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

A Rapid High-Resolution Melting Curve Method to Detect Genotypes of CLN6 Mice

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.