<em>Bst </em>DNA聚合酶大片段在全基因组扩增中的应用

doi:10.13523/j.cb.20160708

中国生物工程杂志

2016, Vol. 36

Issue (7): 48-54 DOI: 10.13523/j.cb.20160708

技术与方法

Bst DNA聚合酶大片段在全基因组扩增中的应用

张心愿^1,2, 邓佳^1,2, 刘馨^1,2, 陈琳^1,2, 林峻^1,2, 蔡伟文^1,2

1 福州大学应用基因组学研究所福州 350108;
2 福州大学生物科学与工程学院福州 350108

Bst DNA Polymerase Large Fragment for Whole Genome Amplification

ZHANG Xin-yuan^1,2, DENG Jia^1,2, LIU Xin^1,2, CHEN Lin^1,2, LIN Jun^1,2, CAI Wei-wen^1,2

1 Institute of Applied Genomics, Fuzhou University, Fuzhou 350108, China;
2 College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, China

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摘要：

Bst DNA聚合酶大片段作为一种常用的DNA聚合酶，因其独特的特点：能引发链置换反应、高保真、耐高温等，而成为一种重要的DNA多重置换扩增酶。目的：为减少成本，设计一种高产，方便且扩增活性高的Bst DNA聚合酶大片段表达体系；探究该酶应用于胃癌石蜡包埋组织基因组DNA的扩增条件。方法：采用pTWIN1质粒作为载体克隆表达Bst DNA聚合酶大片段，应用几丁质亲和层析柱纯化该酶，使用该酶对人类基因组DNA进行不同温度下扩增，探究其最适反应温度，并据此对胃癌石蜡包埋组织基因组DNA进行扩增。结果：由此得到的Bst DNA聚合酶大片段能运用于胃癌石蜡包埋组织基因组DNA的扩增，扩增效率可达200倍，并能应用于aCGH芯片。结论：扩增得到保真性高，覆盖基因组范围大的DNA扩增产物。该应用与aCGH结合，使得对少量的癌症石蜡包埋组织DNA样本进行全基因组扩增，并进行其基因拷贝数变异研究成为可能。

关键词： 多重置换扩增; 石蜡包埋组织; Bst; 全基因组扩增; DNA聚合酶大片段

Abstract:

As a common DNA polymerase, Bst DNA polymerase large fragment has been a kind of important DNA multiple displacement amplification enzyme, which can cause chain reaction, high fidelity, high temperature resistance and so on. Objective:Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. In order to reduce cost, a high yield, convenient and high activity of Bst DNA polymerase large fragment expression system had been designed, and the conditions in the amplification of the gastric cancer genomic DNA from FFPE tissues were explored. Methods:Bst DNA polymerase large fragment was cloned and expressed by the PTWIN1 plasmid, and purified by the chintin column. It was used to amplify the Human genome DNA at different temperature to explore the optimum temperature. Then the gastric cancer genomic DNA from FFPE tissues was amplified on the optimum condition. Results:The Bst DNA polymerase large fragment yielded a median 200-fold of DNA amplification. Conclusion:Bst DNA polymerase large fragment is suitable for WGA of DNA extracted from FFPE tissues. Amplified DNA may be used for the detection of gene copy number changes and genome profiling by array CGH.

Key words: Multiple displacement amplification Bst DNA polymerase large fragment Formalin-fixed Whole genome amplification paraffin-embedded

收稿日期: 2015-12-28 出版日期: 2016-03-02

ZTFLH:

Q789

基金资助:

国家自然科学基金（31371287，31301537，31571300），福建省科技重大专项（2013YZ0002-1，2013YZ0002-2）资助项目

通讯作者: 蔡伟文 E-mail: caiww@fzu.edu.cn

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引用本文:

张心愿, 邓佳, 刘馨, 陈琳, 林峻, 蔡伟文. Bst DNA聚合酶大片段在全基因组扩增中的应用[J]. 中国生物工程杂志, 2016, 36(7): 48-54.

ZHANG Xin-yuan, DENG Jia, LIU Xin, CHEN Lin, LIN Jun, CAI Wei-wen. Bst DNA Polymerase Large Fragment for Whole Genome Amplification. China Biotechnology, 2016, 36(7): 48-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160708 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I7/48

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