippet: Restriction endonucleases, DNA-modifying enzymes, T4 DNA polymerase, and Taq DNA polymerase were purchased from New England Biolabs or BRL; Pfu Turbo DNA polymerase was purchased from Stratagene; and DNaseI was purchased from Sigma. .. Plasmid DNA was isolated with a QIAprep Spin Miniprep kit (Qiagen Inc.).

    Article Title: The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity
    Article Snippet: Materials All restriction enzymes, DNA-modifying enzymes, and DNA ladders were obtained from New England Biolabs. .. Plasmid pETM-41 and TEV protease were kindly provided by Dr Amit Sharma (ICGEB, New Delhi).

    Article Title: Switching the Post-translational Modification of Translation Elongation Factor EF-P
    Article Snippet: Paragraph title: Plasmid and Strain Construction ... All restriction enzymes, DNA modifying enzymes and the Q5® high fidelity DNA polymerase for PCR amplification were purchased from New England BioLabs.

    Quantitation Assay:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: Virus quantitation was performed by plaque assay on Vero cells. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Staining:

    Article Title: Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion
    Article Snippet: After incubation for 5 days at 37°C, the cells were fixed in 10% formaldehyde in PBS for 30 min, the agarose plugs were removed, and the monolayers were stained with 0.1% crystal violet in 30% methanol. .. Restriction enzymes, DNA-modifying enzymes, the Q5 High Fidelity PCR kit, the ProtoScript II First Strand cDNA synthesis kit, endo H, and PNGase F were obtained from New England BioLabs (Ipswich, MA).

    Variant Assay:

    Article Title: The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2a Decreases Receptor Expression and Prevents Gi-mediated Inhibition of Adenylyl Cyclase Activity *
    Article Snippet: DNA-modifying enzymes, PNGaseF and EndoH, were from New England Biolabs (Frankfurt am Main, Germany). .. Vector plasmid pEGFP-N1 (encoding the enhanced variant of GFP) and the monoclonal anti-GFP antibody were from Clontech.

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    New England Biolabs dna modifying enzymes
    Genome-wide base composition bias curves in Illumina reads from PCR-free human <t>DNA</t> libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or <t>NEBNext</t> Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Dna Modifying Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Article Snippet: Materials All DNA modifying enzymes and NEBNext Ultra II Library Prep Kit were provided by New England Biolabs (NEB) (Ipswich, MA).

    Techniques: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    Lagging-strand DNA synthesis creates a DNA topoisomerase requirement for recombination-dependent DNA synthesis on supercoiled, but not on linear DNA templates. (A) Amount of DNA synthesized when the double-stranded DNA template is supercoiled. (B) Amount of DNA synthesized under exactly the same conditions when the double-stranded DNA template is linear. (C) Alkaline agarose gel electrophoresis of the radioactive products from the reactions in A and B. Note that DNA products much longer than the supercoiled template molecule are formed in the absence of gp61 and topoisomerase (reaction 1), due to rolling-circle DNA synthesis from a small moving bubble ( Formosa and Alberts, 1986 ). But when gp61 is added to complete the primosome, lagging-strand synthesis occurs on the supercoiled template. Now all of the products are short without topoisomerase present (reaction 2). When topoisomerase is added, long DNA products are synthesized on the supercoiled template like those in reaction 1, along with products resembling Okazaki fragments and a conspicuous band at 7.25 kb (reaction 3). In contrast, there is no effect of topoisomerase on the products made on the linear template in the presence of 61 protein (compare reactions 5 and 6). See Figure 5 for diagrams that illustrate these results. As described in Materials and Methods , M13MP19 double-stranded DNA, either supercoiled (form I) or Bgl II-linearized (form III), was preincubated with the DNA polymerase holoenzyme, gp32, gp41, gp59, UvsX, UvsY, and the dda DNA helicase. The gp61 DNA primase (61 protein) and the T4 DNA topoisomerase were also present where indicated. After temperature equilibration at 37°C, recombination-dependent DNA synthesis was started by the addition of the homologous 1623 nucleotide single-stranded DNA primer and the nucleotide substrates. After 7 and 14 min of DNA synthesis, the amount of product was determined from the incorporation of radioactively labeled [α- 32 P]dTTP precursor (panels A and B), and aliquots were analyzed by alkaline gel electrophoresis through 0.6% agarose followed by autoradiography (panel C).

    Journal: Molecular Biology of the Cell

    Article Title: In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

    doi: 10.1091/mbc.E18-06-0386

    Figure Lengend Snippet: Lagging-strand DNA synthesis creates a DNA topoisomerase requirement for recombination-dependent DNA synthesis on supercoiled, but not on linear DNA templates. (A) Amount of DNA synthesized when the double-stranded DNA template is supercoiled. (B) Amount of DNA synthesized under exactly the same conditions when the double-stranded DNA template is linear. (C) Alkaline agarose gel electrophoresis of the radioactive products from the reactions in A and B. Note that DNA products much longer than the supercoiled template molecule are formed in the absence of gp61 and topoisomerase (reaction 1), due to rolling-circle DNA synthesis from a small moving bubble ( Formosa and Alberts, 1986 ). But when gp61 is added to complete the primosome, lagging-strand synthesis occurs on the supercoiled template. Now all of the products are short without topoisomerase present (reaction 2). When topoisomerase is added, long DNA products are synthesized on the supercoiled template like those in reaction 1, along with products resembling Okazaki fragments and a conspicuous band at 7.25 kb (reaction 3). In contrast, there is no effect of topoisomerase on the products made on the linear template in the presence of 61 protein (compare reactions 5 and 6). See Figure 5 for diagrams that illustrate these results. As described in Materials and Methods , M13MP19 double-stranded DNA, either supercoiled (form I) or Bgl II-linearized (form III), was preincubated with the DNA polymerase holoenzyme, gp32, gp41, gp59, UvsX, UvsY, and the dda DNA helicase. The gp61 DNA primase (61 protein) and the T4 DNA topoisomerase were also present where indicated. After temperature equilibration at 37°C, recombination-dependent DNA synthesis was started by the addition of the homologous 1623 nucleotide single-stranded DNA primer and the nucleotide substrates. After 7 and 14 min of DNA synthesis, the amount of product was determined from the incorporation of radioactively labeled [α- 32 P]dTTP precursor (panels A and B), and aliquots were analyzed by alkaline gel electrophoresis through 0.6% agarose followed by autoradiography (panel C).

    Article Snippet: Reagents and enzymes All restriction nucleases and most DNA-modifying enzymes (including T4 DNA ligase) were purchased from New England Biolabs in the early 1990s, when all of the reported experiments were performed.

    Techniques: DNA Synthesis, Synthesized, Agarose Gel Electrophoresis, Labeling, Nucleic Acid Electrophoresis, Autoradiography

    In the presence of UvsX protein, both gp59 and gp32 (32 protein) are needed to assemble a primosome on single-stranded DNA. RNA-primed DNA synthesis was carried out as described in Materials and Methods , either with (purple) or without (green) the addition of gp59. The single-stranded, circular M13 DNA template was incubated for 1 min at 37°C with the following DNA binding proteins: (A) gp32 (32 protein), (B) UvsX protein, and (C) both gp32 and UvsX proteins. DNA synthesis was then initiated by the addition of the T4 DNA polymerase holoenzyme and the T4 primosome (the DNA primase, gp61, plus the DNA helicase, gp41). When present, gp32 was present at 1.2 times the amount needed to cover all the single-stranded DNA (62 µg/ml), based on a binding site of 7 nucleotides per gp32 molecule ( Jensen et al. , 1976 ). The UvsX protein was present at 1.3 times the amount needed to cover all the single-stranded DNA (100 µg/ml), based on a binding site of five nucleotides per UvsX protein molecule. In (C) many DNA circles are covered by alternating patches of UvsX and gp32, each in a linear array that reflects each protein's cooperative DNA binding ( Griffith and Formosa, 1985 ). In (D), we diagram the sequence of polynucleotide syntheses in these reactions—the primosome-catalyzed synthesis of a pentaribonucleotide (RNA primer) that then primes DNA synthesis by T4 DNA polymerase and its accessory proteins.

    Journal: Molecular Biology of the Cell

    Article Title: In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

    doi: 10.1091/mbc.E18-06-0386

    Figure Lengend Snippet: In the presence of UvsX protein, both gp59 and gp32 (32 protein) are needed to assemble a primosome on single-stranded DNA. RNA-primed DNA synthesis was carried out as described in Materials and Methods , either with (purple) or without (green) the addition of gp59. The single-stranded, circular M13 DNA template was incubated for 1 min at 37°C with the following DNA binding proteins: (A) gp32 (32 protein), (B) UvsX protein, and (C) both gp32 and UvsX proteins. DNA synthesis was then initiated by the addition of the T4 DNA polymerase holoenzyme and the T4 primosome (the DNA primase, gp61, plus the DNA helicase, gp41). When present, gp32 was present at 1.2 times the amount needed to cover all the single-stranded DNA (62 µg/ml), based on a binding site of 7 nucleotides per gp32 molecule ( Jensen et al. , 1976 ). The UvsX protein was present at 1.3 times the amount needed to cover all the single-stranded DNA (100 µg/ml), based on a binding site of five nucleotides per UvsX protein molecule. In (C) many DNA circles are covered by alternating patches of UvsX and gp32, each in a linear array that reflects each protein's cooperative DNA binding ( Griffith and Formosa, 1985 ). In (D), we diagram the sequence of polynucleotide syntheses in these reactions—the primosome-catalyzed synthesis of a pentaribonucleotide (RNA primer) that then primes DNA synthesis by T4 DNA polymerase and its accessory proteins.

    Article Snippet: Reagents and enzymes All restriction nucleases and most DNA-modifying enzymes (including T4 DNA ligase) were purchased from New England Biolabs in the early 1990s, when all of the reported experiments were performed.

    Techniques: DNA Synthesis, Incubation, DNA Binding Assay, Binding Assay, Sequencing

    Schematic illustration of three possible types of recombination-dependent DNA synthesis. (Left) Conservative DNA synthesis of the type reported by Formosa and Alberts (1986) , in which only a DNA single strand is produced. The dda protein was the T4 DNA helicase used in that earlier work; here we show that we can replace it with a mixture of gp41 DNA helicase and gp59 to drive the reaction shown on the left. (Right) Top, What happens when gp61 is added to form a complete primosome? If the T4 primosome (gp41 plus gp61) is loaded onto the displaced single-stranded tail, conservative DNA synthesis will produce a new DNA double helix in which both of the DNA strands in the new duplex are newly made. Bottom, If the T4 primosome is instead loaded inside the D-loop, DNA synthesis will occur semiconservatively. In that case, as at the standard replication fork, both of the two daughter DNA helices produced contain one old and one new strand.

    Journal: Molecular Biology of the Cell

    Article Title: In vitro reconstitution of DNA replication initiated by genetic recombination: a T4 bacteriophage model for a type of DNA synthesis important for all cells

    doi: 10.1091/mbc.E18-06-0386

    Figure Lengend Snippet: Schematic illustration of three possible types of recombination-dependent DNA synthesis. (Left) Conservative DNA synthesis of the type reported by Formosa and Alberts (1986) , in which only a DNA single strand is produced. The dda protein was the T4 DNA helicase used in that earlier work; here we show that we can replace it with a mixture of gp41 DNA helicase and gp59 to drive the reaction shown on the left. (Right) Top, What happens when gp61 is added to form a complete primosome? If the T4 primosome (gp41 plus gp61) is loaded onto the displaced single-stranded tail, conservative DNA synthesis will produce a new DNA double helix in which both of the DNA strands in the new duplex are newly made. Bottom, If the T4 primosome is instead loaded inside the D-loop, DNA synthesis will occur semiconservatively. In that case, as at the standard replication fork, both of the two daughter DNA helices produced contain one old and one new strand.

    Article Snippet: Reagents and enzymes All restriction nucleases and most DNA-modifying enzymes (including T4 DNA ligase) were purchased from New England Biolabs in the early 1990s, when all of the reported experiments were performed.

    Techniques: DNA Synthesis, Produced

    PCR analyses of M. avium subsp . paratuberculosis and M. avium subsp . avium isolates with the primer pair p19/p20, specific for pig-RDA10 (a); p21/p22, specific for pig-RDA20 (b); p23/p24, specific for pig-RDA30 (c); ISMav2 I/ISMav2 II, specific for M. avium subsp . paratuberculosis (d); MK5/MK6, specific for IS 900 of M. avium subsp. paratuberculosis (e); and (f) MK7/MK8, specific for IS 901 of M. avium subsp. avium . The template DNA used was derived from the nonpigmented type II driver strain (lane D) and the pigmented type I tester strain (lane T). Other template DNA used was from two nonpigmented bovine (lanes 1 and 2), two pigmented (lanes 3 and 4), three nonpigmented ovine (lanes 5 to 7), three nonpigmented caprine (lanes 8 to 10), and one nonpigmented human (lane 11) M. avium subsp . paratuberculosis isolate as well as four M. avium subsp . avium isolates (ATCC 35712, ATCC 25291, DSM 44158, and MAA S4; lanes 12 to 15).

    Journal: Journal of Clinical Microbiology

    Article Title: Characterization of Genetic Differences between Mycobacterium avium subsp. paratuberculosis Type I and Type II Isolates

    doi: 10.1128/JCM.41.11.5215-5223.2003

    Figure Lengend Snippet: PCR analyses of M. avium subsp . paratuberculosis and M. avium subsp . avium isolates with the primer pair p19/p20, specific for pig-RDA10 (a); p21/p22, specific for pig-RDA20 (b); p23/p24, specific for pig-RDA30 (c); ISMav2 I/ISMav2 II, specific for M. avium subsp . paratuberculosis (d); MK5/MK6, specific for IS 900 of M. avium subsp. paratuberculosis (e); and (f) MK7/MK8, specific for IS 901 of M. avium subsp. avium . The template DNA used was derived from the nonpigmented type II driver strain (lane D) and the pigmented type I tester strain (lane T). Other template DNA used was from two nonpigmented bovine (lanes 1 and 2), two pigmented (lanes 3 and 4), three nonpigmented ovine (lanes 5 to 7), three nonpigmented caprine (lanes 8 to 10), and one nonpigmented human (lane 11) M. avium subsp . paratuberculosis isolate as well as four M. avium subsp . avium isolates (ATCC 35712, ATCC 25291, DSM 44158, and MAA S4; lanes 12 to 15).

    Article Snippet: In addition, BLASTX alignments of DNA sequences were performed by using GenBank/EMBL as well as the M. avium subsp . avium (The Institute for Genome Research [TIGR] strain 104) and M. avium subsp. paratuberculosis ) and the University of Minnesota M. avium subsp. paratuberculosis ).DNA-modifying enzymes were purchased from New England Biolabs (Karlsruhe, Germany).

    Techniques: Polymerase Chain Reaction, Derivative Assay

    PCR analysis of M. avium subsp . paratuberculosis isolates of different host groupings by using the internal primer pair I130A/131A and Tsp 45I restriction of the PCR product RD I130. The DNA template used was derived from three nonpigmented bovine (lanes 1 to 3), three pigmented ovine (lanes 4 to 6), three nonpigmented ovine (lanes 7 to 9), three nonpigmented caprine (lanes 10 to 12), and one nonpigmented human M. avium subsp . paratuberculosis isolate (lane 13) as well as the M. avium subsp. avium ATCC 25291 isolate (lane 14). (a) The PCR with the primers I130A/I131A resulted in products of different sizes for the M. avium subsp . paratuberculosis type I and type II isolates. (b) The PCR product RD I130 of the M. avium subsp . paratuberculosis type I isolates was cut into two fragments by digestion with Tsp 45I. The arrows to the left indicate the size of the corresponding PCR products.

    Journal: Journal of Clinical Microbiology

    Article Title: Characterization of Genetic Differences between Mycobacterium avium subsp. paratuberculosis Type I and Type II Isolates

    doi: 10.1128/JCM.41.11.5215-5223.2003

    Figure Lengend Snippet: PCR analysis of M. avium subsp . paratuberculosis isolates of different host groupings by using the internal primer pair I130A/131A and Tsp 45I restriction of the PCR product RD I130. The DNA template used was derived from three nonpigmented bovine (lanes 1 to 3), three pigmented ovine (lanes 4 to 6), three nonpigmented ovine (lanes 7 to 9), three nonpigmented caprine (lanes 10 to 12), and one nonpigmented human M. avium subsp . paratuberculosis isolate (lane 13) as well as the M. avium subsp. avium ATCC 25291 isolate (lane 14). (a) The PCR with the primers I130A/I131A resulted in products of different sizes for the M. avium subsp . paratuberculosis type I and type II isolates. (b) The PCR product RD I130 of the M. avium subsp . paratuberculosis type I isolates was cut into two fragments by digestion with Tsp 45I. The arrows to the left indicate the size of the corresponding PCR products.

    Article Snippet: In addition, BLASTX alignments of DNA sequences were performed by using GenBank/EMBL as well as the M. avium subsp . avium (The Institute for Genome Research [TIGR] strain 104) and M. avium subsp. paratuberculosis ) and the University of Minnesota M. avium subsp. paratuberculosis ).DNA-modifying enzymes were purchased from New England Biolabs (Karlsruhe, Germany).

    Techniques: Polymerase Chain Reaction, Derivative Assay