锅柄聚合酶链反应法扩增贵阳腐霉Pr1基因上游未知序列  

Panhandle PCR Strategy to Amplify the Upstream Unknown Sequence of Pr1 Gene of Pythium guiyangense

作　　者：段斯亮[1] 于声[2] 苏晓庆[3] 

机构地区：[1]广西科技大学医学院病原生物学与免疫学教研室,柳州市545006 [2]广西科技大学医学院检验教研室,柳州市545006 [3]贵阳医学院生物学教研室,贵阳市550004

出　　处：《广西医学》2013年第11期1461-1463,共3页Guangxi Medical Journal

基　　金：广西教育厅科研项目(200103YB154)

摘　　要：目的在灭蚊真菌贵阳腐霉Pr1基因已知序列的基础上,利用锅柄聚合酶链反应法扩增Pr1基因上游未知序列。方法首先采用限制性内切酶BamHⅠ对基因组DNA进行完全酶切,用小牛肠碱性磷酸酶去磷酸化后,与5'磷酸化的寡核苷酸链连接,经过变性、链内退火和聚合酶延伸形成锅柄状,最后进行嵌套式PCR。结果获得了Pr1基因已知序列上游864bp核苷酸序列,GenBank登录号为:JQ975036。结论锅柄聚合酶链反应法可以高度有效地扩增与已知位点相邻的基因组片段,是一种可靠的染色体步移法,有利于Pr1基因全长的克隆。Objective Based on a partial Pr 1 genomic sequence of Pythium guiyangense which has been cloned before ,panhandle polymerase chain reaction （ Panhandle PCR ） strategy was used to amplify the upstream flan-king sequence adjacent to the known sequence of the Pr 1 gene .Methods The genomic DNA was firstly digested with BamHⅠand 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 exten-sion,a pan with a handle was formed ,and lastly the nested PCR was performed .Results A 864bp product was am-plified,which was adjacent to the known sequence of Pr 1 gene .The gene has been accessed by GenBank （ Accession：JQ975036 ） .Conclusion Panhandle PCR is a quick and convenient approach for amplifying and identifying un-known partner genes ,which facilitates cloning full-length Pr1 gene .

关 键 词：锅柄聚合酶链反应 上游调控序列 类枯草菌素蛋白酶 染色体步移 贵阳腐霉 

分 类 号：R349.6[医药卫生—基础医学]