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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 | 2010 Mar

REPOSITORIES: biostudies-literature

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

Nørholm Morten H H MH

BMC biotechnology 20100316

<h4>Background</h4>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.<h4>Results</h4>Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible sit ...[more]

PMID: 20233396

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