放线菌16S rDNA片段扩增方法的优化

Optimization of 16S rDNA amplification methods for actinomycetes strains

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16SrDNA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16SrDNA扩增效果最为理想。结论本实验研究出一种扩增放线菌16SrDNA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。

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 .