DNA聚合酶与引物/模板的相互作用对PCR效率的影响

doi:10.13523/j.cb.20140502

中国生物工程杂志

2014, Vol. 34

Issue (5): 6-13 DOI: 10.13523/j.cb.20140502

研究报告

DNA聚合酶与引物/模板的相互作用对PCR效率的影响

杨奇奇¹, 张俊威¹, 朱坚^1,2, 刘建平¹, 黄强¹

1 复旦大学遗传工程国家重点实验室生命科学学院上海 200433;
2 上海美迪西生物医药有限公司上海 201299

DNA Polymerase Binding to the Primer/template Duplex Affects the Efficiency of PCR

YANG Qi-qi¹, ZHANG Jun-wei¹, ZHU Jian^1,2, LIU Jian-ping¹, HUANG Qiang¹

1 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China;
2 Shanghai Medicilon Inc., Shanghai 201299, China

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摘要：

PCR是体外酶促合成特异DNA片段的一种方法，引物的优劣直接关系到PCR的特异性与成功与否。传统的PCR引物设计软件基本上忽略了DNA聚合酶与引物/模板的亲和性对PCR效率的影响。为揭示DNA聚合酶与引物/模板的相互作用是否对PCR的效率有影响，通过构建Taq DNA 聚合酶与不同序列引物/模板DNA相互作用的三维结构模型，采用MM/GBSA方法计算复合物的结合自由能，以结合自由能为参数，为人血清白蛋白基因(Human Serum Albumin gene，HSA gene)和结核杆菌pyrF基因(Mycobacterium tuberculosis pyrF gene)设计了PCR引物。PCR实验结果表明，引物的PCR效率与结合自由能相关：引物与聚合酶的结合自由能越低，PCR实验的效率相对越高。这说明DNA聚合酶与引物/模板的相互作用对PCR效率有重要影响。因此，引物/模板DNA与聚合酶的结合自由能可以作为PCR引物设计的新参数。

关键词： PCR; DNA聚合酶; 引物设计; 结合自由能; 分子建模

Abstract:

Polymerase chain reaction (PCR) for DNA and RNA amplifications in vitro has a profound impact on modern molecular biology. Since design of proper primers is crucial for the success in PCR, many parameters have been used for primer design. However, the effect of DNA polymerase binding to primer/template duplex on PCR efficiency was not taken into account in conventional primer design programs. To reveal whether or not the DNA polymerase-primer/template binding affects the PCR efficiency, here we built structural models for the Taq DNA polymerase in complex with different primer/template sequences, and designed PCR primers according to relative binding free energies calculated by MM/GBSA method. We verified our primer design approach using Human Serum Albumin (HSA) gene and Mycobacterium tuberculosis pyrF gene, and found that the PCR efficiencies of different designed primers for both tested genes correlated well with the calculated binding free energies. Our finding indicates clearly that the DNA polymerase-primer/template binding affects the PCR efficiency significantly. Thus, the calculated binding free energy could be used as a new parameter to design efficient PCR primers.

Key words: PCR DNA polymerase Primer design Binding free energy Molecular modeling

收稿日期: 2014-03-18 出版日期: 2014-05-25

ZTFLH:

Q819

基金资助:

上海市重点学科建设项目(B111)，上海市自然科学基金(13ZR1402400)资助项目

通讯作者: 刘建平, 黄强 E-mail: jpliu@fudan.edu.cn；huangqiang@fudan.edu.cn

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引用本文:

杨奇奇, 张俊威, 朱坚, 刘建平, 黄强. DNA聚合酶与引物/模板的相互作用对PCR效率的影响[J]. 中国生物工程杂志, 2014, 34(5): 6-13.

YANG Qi-qi, ZHANG Jun-wei, ZHU Jian, LIU Jian-ping, HUANG Qiang. DNA Polymerase Binding to the Primer/template Duplex Affects the Efficiency of PCR. China Biotechnology, 2014, 34(5): 6-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140502 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I5/6

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