Template-blocking PCR: an advanced PCR technique for genome walking.
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.
Bae
Jung-Hoon
JH
Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon 305-333, Republic of Korea.
Sohn
Jung-Hoon
JH
D002480:Cellulase / Q000378:metabolism; D016386:Chromosome Walking / Q000379:methods*; D004247:DNA / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015224:Dideoxynucleosides / Q000378:metabolism; D005784:Gene Amplification; D023281:Genomics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic
D015224:Dideoxynucleosides; D004247:DNA; D010735:Phosphoglycerate Kinase; D004262:DNA Restriction Enzymes; D002480:Cellulase