摘要
Objective： Based on a partialsubtilisin-like protease, Prl genomic sequence ofPythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Prl gene. Methods： The genomic DNA was firstly digested with BamH I and then treated with calf intestinal alkaline phosphatase（CIAP）. Next, a 5＇ phosphorylated oligonucleotide was ligated to the 5＇ ends of BamH I -digested DNA. After denaturation, intmstrand annealing and polymemse extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results： A 864 bp product was amplified,which was adjacent to the known sequence of Prl gene.The gene has been accessed by GenBank （Accession：JQ975036）. Conclusion： Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Prl gene
Objective: Based on a partialsubtilisin-like protease, Pr1 genomic sequence of Pythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Pr1gene. Methods: The genomic DNA was firstly digested with BamHⅠ and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5′ phosphorylated oligonucleotide was ligated to the 5′ ends of BamHⅠ-digested DNA. After denaturation, intrastrand annealing and polymerase extension, a pan with a handle was formed,and lastly the nested PCR was performed. Results: A 864 bp product was amplified,which was adjacent to the known sequence of Pr1 gene.The gene has been accessed by GenBank(Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes,which facilitates cloning full-length Pr1 gene.
基金
Supported by the Guangxi Department of education scientific research funds,China(No.200103YB154)
