摘要
目的建立利用高分辨率熔解曲线(HRM)分析快速鉴定CLN6(神经元蜡样脂褐质沉积症,ceroid—lipofuscinosis,neurona16)小鼠(c2硒基因移码突变)基因型的方法。方法根据NCBI公布的小鼠cln6序列(NC00075)设计HRM引物和测序引物,然后采用HRM技术获得高分辨熔解曲线鉴定实验小鼠基因型,同时通过直接测序法进行验证,评价其灵敏性和准确性。结果181只实验小鼠经HRM检测,共有野生型11只、Cln6基因突变杂合子73只和纯合子97只,HRM结果和直接测序结果完全一致,准确性为100%。结论HRM方法检测DNA微小突变时具有操作简便、快速、灵敏,单管避免污染以及准确度高等优点,值得推广。
Objective To establish a rapid detection method to for genotype CLN6 (ceroid-li- pofuscinosis, neuronal 6) mice with high-resolution melting curve (HRM) analysis. Methods Ac- cording to published mouse Cln6 NCBI sequence ( NC 00075 ) , HRM primers and sequencing prim- ers were designed, then used to obtain high-resolution melting curves identifying mouse genotypes by HRM technique. The sensitivity and accuracy of HRM results were verified by direct sequencing. Results Among 181 mice analyzed, 11 wild-type, 73 heterozygous and 97 homozygous for the Cln6 mutation were identified by HRM, which was completely consistent with direct sequencing. Conclu- sion Using HRM to detect small mutations in DNA is a simple, rapid, sensitive, accurate and sin-gle-tube procedure that may decrease contamination.
出处
《医学分子生物学杂志》
CAS
CSCD
2012年第3期161-165,共5页
Journal of Medical Molecular Biology
基金
国家遗传学重点学科,国家临床重点专科建设项目,上海市重点学科(B204),国家“863”计划项目(No.2007AA022400),上海市曙光计划跟踪项目(No.10GG10)