突变敏感性分子开关检测线粒体DNA A1555G位点的条件优化

Investigations on the optimized conditions of mitochondrial DNA A1555G mutation detected by mutation-sensitive molecular switch technique

目的对高保真酶介导的突变敏感性分子开关技术检测线粒体DNA（mt DNA）A1555G位点条件进行优化。方法利用3＇硫代磷酸化修饰的突变型引物和野生型引物作为下游引物,在其上游设计一条公共引物分别构成突变引物对和野生引物对,以构建好的包含mt DNA A1555G位点的突变型质粒和野生型质粒为模板,进行高保真聚合酶介导的双向引物延伸反应,对PCR体系中的退火温度、引物浓度、模板浓度等条件优化,通过凝胶成像系统对其PCR结果进行分析确定最佳反应条件。结果分子开关技术检测mt DNA A1555G位点的最佳PCR条件：退火温度为61.0℃,引物浓度为0.6μmol/L,检测模板浓度为103~106copies/μL。结论确定了分子开关技术检测mt DNA A1555G位点的最佳反应条件,为该技术在线粒体DNA的突变筛查中的应用提供依据。

Objective To optimize the detection conditions of mitochondrial DNA A1555 G mutation by high fidelity polymerase-mediated mutation-sensitive molecular switch technique. Methods The mutant sequence primer and wild sequence primer modified by 3＇phosphorothioate as the downstream primers,and a common primer as the upstream primer were designed and paired respectively.Then,the mutant plasmid containing well-constructed mt DNA A1555 G locus and wild-type plasmid were selected as templates,and the high fidelity polymerase-mediated bi-directional primer extension reaction was performed. Last,the PCR conditions,including annealing temperature,primer concentration,template concentration and so on,were optimized,and the optimal conditions were determined by the gel imaging of PCR results. Results The optimal PCR conditions of the mt DNA A1555 G detected by mutation-sensitive molecular switch technique were as follows： the optimal annealing temperature,primer concentration and template concentration were 61 ℃,0. 6 μmol / L and 103 to 106copies / μL,respectively. Conclusion The optimal reaction conditions of the mt DNA A1555 G mutation detected by mutation-sensitive molecular switch technique are determined successfully,which may provide evidence for the mutation screening of mitochondrial DNA.