A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.

ABSTRACT:

Background

The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.

Results

Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.

Conclusions

The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

SUBMITTER: Norholm MH 

PROVIDER: S-EPMC2847956 | biostudies-literature |

REPOSITORIES: biostudies-literature