一种改进的SON-PCR基因扩增方法  

An Improved SON-PCR Method for Gene Amplification

作　　者：邓洪渊[1] 王闵霞[1] 孙雪文[1] 马欣荣[1] 曹婷婷[1] 谭红[1] 

机构地区：[1]中国科学院成都生物研究所

出　　处：《应用与环境生物学报》2006年第6期857-860,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家"863"高技术资助项目;中国科学院重要方向项目资助课题(KSCX2-YW-G-016)

摘　　要：单侧寡聚核苷酸嵌套PCR(single oligonucleotide nested PCR,SON-PCR)是一种简便易行的由已知序列克隆其侧翼序列的方法,但该法的第二轮PCR反应引发效率低,而且得到的产物两端含相同的引物序列,不可直接测序.针对该问题,将第二轮PCR改进为两段式扩增,即先以单侧嵌套引物引发5′端做选择性线性扩增,然后再加入第一轮引物特异引发3′端,使特异性和效率都得到很大提高,并可用PCR产物直接测序.分别用已报道的和改进的SON-PCR法克隆Botrytis cinerea羟甲基戊二酰CoA还原酶基因侧翼序列,后者获得期望结果,证明改进的SON-PCR方法行之有效.国内外尚未见同类报道.Single oligonucleotide nested PCR （SON-PCR） is a very convenient and widely applicable method to identify unknown DNA sequence adjacent to known region. But the efficiency of its secondary reaction is low, and the products can not be sequenced directly for the presence of the same primer sequences at both ends. To improve this method, the secondary reaction was divided into two round amplifications as follows ： firstly, single nested primer was used to drive the 5＇ selected linear amplification; then, the primer designed for primary reaction was added to specially bind 3＇ end of the single -stranded DNA and lead to exponential amplification. With the improved SON-PCR, the 5＇ and 3＇ unknown sequences of gene coding Botrytis cinerea 3-hydroxy-3-methylglutaryl coenzyme A reductase （HMGR） were successfully isolated based on available partial sequence, but no positive result was obtained with the reported SON-PCR method. No similar research has been reported. Fig 4, Ref 7

关 键 词：SON—PCR改进的SON-PCR 基因扩增 

分 类 号：Q78[生物学—分子生物学]