| 摘要: |
| 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。 |
| 关键词: 文昌鱼 LIM类同源框基因 逆转录-聚合酶链式反应 |
| DOI： |
| 分类号: |
| 基金项目:国家海洋“863”课题资助项目，863-819-01-03号 |
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| AMPLIFICATION OF LIM CLASS HOMEOBOX GENE FRAGMENT OF AMPHIOXUS BY REVERSE TRANSCRIPTION-PRC |
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WANG Yong1, LANG Gang-Hua2, LIU Zong-Zhu1, ZHANG Pei-Jun1
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1.Experimental Marine Biology Laboratory, Institute of Oceanology, The Chinese Academy of Sc iences, Qingdao, 266071;2.College of Marine Life Sciences, Ocean University of Qingdao, Qingdao 266003
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| Abstract: |
| LIM class homeobox gene is one of the key genes controlling embryonic development of animal. In order to clone the entire sequence of amphioxus LIM class homeobox gene, adult amphioxus was acquired in Shazikou sea field in Qingdao City in June 1997, and the amplification methodology of Amphioxus (Branchiostoma belcheri tsingtauense) LIM class homeobox gene fragment by PCR was studied. The amplification by means of single pair of primers and 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 satisfactory, but amplification by means of three steps-two stages approach and by four steps-two stages method were successful. The sequencing data of PCR product acquired by these two methods show a high accordance with genomic DNA sequencing data of this gene fragment. Therefore, the PCR products, amplified by these two methods, were extremely specific. In comparison with the PCR approaches mentioned, nested-PCR by means of the three-step and two-stage approach and by means of the four-step and two-stage approach were the best to amplify unidentified gene fragments with degenerated primers. |
| Key words: Amphioxus, LIM class homeobox, RT-PCR |
