高AT含量DNA片段PCR扩增体系的优化  

PCR system optimization for the efficient amplification of AT-rich DNA fragments

作　　者：张姝[1] 张永杰[2] 郝爱静 ZHANG Shu;ZHANG Yongjie;HAO Aijing(Institute of Applied Chemistry,Shanxi University,Taiyuan 030006,Shanxi,China;School of Life Science,Shanxi University,Taiyuan 030006,Shanxi,China)

机构地区：[1]山西大学应用化学研究所,山西太原030006 [2]山西大学生命科学学院,山西太原030006

出　　处：《陕西师范大学学报（自然科学版）》2020年第5期71-77,共7页Journal of Shaanxi Normal University：Natural Science Edition

基　　金：国家自然科学基金(31872162);山西省自然科学基金(201601D011065);山西省回国留学人员科研资助项目(2017-015)。

摘　　要：选用4种不同长度(1.0~5.4 kb)、高AT含量(72%~80%)的真菌线粒体DNA片段,通过比较4种PCR添加剂(牛血清白蛋白(BSA)、二甲基亚砜(DMSO)、甲酰胺和镁离子)和4种KOD系列DNA聚合酶(KOD-Plus-、KOD-Plus-Neo、KOD FX和KOD FX Neo)对PCR扩增效果的影响,优化高AT含量DNA片段的PCR扩增体系。结果表明:当PCR体系中单独使用1种添加剂时,只有镁离子能够促进扩增,其他3种添加剂都不能产生预期扩增条带。BSA和DMSO对镁离子的扩增促进作用无负面影响,而甲酰胺会抑制镁离子的扩增促进作用。当镁离子存在时,4种KOD聚合酶的PCR体系均可获得预期的扩增产物;当缺乏镁离子时,使用KOD-Plus-或KOD-Plus-Neo聚合酶的PCR体系未能得到扩增产物,表现出对镁离子的依赖性。镁离子对高AT含量DNA片段的扩增具有促进作用,最佳使用浓度为1.75 mmol/L。研究结果为其他真核生物中高AT含量DNA片段的扩增提供了方法依据。The purpose of this study is to optimize PCR system to achieve efficient amplification for AT-rich DNA fragments.Four additives and four KOD-based high-fidelity DNA polymerases were studied for their amplification effects of four AT-rich fungal mitochondrial DNA fragments(AT contents:72%~80%)with variable lengths of 1.0~5.4 kb.When applied alone,only magnesium ion facilitated the amplification,and the other three additives(bovine serum albumin(BSA),dimethyl sulfoxide(DMSO)and formamide)failed to produce the expected amplicon,which were contrary to their well-known facilitative effects on GC-rich DNA amplification.The presence of BSA or DMSO showed no impact on the amplification effect of magnesium ion,whereas the presence of formamide expressed inhibition effect on the facilitation function of magnesium ion.The four KOD-based polymerases(KOD-Plus-,KOD-Plus-Neo,KOD FX and KOD FX Neo)generated expected amplicons when magnesium ion was present in the PCR system,but KOD-Plus-and KOD-Plus-Neo failed to generate expected amplicon when magnesium ion was absent.This study verified that magnesium ion was critical for the successful amplification of AT-rich DNA fragments,and the optimal concentration was 1.75 mmol/L.The results provide a basis for the amplification of AT-rich DNA fragments in other eukaryotes.

关 键 词：添加剂 PCR扩增 高AT含量DNA片段 镁离子 

分 类 号：Q93-3[生物学—微生物学]