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PCR-RFLP技术检测SUMO4基因163A/G多态性的实验条件研究
引用本文:李斌,李会芳,王玉明,周晰溪,张娴,宋滇平.PCR-RFLP技术检测SUMO4基因163A/G多态性的实验条件研究[J].昆明医学院学报,2010,31(5):4-7.
作者姓名:李斌  李会芳  王玉明  周晰溪  张娴  宋滇平
作者单位:1. 昆明医学院第一附属医院糖尿病科,云南昆明,650032
2. 昆明医学院第一附属医院检验科,云南昆明,650032
基金项目:国家自然科学基金资助资助 
摘    要:目的确定PCR-RFLP技术检测小泛素样修饰蛋白4(SUMO4)163A/G多态性的最适实验条件.方法对影响SUMO4基因PCR反应的主要因素设定系列浓度梯度分别进行研究.结果最适变性温度为94℃;最适退火温度为55℃;最适引物浓度为0.25μmol/L;最适Mg2+浓度为1.5mmol/L;最适dNTP浓度为0.25mmol/L;以2.5U为最适Taq酶量;酶切体系为31μL体系中加10μL产物用10U的酶消化.结论最佳实验条件的摸索是进行糖尿病实验研究的关键.

关 键 词:聚合酶链反应  限制性片段长度多态性  小泛素样修饰蛋白4  实验条件

The Experimental Conditions for SUM04 Gene 163A/G Polymorphism Detection with PCR-RFLP
LI Bin,LI Hui-fang,WANG Yu-ming,ZHOU Xi-xi,ZHANG Xian,SONG Dian-ping.The Experimental Conditions for SUM04 Gene 163A/G Polymorphism Detection with PCR-RFLP[J].Journal of Kunming Medical College,2010,31(5):4-7.
Authors:LI Bin  LI Hui-fang  WANG Yu-ming  ZHOU Xi-xi  ZHANG Xian  SONG Dian-ping
Institution:1) Dept. of Diabetes;2) Dept. of Clinical Laborotory,The 1st Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650032,China)
Abstract:Objective To determine the optimum expermental conditions for the polymorphism of SUMO4 gene 163A/G detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) . Methods The main influential factors were detected by PCR in serial concentration gradient and electrophoresis,the main factors were detected by RFLP in different system.Results The optimum conditions:denaturation temperature was 94 ℃;annealing temperature was 55 ℃;primer concentration was 0.25 umol/L;magnesium concentration was 1.5 mmol/L;concentration of dNTP was 0.25 mmol/L;Taq enzyme was 2.5 U;the effective system was 31 uL including 10 uL DNA solution and 10 U enzyme.Conclution The key way to a large empirical study is to detect the optimal experiment conditions of diabetes.
Keywords:Polymerase chain reaction  Restriction fragment length polymorphism  Small ubiquitin-like modifer 4  Experimental condition
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