高GC含量DNA模板的PCR扩增

PCR Amplification of GC-Rich DNA

目的：探索高GC含量DNA的PCR扩增条件，为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中，使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂，并选择合适的PCR循环程序，优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量，但会降低其特异性；7-deaza-dGTP可以提高扩增产物的特异性及保真度，但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率，增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来，并可扩增出长达6 kb的片段，且序列完全正确，可以进行后续拼接。

Objective： To determine the optimized conditions for PCR amplification of DNA with rich GC in or-der to amplify DNA fragments from daptomycin biosynthetic gene cluster and further for assembly. Methods：DMSO and 7-deaza-dGTP were added to the PCR amplification system and amplification cycling was chosen to optimize the conditions for PCR amplification of DNA with rich GC. Results： 1%~4% DMSO greatly improved tar-get product yield during PCR amplification but the specificity was reduced. 7-deaza-dGTP improved target prod-uct specificity and facilitated subsequent sequencing of GC-rich DNA, although product yield was not increased. Combination touch down PCR with 7-deaza-dGTP had good effect on PCR amplification, and will allow for the production of a wide variety of GC-rich gene constructs. Conclusion： Using this protocol, all the 1 kb DNA frag-ments from daptomycin biosynthetic gene cluster were successfully amplified and long DNA fragments up to 6 kb were also amplified, which will facilitate our thorough understanding to genes and their regulations and functions.