应用PCR方法向基因内部引入序列的策略研究

摘要

应用PCR方法,利用3条引物与2个模板,分别采用一步PCR、两步PCR、三步PCR扩增策略,向cte基因内部引入一段内含子序列,以达到基因改造的目的.结果表明,利用一步PCR的策略,难以扩增得到目的基因,利用二步PCR的策略,虽然可以得到目的条带,但存在非特异扩增;通过三步PCR的策略,即第一步PCR扩增产物作为第二步扩增反应的引物,进行小循环的单引物扩增,第三步以第二步产物为模板进行双引物扩增的方法,可以高效扩增得到目的条带.说明三步PCR策略为基因改造的理想方法.%An intron was introduced into acre gene by one-step PCR, two-step PCR, three-step PCR amplification strategy, respectively, and using three primers and two templates, in order to achieve the purpose of genetic modification. In the study, it was found that by one-step PCR amplication strategy, target genes were amplified difficultly. By two-step PCR strategy, target genes could be found, but existed non-specific amplification. By three-step PCR strategy, a linear PCR was performed using the intron fragments from the above step as the primer, the resulting products from the second step were served as the template to amplify. Target genes could be amplified efficiently. It concluded that three-step PCR strategy was an ideal method.