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Detection of mtATPase6 Point Mutation on Asthenospermia by PCR-RFLP and PCR-SSCP
Long-Jin Jin*, Chun-Ling Zhang, Zhe-Feng Lou1, Li-Ya Zhang2, Lin-Li Chen, Wei Li, Jian-Xin Lu
College of Life Sciences, Key Research Laboratory for Medical Genetics of Zhejiang; 1Central Laboratory of Biology; 2Reproductive Medicine Center, First Affiliated Hospital; Wenzhou Medical College, Wenzhou 325035, China
Abstract: 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? 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.
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