Panhandle PCR strategy to amplify the upstream unknown sequence of the Pr1 gene of pythium guiyangense

Abstract

ObjectiveBased 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 Pr1 gene. MethodsThe genomic DNA was firstly digested with Bam H I and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5′ phosphorylated oligonucleotide was ligated to the 5′ ends of Bam H I -digested DNA. After denaturation, intrastrand annealing and polymerase extension, a pan with a handle was formed, and lastly the nested PCR was performed. ResultsA 864 bp product was amplified, which was adjacent to the known sequence of Pr1 gene. The gene has been accessed by GenBank (Accession:JQ975036). ConclusionPanhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes, which facilitates cloning full-length Pr1 gene.