高分辨率熔解曲线法检测CYP4A118590T＞C单核苷酸多态性

High Resolution Melting Analysis for CYP4A11 8590T＞C Single Nucleotide Polymorphism

目的 建立CYP4A11 8590T＞C单核苷酸多态性（single nucleotide polymorphism,SNP）的高分辨率熔解曲线（high resolution melting,HRM）检测方法.方法先采用温度梯度PCR,确定适宜的退火温度;再利用正交试验,优化引物、DNA模板量和Mg2＋量,最终确定PCR反应体系和反应条件.通过对607例无血缘关系的受试者基因组DNA进行HRM分析,并随机选择50例产物测序.结果 引物最适退火温度为57.8 ℃;PCR最佳反应体系为20 μl,包括2×conc dNTP mix 10 μl,上下游引物（10 μmol/L）各0.5 μl,DNA溶液（30 ng/μl）1.0 μl,Mg2＋（2.5 mmol/L）1.5 μl和灭菌水6.5 μl.607例受试者中CYP4A11 8590TT、TC和CC基因型频率分别为54.7 %、37.6 %和7.7 %.结论该正交试验优化的HRM技术可用于检测CYP4A11 8590T＞C单核苷酸多态性,且其分析结果和测序结果一致.

Objective To establish the optimal reaction system and reaction conditions of high resolution melting for CYP4A11 8590T 〉 C single nucleotide polymorphism. Methods The annealing temperature was determined according to Tm value of primer in order to make sure the appropri- ate annealing temperature. The orthogonal test was used to optimize PCR reaction system in the three factors including Primer, Mg2＋ and DNA template. The 8590T 〉 C genetic polymorphism of 607 subjects detected by HRM, and 50 subjects direct sequencing was done randomly to confirm the results. Results The optimal annealing temperature was 57.8 ℃. The optimization of20pd PCR reaction system including 2 × cono dNTP mix 10 μl, Primer 0. 5 μl （ 10 μmol/L）, DNA template 1.0 μl （ 30 ng/μl ）, Mg2 × 1.5 μl （ 2. 5mmol/L ） and H20 6. 5μl. 607 subjects of CYP4A11 8590TY, TC and CC genotype frequencies were 54. 7 %, 37.6 % and 7.7 %. Conclusion The HRM reaction system we set up by orthogonal test can be used to analysis CYP4A11 8590T 〉 C single nucleotide polymorphism, and the results between HRM and direct sequencing were consistent.