突变敏感性分子开关检测线粒体DNA A1555G位点的条件优化

发布时间：2018-06-11 23:34

  本文选题：突变敏感性分子开关 + 氨基糖苷类耳聋　； 参考：《临床检验杂志》2016年03期

【摘要】：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。
[Abstract]:Objective to optimize the conditions for the detection of mtDNA A1555G site in mitochondrial DNA by high fidelity enzyme mediated mutagenic molecular switch technique. Methods 3'thiophosphorylation modified mutant primers and wild primers were used as downstream primers, and a common primer was designed to form mutant primer pair and wild primer pair, respectively. The mutant plasmids and wild type plasmids containing mt DNA A1555G site were used as templates to carry out bidirectional primer extension mediated by high fidelity polymerase. The conditions of annealing temperature, primer concentration and template concentration in PCR system were optimized. The PCR results were analyzed by gel imaging system to determine the optimal reaction conditions. Results the optimal conditions for detecting mt DNA A1555G locus by molecular switch technique were as follows: annealing temperature was 61.0 鈩，

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