摘要
利用聚合酶链式反应 (PolymeraseChainReaction ,PCR)与限制性内切酶相结合的方法 ,设计 4条含有限制性酶切位点和相应突变的引物。以马铃薯X病毒 (PotatoVirusX ,PVX)外壳蛋白cp基因为模板 ,扩增出相应的片段 ,相应酶切后通过三片段连接构建到克隆载体pBlueKS(+/ - )上。随机挑选重组子测序表明 ,利用三片段拼接成功地在PVX外壳蛋白基因的不同部位产生了突变。实验结果说明利用三片段接可以大大提高筛选得到突变子的效率 ,从而节省人力、物力和时间。
Target sequences were amplified using PCR (Polymerase Chain Reaction) using a high-fidelity DNA polymerase with primers contained mutated nucleotides and restriction endonuclease recognition sequences. The PCR products be cut with corresponding restriction endonuclease, and ligated with the other part sequence of Potato Virus X coat protein gene and clone vector pBlueKS(+/-)(Three-Fragment-Ligation, TFL), then recombinants be recognized by blue/white color identification. Sequencing results showed that all recombinants were all mutated successfully, which indicated that the TFL ligation method could enhance the efficiency of getting interested recombinants, and save the time and cost.
出处
《生物技术通报》
CAS
CSCD
2003年第4期29-32,共4页
Biotechnology Bulletin
基金
国家重点基础研究发展计划 973 (批准号 :G2 0 0 0 0 162 0 5 )
国家自然科学基金重点资助项目 (批准号 :3 99890 0 1)
