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

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.