CAJ | 학술논문

于1997年6月在青岛市沙子口海域采集文昌鱼样品，采用RT-PCR方法进行青岛文昌鱼LIM类同源框基因片段的扩增研究。结果表明，用一对引物一次PCR扩增的方法未能得到特异的PCR产物，用一对引物两次PCR扩增的方法也同样未果。在对套式PCR进行研究时发现，用普遍PCR方法进行的套式PCR结果不够理想，而用三步两段法和四步两段法套式PCR则可进行成功的扩增。用这两种方法扩增的PCR产物具有高度特异性。相比之下，用简并的引物对未知的基因片段进行PCR扩增时，三步两段法和四步两段法的套式PCR应是首选方法。
우1997년6월재청도시사자구해역채집문창어양품，채용RT-PCR방법진행청도문창어LIM류동원광기인편단적확증연구。결과표명，용일대 인물일차PCR확증적방법미능득도특이적PCR산물，용일대인물량차PCR확증적방법야동양미 과。재대투식PCR진행연구시발현，용보편PCR방법진행적투식PCR결과불구이상，이용삼보 량단법화사보량단법투식PCR칙가진행성공적확증。용저량충방법확증적PCR산물구유고도특 이 성。상비지하，용간병적인물대미지적기인편단진행PCR확증시，삼보량단법화사보량단법 적투식PCR응시수선방법。
LIM class homeobox gene is one of the key gene s controlling embryonic development of animal. In order to clone the entire sequ ence of amphioxus LIM class homeobox gene, adult amphioxus was acquired in Shazi k ou sea field in Qingdao City in June 1997, and the amplification methodology of Amphioxus (Branchiostoma belcheri tsingtauense) LIM class homeobox gene frag ment by PCR was studied. The amplification by means of single pair of primers an d single PCR operation failed to produce specific PCR product, and amplification by means of single pair of primers and twice PCR operations failed, too. In the study of nested-PCR, amplification by means of general PCR approach was not sat isfactory, but amplifications by means of three steps-two stages approach and by four steps-two stages method were successful. The sequencing data of PCR produc t acquired by these two methods show a high accordance with genomic DNA sequenci n g data of this gene fragment. Therefore, the PCR products, amplified by these tw o methods, were extremely specific. In comparison with the PCR approaches mentio ned, nested-PCR by means of the three-step and two-stage approach and by means o f the four-step and two-stage approach were the best to amplify unidentified gen e fragments with degenerated primers.