应用改良PCR法构建高GC含量启动子多位点定点突变载体

首都医科大学学报 ›› 2010, Vol. 31 ›› Issue (1): 88-92.

应用改良PCR法构建高GC含量启动子多位点定点突变载体

姜鸾¹, 邵蕾², 胡旸³. 董凌月⁴

1. 首都医科大学2005级临床医学专业七年制3班;2. 首都医科大学2005级临床医学专业五年制3班;3. 首都医科大学2005级临床医学专业五年制4班; 4. 首都医科大学基础医学院细胞生物学系

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-02-21 发布日期:2010-02-21
通讯作者: 董凌月

Improvement of a PCRbased Method for Vector Construction of Multisitedirected Mutations in GC-rich Promoter

JIANG Luan¹, SHAO Lei², HU Yang³, DONG Ling-yue⁴

1. Class No. 3 of Grade 2005 in Department of 7Year Clinical Medicine, Capital Medical University; 2. Class No. 3 of Grade 2005 in Department of 5Year Clinical Medicine, Capital Medical University; 3. Class No. 4 of Grade 2005 in 5 Year's Clinical Medicine, Capital Medical University; 4. Department of Cell Biology, Basic Medical Sciences, School of Basic Medical Sciences, Capital Medical University

Received:1900-01-01 Revised:1900-01-01 Online:2010-02-21 Published:2010-02-21
Contact: DONG Ling-yue

摘要/Abstract

摘要：

目的利用StrataGene公司的QuickChangeTM定点突变试剂盒对高GC含量的人肝刺激因子启动子进行多位点定点突变。方法采用含有多位点突变的引物扩增启动子片段，通过加入不同浓度的DMSO, 降低高GC含量模板及引物的解链温度，利用乙醇沉淀提高酶切产物的浓度。结果经测序鉴定成功构建5个含有不同突变位点的hHSS启动子突变载体。结论这种改良的PCR方法提高了对高GC含量启动子进行扩增的效率，具有快速、简便、经济、成功率高的特点，是值得推广的多位点定点突变载体构建方法。

关键词: 定点突变, PCR, DMSO, 人肝刺激因子

Abstract:

Objective To construct several vectors in which multi-site mutation occurs within a GC-rich hHSS promoter. Methods An interested genomic region of human hepatic stimulator substance(hHSS) was multi-site-directly mutated and amplified. During amplification, the melting temperature was reduced by adding certain concentration of DMSO and the concentration of the enzyme-digested products was increased through ethanol precipitation. Results The results showed that five different hHSS promoter vectors containing multipoint mutations were effectively constructed and confirmed by DNA sequencing. Conclusion This improved method for PCR amplification of GC-rich promoter was efficient, which provides a rapid, simple and economic approach to analysis of genomic DNA.

Key words: site directed mutagenesis, polymerase chain reaction, dimethyl sulfoxide, human hepatic stimulator substance

中图分类号:

Q 782

姜鸾;邵蕾;胡旸;董凌月. 应用改良PCR法构建高GC含量启动子多位点定点突变载体[J]. 首都医科大学学报, 2010, 31(1): 88-92.

JIANG Luan;SHAO Lei;HU Yang;DONG Ling-yue. Improvement of a PCRbased Method for Vector Construction of Multisitedirected Mutations in GC-rich Promoter[J]. Journal of Capital Medical University, 2010, 31(1): 88-92.

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