q5  (New England Biolabs)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: DNA synthesis from diphosphate substrates by DNA polymerases

doi: 10.1073/pnas.1712193115

Figure Lengend Snippet: Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

Article Snippet: Taq DNA polymerase, Phusion High-Fidelity DNA polymerase, DeepVent DNA polymerase, Vent DNA polymerase, Q5 DNA High-Fidelity polymerase, Bst polymerase, Bsu DNA polymerase (large fragment), and ThermoPol Reaction Buffer [1×: 20 mM Tris⋅HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton–X–100, pH 8.8 at 25 °C] were purchased from New England Biolabs.

Techniques: Polymerase Chain Reaction, Amplification

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: Optimization of EvaGreen- and Q5 DNA polymerase-based dNTP quantification. ( A-F ) Each critical reaction component and reaction temperature were varied and the outcome evaluated. ( G ) Assay conditions in graphs A to F . The standard curves were generated from end-point (1h) baseline-corrected fluorescence values.

Article Snippet: Based on the lowest background signal, and reported ( ) inhibitor resistance in DNA extraction-free PCR, we chose to continue the optimization with Q5 DNA polymerase.

Techniques: Generated, Fluorescence

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: Based on the lowest background signal, and reported ( ) inhibitor resistance in DNA extraction-free PCR, we chose to continue the optimization with Q5 DNA polymerase.

Techniques: Hydrolysis Assay, Generated, Fluorescence

Journal: Bio-protocol

Article Title: CRISPR/Cas9 Editing of the Bacillus subtilis Genome

doi: 10.21769/BioProtoc.2272

Figure Lengend Snippet: PCR based verification of genome edit A. A schematic representation showing the locations of PCR primers used in panel B; B. Agarose gel stained with ethidium bromide of amplified PCR products using primers specific to the point mutation (H743A, for which we changed CAC→GCT) and the B. subtilis chromosome (left panel), and using primers specific to the editing plasmid (right panel). Of twelve isolates screened all were found to have the predicted point mutation, and two of twelve retained resistance to spectinomycin (asterisks). The slow migrating band in the PY79 lane corresponds to nonspecific PCR amplification in the wild type background. The editing plasmid was detectable in spectinomycin resistant isolates, but not in the remaining ten. C. Agarose gel stained with ethidium bromide of PCR reactions using primers specific for deletion of four B. subtilis genes. Primers were designed outside of the editing template, and are therefore specific to chromosomal DNA. For all four deletions, all isolates were found to be spectinomycin sensitive, and PCR genotyping of three isolates for each deletion found that all isolates tested had the intended deletion. Note that for the control reaction for ponA , the PCR product is faint because the amplicon is approximately 5 kb and the extension time of the PCR was 3 min, which is near the limit for complete extension for Q5 DNA polymerase; the other three control amplicons are no larger than 3.4 kb.

Article Snippet: The PCR program used to linearize pPB41 is: PCR amplify CRISPR/Cas9 using plasmid generated in step B1 ( ) Amplify plasmid generated in step B1 (pPB43 in the example) via PCR using Q5 DNA polymerase (NEB) and primers oPEB232 and oPEB234.

Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Mutagenesis, Plasmid Preparation

Journal: Molecular Oncology

Article Title: Correlation between circulating cell‐free PIK3CA tumor DNA levels and treatment response in patients with PIK3CA‐mutated metastatic breast cancer

doi: 10.1002/1878-0261.12305

Figure Lengend Snippet: Assay specificity for PIK 3 CA mutations. Comparison of mutant PIK 3 CA detection in human control genomic DNA ( gDNA ) and cell‐free plasma DNA following pre‐amplification or no pre‐amplification using Q5 High‐Fidelity DNA Polymerase (New England BioLabs) or TaqMan ® PreAmp Master Mix (Life Technologies). The level of blank (LoB) was 0.04% for the mutation E545K and for H1047R, 0.02% for E542K, and less than 0.01% for H1047L as shown by the graphs.

Article Snippet: To increase the sensitivity of the analysis, the remaining 75 μL serum DNA was subjected to 12 cycles of PCR pre‐amplification with Q5 High‐Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) using a multiplex PIK3CA primer mix.

Techniques: Mutagenesis, Amplification