一种快速检测CLN6模型小鼠基因型的方法——高分辨率熔解曲线分析法

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,neuronal 6)小鼠(Cln6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(NC 00075)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 CLN 6 ( ceroid-li-pofuscinosis , neuronal 6) mice with high resolution melting curve (HRM) analysis. Methods According to published mouse Cln6 NCBI sequence (NC 00075) , HRM primers and sequencing primers 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 . Conclusion Using HRM to detect small mutations in DNA is a simple , rapid, sensitive, accurate and sin gle-tube procedure that may decrease contamination.