dntp  (New England Biolabs)


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    Structured Review

    New England Biolabs dntp
    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and <t>antisense</t> MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and <t>dNTP.</t> The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
    Dntp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm017

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

    Techniques Used: Synthesized, Molecular Weight, Clone Assay

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Evolution of population genetic structure of the British roe deer by natural and anthropogenic processes (Capreolus capreolus)
    Article Snippet: .. PCR reactions (20 μL) contained 0.2 pmol/L/μL each primer, 0.2 mmol/L each dNTP, 10 mmol/L Tris-HCL, pH 9.0, 1.5 mmol/L MgCl2 , and 0.4 units of Taq polymerase (New England Biolabs, Hitchin, UK) with cycle conditions: 95°C for 5 min; 35 cycles at 94°C for 45 sec, 51°C for 45 sec; and 72°C for 45 sec; 72°C for 5 min. PCR products were purified using Qiagen columns (Qiagen, Inc) and directly sequenced using an ABI 377 automated sequencer. ..

    Article Title: Spinal muscular atrophy within Amish and Mennonite populations: Ancestral haplotypes and natural history
    Article Snippet: .. A 25 μL reaction was prepared with 1U of Taq polymerase (New England Biolabs [NEB], Ipswich, MA), 200mM each of dNTP, 2.5μL of 10X PCR buffer (NEB), 1.6 μmol/L of primers, and 100ng of genomic DNA. .. An ABI Veriti thermocycler was used to amplify products under the following conditions: initial denaturation (3 minutes at 96°C), thermal cycling (30 sec at 96°C, 15 sec at 60°C, 90 sec at 72°C), and terminal incubation (10 minutes at 72°C).

    Article Title: Genetic characterization of fall armyworm infesting South Africa and India indicate recent introduction from a common source population
    Article Snippet: .. PCR amplification for all segments was performed in a 30-μl reaction mix containing 3 μl 10X manufacturer’s reaction buffer, 1 μl 10mM dNTP, 0.5 μl 20-μM primer mix, 1 μl DNA template (between 0.05–0.5 μg), 0.5 unit Taq DNA polymerase (New England Biolabs, Beverly, MA). .. Typically 96 PCR amplifications were performed at the same time using either 0.2-ml tube strips or 96 well microtiter plates.

    Article Title: Microbial Co-Occurrence Patterns and Keystone Species in the Gut Microbial Community of Mice in Response to Stress and Chondroitin Sulfate Disaccharide
    Article Snippet: .. The PCR reaction included 2.5 units of AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) in a 50 μL reaction buffer containing 200 nM primers, 200 nM dNTP, 60 mM Tris-SO4 , 18 mM (NH4)2 SO4 , 2.0 mM MgSO4 , 1% glycerol, and 100 ng/uL bovine serum albumin (New England BioLabs, Ipswich, MA, USA). .. PCR was performed using the following cycling profile: initial denaturing at 95 °C for 2 min followed by 20 cycles of 95 °C 30 s, 60 °C 30 s, and 72 °C 60 s. Amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter Genomics, Danvers, MA, USA), quantified using a BioAnalyzer high-sensitivity DNA chip, and pooled at equal molar ratios.

    Amplification:

    Article Title: Lack of specificity associated with using molecular beacons in loop mediated amplification assays
    Article Snippet: .. The reaction chemistry for LAMP and amplification detection was 1X isothermal buffer (NEB, Massachusetts, United States), 300 micromolar each dNTP, 0.8 micromolar Betaine, 0.5 micromolar SYTO9 Green, 0.32 units per microlitre Bst polymerase v2.0 warm start (NEB), 0.8 micromolar each LAMP primer, 0.4 micromolar each Loop primer, 0.2 micromolar each displacement primer and molecular grade water for a reaction volume of 20 microlitre. ..

    Article Title: Genetic characterization of fall armyworm infesting South Africa and India indicate recent introduction from a common source population
    Article Snippet: .. PCR amplification for all segments was performed in a 30-μl reaction mix containing 3 μl 10X manufacturer’s reaction buffer, 1 μl 10mM dNTP, 0.5 μl 20-μM primer mix, 1 μl DNA template (between 0.05–0.5 μg), 0.5 unit Taq DNA polymerase (New England Biolabs, Beverly, MA). .. Typically 96 PCR amplifications were performed at the same time using either 0.2-ml tube strips or 96 well microtiter plates.

    Article Title: The Genome of Chelonid Herpesvirus 5 Harbors Atypical Genes
    Article Snippet: .. DNA was amplified with the primers listed in in the presence of 10 nm dNTP, DMSO, 5x Phusion GC buffer and 2 U of Phusion polymerase (New England Biolabs, Ipswich, MA). .. Synthetic oligonucleotides were obtained from Microsynth (Balgach, Switzerland).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing
    Article Snippet: .. Pre-amplification was carried out in 25-μL reactions that contained 50 ng of cell line DNA or a variable quantity (50–250 ng) of DNA from FFPE samples, 1.5 mM MgCl2 , 200 mM dNTP, 500 nM primers and 1 unit Phusion Hot Start Flex DNA polymerase (New England Biolabs, Ipswich, USA). .. An amplification programme was started by an initial activation of the enzyme at 98 °C for 30 s. The initial amplification cycle was denaturation at 98 °C for 10 s, annealing at 72 °C for 30 s and elongation at 72 °C for 30 s. Amplification was continued for ten cycles, reducing the annealing temperature by 1 °C each cycle, followed by 40 cycles of 10 s denaturation at 98 °C, 30 s annealing at 62 °C and 30 s elongation at 72 °C.

    Size-exclusion Chromatography:

    Article Title: Evolution of population genetic structure of the British roe deer by natural and anthropogenic processes (Capreolus capreolus)
    Article Snippet: .. PCR reactions (20 μL) contained 0.2 pmol/L/μL each primer, 0.2 mmol/L each dNTP, 10 mmol/L Tris-HCL, pH 9.0, 1.5 mmol/L MgCl2 , and 0.4 units of Taq polymerase (New England Biolabs, Hitchin, UK) with cycle conditions: 95°C for 5 min; 35 cycles at 94°C for 45 sec, 51°C for 45 sec; and 72°C for 45 sec; 72°C for 5 min. PCR products were purified using Qiagen columns (Qiagen, Inc) and directly sequenced using an ABI 377 automated sequencer. ..

    Purification:

    Article Title: Evolution of population genetic structure of the British roe deer by natural and anthropogenic processes (Capreolus capreolus)
    Article Snippet: .. PCR reactions (20 μL) contained 0.2 pmol/L/μL each primer, 0.2 mmol/L each dNTP, 10 mmol/L Tris-HCL, pH 9.0, 1.5 mmol/L MgCl2 , and 0.4 units of Taq polymerase (New England Biolabs, Hitchin, UK) with cycle conditions: 95°C for 5 min; 35 cycles at 94°C for 45 sec, 51°C for 45 sec; and 72°C for 45 sec; 72°C for 5 min. PCR products were purified using Qiagen columns (Qiagen, Inc) and directly sequenced using an ABI 377 automated sequencer. ..

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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

    Overview of Nick-seq and data analysis workflow. ( A ) Nick-seq library preparation. Briefly, genomic DNA is first subjected to sequencing-compatible fragmentation; the resulting 3′-OH ends are blocked with dideoxyNTPs; the DNA modification is converted to a strand-break by enzymatic or chemical treatment; capture of the 3′- and 5′-ends of resulting strand-breaks using two complementary strategies: one portion of DNA is subjected to nick translation (NT) with α-thio-dNTPs to generate phosphorothioate (PT)-containing oligonucleotides that are resistant to subsequent hydrolysis of the bulk of the genomic DNA by exonuclease III and RecJ f . The purified PT-protected fragments are used to generate an NGS library with the modification of interest positioned at the 5′-end of the PT-labeled fragment. A second portion of the same DNA sample is used for terminal transferase (TdT)-dependent poly(dT) tailing of the 3′-end of the strand-break, with the tail used to create a sequencing library by reverse transcriptase template switching ( 9 ). Subsequent NGS positions the modification of interest 5′-end of the poly(dT) tail. ( B ) Processing of the Nick-seq data includes: raw NGS reads are aligned to the reference genome for read coverage calculation; the genome sites with reads coverage ≥5 are then filtered for nick site calling with three parameters: x = the read coverage at position N/coverage at N – 1; y = coverage at position N/coverage at N + 1; z = coverage at position N /coverage at N of negative control sample. The site N is defined as a nick site if its x > 1, y > 1, z > 1 for NT reads and x > 2, y > 2, z > 2 for TdT reads.

    Journal: Nucleic Acids Research

    Article Title: Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

    doi: 10.1093/nar/gkaa473

    Figure Lengend Snippet: Overview of Nick-seq and data analysis workflow. ( A ) Nick-seq library preparation. Briefly, genomic DNA is first subjected to sequencing-compatible fragmentation; the resulting 3′-OH ends are blocked with dideoxyNTPs; the DNA modification is converted to a strand-break by enzymatic or chemical treatment; capture of the 3′- and 5′-ends of resulting strand-breaks using two complementary strategies: one portion of DNA is subjected to nick translation (NT) with α-thio-dNTPs to generate phosphorothioate (PT)-containing oligonucleotides that are resistant to subsequent hydrolysis of the bulk of the genomic DNA by exonuclease III and RecJ f . The purified PT-protected fragments are used to generate an NGS library with the modification of interest positioned at the 5′-end of the PT-labeled fragment. A second portion of the same DNA sample is used for terminal transferase (TdT)-dependent poly(dT) tailing of the 3′-end of the strand-break, with the tail used to create a sequencing library by reverse transcriptase template switching ( 9 ). Subsequent NGS positions the modification of interest 5′-end of the poly(dT) tail. ( B ) Processing of the Nick-seq data includes: raw NGS reads are aligned to the reference genome for read coverage calculation; the genome sites with reads coverage ≥5 are then filtered for nick site calling with three parameters: x = the read coverage at position N/coverage at N – 1; y = coverage at position N/coverage at N + 1; z = coverage at position N /coverage at N of negative control sample. The site N is defined as a nick site if its x > 1, y > 1, z > 1 for NT reads and x > 2, y > 2, z > 2 for TdT reads.

    Article Snippet: Materials Nicking endonucleases Nb.BsmI, Nb.BspQI, Nb.BsrDI, endonuclease IV, DNA polymerase I, OneTaq DNA polymerase, dNTPs, Nci I, exonuclease III and RecJf were purchased from New England Biolabs.

    Techniques: Sequencing, Modification, Nick Translation, Purification, Next-Generation Sequencing, Labeling, Negative Control