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

被引:23
作者
Bae, Jung-Hoon [1 ]
Sohn, Jung-Hoon [1 ]
机构
[1] KRIBB, Ind Biotechnol & Bioenergy Res Ctr, Taejon 305333, South Korea
基金
新加坡国家研究基金会;
关键词
Template-blocking PCR; Cassette ligation-mediated PCR; Genome walking; POLYMERASE-CHAIN-REACTION; MEDIATED PCR; DNA; CLONING; GENES; TRANSFORMATION; AMPLIFICATION; CASSETTE;
D O I
10.1016/j.ab.2009.11.003
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2009 Elsevier Inc. All rights reserved.
引用
收藏
页码:112 / 116
页数:5
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