Mn^(2+)驱动的不同长度DNA片段PCR随机突变研究

PCR-driven Random Mutagenesis of Different Length DNA Fragments by Manganese Modification

针对倾向错误的PCR技术中不同目的、不同长度基因的诱变种类一直缺乏准确参考的问题,以酵母spt15基因为研究对象,在标准的PCR反应体系中加入不同Mn2+摩尔浓度(0.125,0.250和0.500mmol.L-1)进行倾向错误的PCR,扩增spt15不同长度(分别约为200,500和700bp)的DNA片段,研究其突变频率,突变碱基数目及突变类型,以分析不同种类的突变所需的最佳条件.研究结果表明:长片段DNA的PCR突变率对Mn2+摩尔浓度更敏感;模板的长度对DNA PCR突变碱基个数也有较为明显的影响,低Mn2+摩尔浓度有利于单突变;Mn2+驱动的突变有明显的倾向性,其中A→G和T→C的突变居多.

To analyze the best conditions for different types of mutations required in error-prone PCR system,this paper tried to process error-prone PCR by adding different concentrations of manganese（0.125,0.250and 0.500mmol/L）into normal PCR system.Different lengths of DNA fragments of S.cerevisiae spt15（about 200,500and 700bp）were amplified to study their mutation ratios,spectrum of mutations and the distribution of base substitutions.The results have shown that the error-prone PCR mutation rate of longer fragment DNA is more sensitive to manganese ions concentration.The mutation number of bases has a significant impact on the length of template DNA.Lower manganese ions concentration is conducive to a single mutation.The base substitution distribution of mutation driven by manganese ions has obvious bias towards the transition of A → G and T → C.