富含GC DNA PCR扩增条件的优化

Optimizing conditions for PCR amplification of DNA with rich GC

目的 :探索富含GCDNA的PCR扩增条件 ,为大环内酯类化合物组合生物合成中模块和酶域的任意组合奠定基础。方法 :在PCR扩增体系中 ,添加甲酰胺、甘油、二甲亚砜 (DMSO)、Mg2 + 等增强剂 ,并选择合适的扩增体系 ,优化富含GCDNAPCR扩增条件。结果 :甲酰胺对富含GCDNAPCR扩增没有影响 ,而甘油和DMSO均可提高富含GCDNAPCR产物的特异性和产率 ,以 5 %～ 10 %DMSO效果最好。使用Roche长片段DNA扩增系统 ,在使用缓冲液 2反应体系中添加 10 %DMSO和提高 0 .2 5mmol LMg2 + 浓度时 ,可以扩增长度为 5kb、GC含量为 73%的DNA片段。结论 :应用优化的PCR扩增条件 ,可以扩增长达 5kb富含GC的DNA片段 。

Objective:To determine the optimized conditions for PCR amplification of DNA with rich GC in order to freely assemble modules or domains of polyketide synthase (PKS) in macrolide combinatorial biosynthesis.Methods:Formamide, glycerol, DMSO and Mg 2+ were added to the PCR amplification system and amplification system was chosen to optimize the conditions for PCR amplification of DNA with rich GC.Results:Formamide had no influence on PCR amplification of DNA with rich GC, while both glycerol and DMSO promoted output and specificity of PCR product, with 5%-10% DMSO showing the best effectiveness. When Roche Expand Long Template PCR System was used and 10% DMSO and extra 0.25?mmol/L Mg 2+ were added to buffer 2, 5?kb DNA with 73% GC content was amplified.Conclusion:With addition of DMSO and extra Mg 2+ to PCR system, at least 5?kb DNA with rich GC could be amplified, and that can meet the need to assemble any modules or domains in macrolide combinatorial biosynthesis.