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BMC Biotechnol. 2010 Mar 16;10:21. doi: 10.1186/1472-6750-10-21.

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

Author information

1
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden. norholm@gmail.com

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.

PMID:
20233396
PMCID:
PMC2847956
DOI:
10.1186/1472-6750-10-21
[Indexed for MEDLINE]
Free PMC Article

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