Template-blocking PCR: an advanced PCR technique for genome walking

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Title
Template-blocking PCR: an advanced PCR technique for genome walking
Author(s)
Jung Hoon BaeJung Hoon Sohn
Bibliographic Citation
Analytical Biochemistry, vol. 398, no. 1, pp. 112-116
Publication Year
2010
Abstract
This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.
Keyword
Cassette ligation-mediated PCRGenome walkingTemplate-blocking PCR
ISSN
0003-2697
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/j.ab.2009.11.003
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
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