Many proofreading polymerases derived from archaeal Family B DNA polymerases stall replication in response to uracil bases in DNA templates (Wardle, J. et al. (2008) Nucleic Acids Res. 36 (3), 705-711). Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases. This feature is useful for amplifying bisulfite-converted, deaminated, or damaged DNA, preventing carryover contamination in PCR (when used with dUTP and UDG), and in USER cloning methods.Q5U is a hot start polymerase containing a unique aptamer selected for polymerase inhibition at room temperature and optimal amplification during typical PCR conditions.Figure 1: Common applications enabled by Q5U Hot Start High-Fidelity DNA PolymeraseArchaeal family B-type polymerases can incorporate/tolerate a variety of modified nucleotides but will stall upon encountering uracil and inosine residues. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase which efficiently incorporates dUTP and amplifies uracil-containing templates. Common applications enabled by Q5U Hot Start High-Fidelity DNA Polymerase are illustrated above.Figure 2: Q5U Hot Start High-Fidelity DNA Polymerase provides superior amplification of bisulfite-converted DNAAmplification of bisulfite-converted human genomic DNA targets using Q5U Hot Start High-Fidelity DNA Polymerase (Q5U), Phusion U Hot Start DNA Polymerase (P) and KAPA Hifi HotStart Uracil+ ReadyMix PCR Kit (K). Amplicon name and sizes are indicated above the gel. Each 25 μl reaction contained 12 ng of bisulfite-converted DNA (Qiagen). All reactions were conducted according to manufacturer’s recommendations using 35 cycles and visualized by microfluidic LabChip® analysis.Figure 3: Q5U Hot Start High-Fidelity DNA Polymerase enables robust amplification of FFPE normal lung DNAAmplification of FFPE normal lung DNA (Biochain). Amplicon sizes are indicated above the gel. Each 25 μl reaction contained 18 ng of FFPE DNA (Biochain). Cycling conditions were consistent with recommendations for this sample type and visualized by microfluidic LabChip analysis.Figure 4: Q5U Hot Start High-Fidelity DNA Polymerase enables robust amplification of enzymatically deaminated DNAAmplification of enzymatically deaminated (APOBEC) genomic DNA targets using Q5U Hot Start High-Fidelity DNA Polymerase (Q5U), Phusion U Hot Start DNA Polymerase (P) and KAPA Hifi HotStart Uracil+ ReadyMix PCR Kit (K). Amplicon name and sizes are indicated above the gel. Each 50 μl reaction contained 12 ng of enzymatically deaminated DNA. All reactions were conducted according to manufacturer’s recommendations using 35 cycles and visualized by microfluidic LabChip analysis.Figure 5: USER® cloning workflowIn USER cloning, target DNA molecules and cloning vectors are generated by PCR with 8–12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and/or deletions. We recommend using the GeneDesign software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 μM.Figure 6: Q5U Hot Start High-Fidelity DNA Polymerase enables highly efficient and accurate USER cloning as compared to Hot Start Taq DNA PolymeraseUSER cloning was performed to ligate a 3 kb lacZ gene into a pET21a vector backbone with Q5U Hot Start High-Fidelity DNA Polymerase and Hot Start Taq DNA polymerase. Reactions containing Q5U resulted in a higher assembly efficiency and better accuracy as evidenced by the total number of colonies obtained (A) and percentage of blue colonies (B). Accuracy was further examined using Sanger sequencing which revealed a number of point mutations in the blue colonies obtained from the Taq assembly and no mutations from the 4 clones sequenced from the Q5U experiments (C). The “X”s indicate the number of additional mutations found in the clones produced with Taq, consistent with previous results demonstrating the high mutation tolerance of lacZ. (Barnes, W. M. (1992) Gene, 112, 29-35.)
This product is related to the following categories:
Q5® High-Fidelity DNA Polymerases Products,
PCR, qPCR & Amplification Technologies Products
This product can be used in the following applications:
Applications of USER? and Thermolabile USER II Enzymes,
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