高GC含量DNA模板的PCR扩增  

PCR Amplification of GC-Rich DNA

作　　者：邢玉华[1,2] 戴素琴[1,3] 刘体颜[1,3] 王微[1,2] 谭俊杰[1] 曲国龙[1] 刘刚[1] 陈惠鹏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]吉林大学生命科学学院,吉林长春130012 [3]安徽大学生命科学学院,安徽合肥230039

出　　处：《生物技术通讯》2013年第5期645-649,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划子课题(2012AA022001-03D)

摘　　要：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.

关 键 词：达托霉素生物合成基因 高GC含量DNA PCR添加剂 touch down PCR 

分 类 号：Q78[生物学—分子生物学]