关键词: HRM, 巢式 PCR, ob / ob 鼠, db / db 鼠, 基因分型, 点突变模式小鼠

Abstract: Objective Applying nested PCR with high resolution melting ( HRM) technology to improve the genotyping detection method of point mutation model mice. Method Taking ob / ob mice and db / db mice as samples, the mouse toes were cut for numbering and used for extraction of genomic DNAs. Nested PCR primers were designed according to the reference sequence of the mutation site. After two rounds of PCR amplification, the products were directly detected under HRM analysis to obtain genotyping result. The accuracy of the genotyping result was verified by PCR-SBT and PCR-RFLP. Result 80 ob / ob mice and 65 db / db mice were tested, and the nested PCR with HRM method could accurately distinguish the three genotypes. Among ob / ob mice, 21, 47 and 12 were identified as homozygotes, heterozygotes and wild-type mice, respectively. In db / db mice, the numbers of homozygotes, heterozygotes and wild-type were 23, 33 and 9, respectively. The genotyping result of the nested PCR with HRM method were highly consistent with the result of PCR-SBT and PCR-RFLP. Besides, the genotyping result were consistent with the phenotypes of mice. Conclusion The established nested PCR with HRM method is a fast, simple to perform and highly accurate genotyping method, which is suitable for the genotyping of a large number of point mutation model mice.

Key words: HRM, nested PCR, ob / ob mice, db / db mice, genotyping, point mutation model mice