串联表达载体中短重复序列(BmKb1)n模板的PCR效率及其优化条件研究

江汉大学学报（自然科学版） ›› 2012, Vol. 40 ›› Issue (5): 71-75.

串联表达载体中短重复序列(BmKb1)n模板的PCR效率及其优化条件研究

范丽梅，李正，余腾，刘燕群，罗锋

江汉大学医学院，湖北武汉430056

收稿日期:2012-05-06 出版日期:2012-10-20 发布日期:2013-11-07
通讯作者: 罗锋(1975—)，男，讲师，博士，研究方向:毒素分子生物学。E－mail:lf7954＿cn＠sina.com
作者简介:范丽梅(1989—)，女，研究方向:分子生物学。
基金资助:
武汉市科技局青年科技晨光计划项目(200950199019－03)；武汉市市属高等学校科学研究重点项目(20070705)；湖北省高等学校优秀中青年科技创新团队项目(T2012)

PCR Efficiency of Short Repetitive Sequences (BmKb1)n in Tandem Expression Vector and Optimization of Its PCR Conditions

FAN Li－mei,LI Zheng,YU Teng,LIU Yan－qun,LUO Feng

School of Medicine,Jianghan University,Wuhan 430056,Hubei,China

Received:2012-05-06 Online:2012-10-20 Published:2013-11-07

摘要/Abstract

摘要： 采用OverlapPCR法获取BmKb1基因，构建载体pGEX－6p－1／BmKb1，同尾酶技术用于构建串联表达载体pGEX－6p－1／(BmKb1)n(n＝2，4，6，8，10)。以pGEX－6p－1／(BmKb1)n为模板，使用标准PCR程序研究短重复序列PCR效率;以BmKb1八倍体为模板，分别研究PCR循环数、引物终浓度及退火温度对短重复序列PCR效率及特异性的影响。结果表明串联重复序列数目增加，则非特异性条带增加，特异性条带含量减少;BmKb1基因串联体PCR扩增在18～20循环数、引物终浓度为0.05～0.1μM、60～62℃较高退火温度时，可获得相对高产量高特异性BmKb1基因串联体PCR片段。本研究将为串联表达载体克隆、筛选、测序及基因组STRs序列克隆测序提供技术参考。

关键词: 重复序列, PCR, BmKb1, 循环数

Abstract: After acquiring BmKb1 gene by Overlap PCR, pGEX－6p－1／BmKb1 vector was constructed. The series recombinant pGEX－6p－1／(BmKb1)n (n ＝ 2, 4, 6, 8, 10) plasmids were build with isocaudamer technology. With the template of pGEX－6p－1／(BmKb1)n, standard PCR procedure was used to study PCR efficiency of the short tandem repeats; The BmKb1 octoploid template was used to study the effects of the number of PCR cycles, the final concentration of primers and the annealing temperature on PCR efficiency and specificity of the short sequence repeat, respectively. The results showed thatwith the number of tandem repeat sequences increasing, the number of non－specific bands will increase, and the content of specific bands will decrease; Amplifying BmKb1 gene tandem by PCR in the 18-20 cycle numbers, with the primer final concentration of 0.05-0.1 μM, as well as at higher annealing temperature of 60-62 ℃, the relatively high yield and high the specificity PCR fragments of BmKb1 gene concatemer will be available. This study will provide a technology basis for the cloning, PCR screening, sequencing of tandem expression vector and genome STRs.

Key words: tandem repeats, PCR, BmKb1, cycle number

中图分类号:

Q785

范丽梅，李正，余腾，刘燕群，罗锋. 串联表达载体中短重复序列(BmKb1)n模板的PCR效率及其优化条件研究[J]. 江汉大学学报（自然科学版）, 2012, 40(5): 71-75.

FAN Li－mei,LI Zheng,YU Teng,LIU Yan－qun,LUO Feng. PCR Efficiency of Short Repetitive Sequences (BmKb1)n in Tandem Expression Vector and Optimization of Its PCR Conditions[J]. Journal of Jianghan University(Natural Science Edition), 2012, 40(5): 71-75.

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