Reconstruction of plasmid DNA with Red-mediated homologous recombination combined with restriction digestion
- 厦门大学－已发表论文 
目的:用Red介导的单链寡核苷酸体内同源重组结合限制酶切方法对pBR322-Red质粒中的DNA序列进行精确的缺失突变。方法:用合成的单链寡核苷酸打靶分子电击转化W3110(pBR322Red)宿主菌,利用Red提供的同源重组功能敲除从kil基因至N基因长约2100bp的DNA序列,并用kilN序列中含有的单一酶切位点XhoⅠ对混合质粒进行酶切,取酶切产物转化W3110感受态细胞,菌落PCR方法筛选重组阳性克隆。结果:在没有任何筛选标记的情况下,用菌落PCR方法从10个菌落中成功挑选出1个重组阳性克隆。结论:该方法操作简单、精确,能够避免引入新突变,为DNA序列的缺失突变提供了一种新方法。Objective:To precisely delete a 2 100 bp DNA sequence in pBR322-Red plasmid using Red mediated single-stranded oligonucleotides in vivo homologous recombination combined with restriction digestion strategy.Methods:A single-stranded oligonucleotides DNA target cassette was introduced into W3110(pBR322-Red) host strain by electroporation,a 2 100 bp DNA fragment from kil to N gene that contains a unique XhoI site in pBR322-Red plasmid was deleted by Red mediated in vivo homologous recombination. The recombinant plasmid DNA was screened from the plasmid DNA mixture with XhoⅠ restriction endonuclease digestion,and retransformed into W3110 competent cells. The positive clones were further (identified) by colony PCR assay.Results: One positive clone out of ten clones was successfully identified without any selectable marker. Conclusion:This new method of DNA sequence deletion mutation is simple and accurate. It could avoid new mutation in PCR process of traditional DNA manipulation method.