摘要
分子进化研究中所遇到的一个常见问题是某些基因的目标区域进化较快而难以用特定引物在不同种属间进行有效扩增,而这将影响到整个实验的进程和全部结果的综合分析。虽然巢式和半巢式PCR等能显著提高扩增的特异性,但应用于高变异区的扩增结果仍是杂带多或涂抹严重,不能满足后续实验需要。在果蝇Fak56D基因的研究中需要扩增不同属及黑腹果蝇种组不同种亚组的相关片段,由于该DNA区域变异较显著,常用扩增方法对大部分材料的效果都很不理想。实验创造性组合了一套经济实用的扩增方法,即巢式或半巢式PCR结合定向DNA片段胶回收技术后再扩增的三步扩增法,得到了令人满意的扩增结果,为下一步的克隆、测序等研究创造了必要条件。
A common problem in research of molecular evolution is difficult to efficiently amplify quick evolving target sequence of genes in different species or genus using specific primers, thus making experimental process and final (analysis of) total results delayed. Although using nested or semi-nested PCR can prominently increase PCR specificity, it really cannot efficiently amplify quick evolving region of DNA in our research of gene Fak56D. In this research, we need PCR products corresponding to gene Fak56D of different species of Drosophila melanogaster or other genus of Drosophila. For the high evolutionary rate,most materials did not produce qualified PCR products. To solve this problem, we initially used a combination method of three steps, i.e. semi-nested PCR taken together with orientational gel extraction, which satisfactorily met the demands of next cloning and sequencing steps.
出处
《遗传》
CAS
CSCD
北大核心
2005年第3期442-446,共5页
Hereditas（Beijing)
基金
国家自然科学基金重点项目(编号:39930100)~~
关键词
高变异区
PCR
三步法
quick evolving region
PCR
three steps
