重叠延伸PCR法构建UGRP1基因启动子的点突变表达载体  

Overlap-extension PCR Based Site-directed Mutants of UGRP1 Gene Promoter

作　　者：程烽[1] 盛燕[2] 施静艺[2] 陈漪[2] 徐潮[2] 刘智[2] 梁军[2] 潘春明[2] 朱忠勇[1] 宋怀东[2] 

机构地区：[1]南京军区福州总医院全军医学检验中心实验科,福州350025 [2]上海交通大学附属瑞金医院人类基因组国家重点实验室上海血液学研究所,上海200025

出　　处：《癌变．畸变．突变》2007年第1期8-10,共3页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金项目(No30530370)

摘　　要：背景与目的:构建UGRP1基因启动子的定点突变表达载体。材料与方法:以插入UGRP1基因正常启动子的质粒pGL3-UGRP1(-112G)为模板,用重叠延伸PCR定点诱变技术,对-112位点的碱基进行定点突变,并构建定点突变表达载体。结果:DNA测序表明,UGRP1基因启动子-112处的碱基已由G突变为A,成功实现定点诱变。结论:重叠延伸PCR定点诱变技术高效、简便。pGL3-UGRP(-112A)的成功构建,为进一步研究-112G/A多态性对该基因转录活性的影响奠定了基础。BACKGROUND ＆ AIM： To construct sited-directed mutants of human UGRP1 gene promoter. MATERIALS AND METHODS： Mutants were constructed by overlap extention method. The plasmid pGL3-UGRP1（-112G） was used as the template, site-directed mutagenesis was performed by overlap- extention PCR methed at -112 in UGRP1 gene promoter, and the expression vector of mutant at - 112 was then constructed. RESULTS： By DNA sequencing, human UGRP1 gene promoter was successfully changed from G to A at - 112 bp and the expression vector with the site-directed mutants were constructed. CONCLUSION： Overlap-extension PCR method was convenient and efficient for site-directed mutagenesis. Contruction of desired mutant pGL3-UGRPI（- 112A） may provide a basis for the research on regulating transcriptional activity of G/A polymorphism at -112 bp of human UGRPI gene promoter.

关 键 词：子宫球蛋白相关蛋白1基因(UGRP1) 启动子 重叠延伸PCR 定点诱变 

分 类 号：Q782[生物学—分子生物学]