用锅柄聚合酶链反应法克隆卡地腐霉Pr1基因的未知片段

目的：分离和克隆灭蚊真菌卡地腐霉类枯草杆菌蛋白酶基因已知片段的3′端未知序列。方法：采用PANHANDLE PCR，以基因组DNA为模板，通过链内退火及延伸形成一个类似锅柄形状的结构，经嵌套式PCR扩增得到未知目的片段。通过已知cDNA片段设计引物，PCR扩增得到该蛋白酶部分基因组序列，采用限制性内切酶BamH Ⅰ消化卡地腐霉基因组DNA，经去磷酸化处理后，在消化片段3′端连接一段与卡地腐霉类枯草杆菌蛋白酶基因5′端已知序列互补的5′端磷酸化寡核苷酸链NA，经链内退火及聚合酶延伸形成一锅柄状结构。结果：获得一大小为876bp的PCR产物，经序列分析验证，该panhandle PCR产物包含了已知卡地腐霉类枯草杆菌蛋白酶基因3′端未知的853bp核苷酸序列。结论：这种特殊的PCR方法可以高度有效地克隆和鉴定已知片段两侧的未知基因片段。

Cloning of Unknown Partner Sequence of the Subtilisin-like Protease Gene of Pythium carolinianum with Panhandle PCR Technique

Objective: To explore the DNA sequence of the subtilisin-like protease (Pr1) gene of Pythium carolinianum, a mosquito-killing fungus. Methods: A new approach called panhandle polymerase chain reaction (PPCR) was used to clone a 3′unknown sequence adjacent to a known sequence of Pr1 gene of P. carolinianum. The template was a segment of DNA with an intra-strand loop schematically shaped like a pan with a handle. Based on a partial Pr1 cDNA sequence of the fungus cloned previously, a 530 bp genomic sequence was cloned by designing a pair of specific primers. The genomic DNA was firstly digested to create a 5′overhang end with restriction enzyme BamH Ⅰ and then treated with calf intestinal alkaline phosphatase. Next, a 32-nucleotide single-stranded 5′phosphorylated oligonucleotide was ligated to the 3′ends of BamHⅠ-digested DNA. After denaturation, intra-strand annealing and polymerase extension, a panhandle was formed and then nested PCR was performed. Results: A 876 bp panhandle PCR product was subsequently subcloned and the sequence analysis indicated that it contains the 3′unknown partner sequence adjacent to the known sequence of Pr1 gene. Conclusion: Panhandle PCR is a quick and convenient new technique for cloning and identifying unknown partner genes.