摘要
目的利用巢式PCR结合高分辨率熔解曲线(HRM)技术,改进点突变模式小鼠的基因分型方法。方法以ob/ob鼠和db/db鼠为样本,剪小鼠脚趾编号并提取基因组DNA。根据突变位点参考序列设计巢式PCR引物并进行两轮扩增后,对产物进行HRM分析,获得基因分型结果。同时,通过PCR-SBT法和PCR-RFLP法对分型结果的准确性进行验证。结果对80只ob/ob鼠和65只db/db鼠进行了检测,巢式PCR-HRM法能够准确地分辨出3种基因型。在ob/ob鼠中,鉴定得到的纯合子、杂合子和野生型分别为21、47和12只。而在db/db鼠中,纯合子、杂合子和野生型分别为23、33和9只。巢式PCR-HRM法的分型结果与PCR-SBT法和PCR-RFLP法的结果高度一致,且分型结果与小鼠表型一致。结论建立的巢式PCR-HRM法是一种快速、简便、准确度高的基因分型方案,适用于大批量点突变模式小鼠的基因分型鉴定。
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.
作者
刁戈
田敏
李润博
郭建新
韩健
DIAO Ge;TIAN Min;LI Runbo;GUO Jianxin;HAN Jian(Department of Obstetrics and Gynecology,Daping Hospital,Army Medical University/Third Military Medical University,Chongqing 400042,China)
出处
《实验动物科学》
2023年第3期24-30,共7页
Laboratory Animal Science
基金
重庆市自然科学基金(cstc2019jcyj-msxmX0445)
军队计生专项(21JSZ03)。
