FAQ: Can I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods?

Yes. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase which efficiently amplifies uracil-containing templates and primers. For USER cloning methods, target DNA molecules and cloning vector 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. In comparison to Taq-based enzymes, Q5U Hot Start High-Fidelity DNA Polymerase makes significantly fewer errors, streamlining any cloning protocol (for example, see data below) 

USER 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.)