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

采用Overlap PCR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。

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

After acquiring BmKbl gene by Overlap PCR, pGEX-6p-1/BmKbl vector was constructed. The series recombinant pGEX-6p-1/（BmKbl）n （n = 2, 4, 6, 8, 10） plasmids were build with isocaudamer technology. With the template of pGEX-6p-1/（BmKbl）n standard PCR procedure was used to study PCR efficiency of the short tandem repeats; The BmKbl 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 re- peat, respectively. The results showed that： with the number of tandem repeat sequences increas- ing, the number of non-specific bands will increase, and the content of specific bands will de- crease; Amplifying BmKbl gene tandem by PCR in the 18-20 cycle numbers, with the primer final concentration of 0.05-0.1μM, as well as at higher annealing temperature of 60-62℃, the relatively high yield and high the specificity PCR fragments of BmKbl 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.