长片断引物反向PCR方法构建重复序列的重组质粒

摘要

构建重复序列的重组质粒比较困难,因为扩增重复序列会产生多聚物.利用长片断引物反向PCR方法构建重组质粒pET20b-C1-Xyn-C1,将碳水化合物结合模块C1连接在木聚糖酶Xyn两端,为类似质粒的构建提供新方法.第一步,以C1特异性正向引物LF、载体特异性反向引物VRP从pET20b-Xyn-C1模板上扩增C1-pET20b长片断DNA,作为第二步的正向引物;第二步,以pET20b-C1-Xyn为模板,Xyn特异性反向引物RX,正向引物与模板上pET20b同源区域匹配,阻止了模板上C1同源区域的匹配,从而抑制多聚物产生;反向PCR扩增得到线性重组质粒C1-pET20b-C1-Xyn,将此线性产物用T4 DNA连接酶连接后转化DH5α感受态细胞,得到含重复序列的pET20b-C1-Xyn-C1转化子.%It is difficult to construct recombinant plasmid containing repeated DNA sequences,because multimer would be produced in PCR amplification. Megaprimer reverse PCR was used to construct recombinant plasmid pET20b-C1-Xyn-C1,which contained a xylanase(Xyn)DNA flanked by repeated DNA sequence,a Carbohydrate binding module C1. Firstly,megaprimer C1-pET20b was amplified using forward primer LF for C1,reverse primer VRP for pET20b,and pET20b-Xyn-C1 template. Secondly,the linear recombinant plasmid C1-pET20b-C1-Xyn DNA was amplified by using reverse primer RX for Xyn,pET20b-C1-Xyn template,and the amplified C1-pET20b as forward megaprimer. The forward megaprimer annealed to pET20b instead of region of the template. The linear recombinant plasmid was ligated with T4 DNA ligase and transformed to DH5α competent cell. The recombinant pET20b-C1-Xyn-C1 containing repeated sequences C1 was constructed.