PCR-RFLP和PCR-SSCP方法对弱精子症精子mtATPase6基因突变的检测  

Detection of mtATPase6 Point Mutation on Asthenospermia by PCR-RFLP and PCR-SSCP

作　　者：金龙金[1] 张春玲[1] 楼哲丰[2] 张李雅[3] 陈林丽[1] 李玮[1] 吕建新[1] 

机构地区：[1]温州医学院生命科学学院,浙江省医学遗传学重点实验室 [2]温州医学院生命科学学院,生物学实验中心 [3]温州医学院附属第一医院生殖医学中心,温州325035

出　　处：《细胞生物学杂志》2007年第4期573-578,共6页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金(No.Y206582);温州市科技发展计划项目(No.Y20060063)资助~~

摘　　要：为了检测弱精子症精子mtATPase6基因突变,采用PCR方法扩增了17例弱精子症mtDNA8602～9416区域815bp目的片段,用MspI和HaeⅢ限制性内切酶酶切,分别用琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和单链构像多态性(SSCP)筛查突变。筛选出3例突变标本,经测序确认突变位点和突变性质。结果显示17例标本全部扩增出mtDNA8602～9416的815bp目的片段,PCR产物经MspI限制性内切酶酶切后电泳结果与剑桥序列预期酶切图谱相一致。PCR产物经HaeⅢ酶切,聚丙烯酰胺凝胶电泳图谱上出现泳动带的异常,在17例弱精子症中共筛选出2例酶切片段异常标本。PCR-HaeⅢ酶切产物的SSCP电泳图谱分析结果显示,在PCR-RFLP(HaeⅢ)电泳图谱正常的15例弱精子症标本中筛选出1例异常。两种方法在弱精子症精子标本中筛选出mtATPase6基因突变3例(3/17,17.7%)。3例测序结果发现A8701G、C8943T、C8964T、T8966C、G9053A、C9060A、C9075T、A9120G、C9296T共9个点突变,其中A8701G、T8966C、G9053A三个突变为错义突变,其余均为同义突变。上述结果提示,弱精子症精子mtATPase6基因存在较高突变率;PCR-RFLP和PCR-SSCP能简单、快速、灵敏地筛选mtATPase6基因突变。The aim of the research is to detect the relativity between mtATPase6 point mutation in sperms and asthenospermia. The 815 bp fragment of mtDNA 8 602-9 416 was amplified by PCR for 17 asthenospermia cases. Detection of mutation was performed using PCR-RFLP （restriction fragment length polymorphism） with restriction enzymes MspI or HaeⅢ and PCR-SSCP （single strand conformation polymorphism） analysis. Three mutant cases were detected and confirmed by DNA sequencing with their mutant sites. The result of PCR-RFLP with MspI is consistent with the forecasted restriction map in all 17 cases. But 3 cases with mutations were detected by PCR-RFLP with HaeⅢ and PCR-SSCP. There are several types of mtATPase6 point mutations： A8701G, C8943T, C8964T, T8966C, G9053A, C9060A, C9075T, A9120G and C9296T. Three of them （A8701G, T8966C, G9053A） were missense mutation. There is higher mutation rate of mtDNAase6 in asthenospermia sperms. PCR-RFLP and PCR-SSCP are two simple and quick methods for detecting gene mutation.

关 键 词：弱精子症 mtATPase6 点突变 PCR-RFLP PCR-SSCP 

分 类 号：R698.2[医药卫生—泌尿科学]