PCR-RFLP检测Apo CIII基因T-455C多态性的反应条件优化

目的:探讨聚合酶链反应-限制性片段长度多态性(PCR-RFLP)反应体系中各种成分的优化,建立检测载脂蛋白CIII(Apo CIII)T-455C多态性的方法。方法:采用PCR-RFLP检测ApoCIII基因T-455C多态性,同时通过对参与反应体系的成分,在一定范围内设置不同的浓度或参数组,进行正交试验和单因素逐项试验,观测各成分在不同条件下对结果的影响。结果:Apo CIII基因检测到3种基因型。PCR反应体系中不同模板含量、引物浓度、Taq DNA聚合酶用量、dNTP浓度、Mg2+浓度、退火温度和酶切体系中PCR产物量、内切酶量、酶切时间等对反应结果均有不同程度的影响。结论:该研究建立的PCR-RFLP体系检测Apo CIII基因T-455C多态性技术,是一种高效快速、简单敏感、准确可靠的分析方法,适合常规实验室开展。

Optimization of Conditions for Detection of Apolipoprotein CIII T-455C Polymorphism by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

Objective:To establish a method of polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) to analyze the polymorphism of Apolipoprotein CIII T-455C(Apo CIII) by optimizing the factors which can affect the profiles of experiment.Methods:PCR-RFLP technique was performed to detect the T-455 Cpolymorphism of Apo CIII.The compositions that join the PCR-RFLP system within certain different concentration or parameters were set up and its results were obsered.Results:Three kinds of genotypes at Apo CIII-455 locus were found.Concentration of primer,magnesium chloride,dNTP,template DNA,Taq DNA polymerase and annealing temperature in PCR and the quantity of PCR product and endonuclease and digesting time in digestion process affect the profiles of the whole experiment.Conclusion:PCR-RFLP is a simple,sensitive and rapid reliable method to investigate T-455C polymorphism in Apo CIII gene promoter,and it is suitable for the routine or study work in ordinary laboratory.