放线菌16S rDNA 片段扩增方法的优化

摘要

Objective To optimize the most suitable polymerase chain reaction (PCR) amplifica-tion condition for actinomycetes strains with the adjustment and testing of different experimental meth-ods .Methods Extracted genomic DNA of actinomycetes strains and amplify their 16S rDNA gene un-der various annealing temperatures .Changed the concentration of template DNA and add extra Mg2+in PCR reaction system .DMSO was added in the reaction system .Results In order to amplify 16S rD-NA (about 1 500bp) of actinomycete ,the most ideal condition was decided as 10 × template DNA , 0 .5 μL Mg2+ and 5% DMSO ,with annealing temperature of 59 ℃ .Conclusion The method is effi-cient ,convenient ,fast and economical for identification and taxonomy of actinomycetes strains in large quantities .%目的：通过对放线菌PCR扩增方法的综合改进，找到某些用常规手段难扩增的放线菌16S rDNA的最佳PCR扩增条件。方法（1）提取放线菌基因组DNA并对其进行PCR扩增以找到最适退火温度；（2）改变PCR反应体系中模板DNA和Mg2＋的浓度；（3）在反应体系中加入适量的二甲基亚砜。结果在25μL的PCR反应体系中，模板DNA浓度为10×、加入0．5μL的Mg2＋和5％的二甲基亚枫、退火温度为59℃时对放线菌16S rDNA扩增效果最为理想。结论本实验研究出一种扩增放线菌16S rDNA的优化条件，为今后大批量的放线菌的分子生物学研究提供了更加高效、方便、快捷、经济的实验方法。