关键词: 三步法, 高变异区, PCR

Abstract: 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.