应用PCR-RFLP技术检测二甲基精氨酸二甲胺水解酶2基因-449G/C多态性的实验研究  

The experimental conditions for dimethyl arginine dimethylamine hydrolase 2 gene-449G/C polymorphism detaction with PCR-RFLP

作　　者：杨晶晶[1,2] 石柔[1] 宋滇平[1] 陈洪艳[3] 

机构地区：[1]昆明医科大学第一附属医院糖尿病科,650032 [2]云南省第一人民医院 [3]昆明医科大学第一附属医院超声科,650032

出　　处：《中国老年保健医学》2015年第3期30-32,共3页Chinese Journal of Geriatric Care

基　　金：云南省应用基础研究(昆医联合专项)基金(项目编号2014FZ014);云南省教育厅科研基金(项目编号2012Y023)

摘　　要：目的确定聚合酶链反应-限制性片段长度多态性(Polymerase chain reaction-restricted fragment length polymorphism,PCR-RFLP)技术检测二甲基精氨酸二甲胺水解酶2(dimethyl arginine dimethylamine hydrolase 2,DDAH2)基因-449G/C的最适实验条件。方法对影响DDAH2基因PCR反应的主要因素进行研究。结果最适变性温度为95℃,最适退火温度为58℃,最适引物浓度为0.25μmol/L,最适2X Power Taq PCR Master Mix浓度为12.5μl,酶切体系为15.5μl体系中加5.0μl产物用5U的限制性内切酶Sma I消化。结论应用PCR-RFLP技术建立的DDAH2基因-449G/C位点多态位点检测方法简便、经济、快速。Objectives To determine the optimum experimental conditions for DDAH2 gene -449G/C polymorphism by Polymerase chain reaction-restricted fragment length polymorphism （PCR-RFLP）. Methods The main influential factors were detected by PCR in electrophoresis, the main factors were detected by RFLP in different system. Results The optimum conditions： denaturation temperature was 95℃, annealing temperature was 58℃, Stretching temperature was 72℃, primer concentration was O. 25μmoL/L, 2X Power Taq PCR Master Mix was 12. 5μl, the effective system was 15.5μl including 5μl DNA solution and 5U enzyme. Conclusions It suggested that the method of detecting the dimethyl arginine dimethylamiue hydrolase 2 gene -449G/C polymorphism based on PCR-RFLP theory is facilitated, economic, and rapid.

关 键 词：聚合酶链反应 限制性片段长度多态性 二甲基精氨酸二甲胺水解酶2 实验条件 

分 类 号：R587.2[医药卫生—内分泌]