限制性内切酶介导的重叠延伸在人环氧合酶-1(COX-1)基因克隆中的应用

Application of Overlap Extension Mediated by Restriction Endonuclease in Cloning of Human Cyclooxygenase-1 (COX-1) Gene

建立一种用于克隆全长基因的、限制性内切酶介导的重叠延伸法 .对全长基因进行分段扩增 ,并利用适当的限制性内切酶对基因序列内相应的限制性位点进行酶切 ,从而使分段扩增片段得以重叠并互为模板 ,在DNA聚合酶的作用下延伸获得全长基因 .将环氧合酶 1 (COX 1 )基因的外显子 9巧妙地拼接到了缺失外显子 9的COX 1cDNA片段中 ,获得了COX 1基因的全长cDNA .该方法分 3步进行 .首先 ,通过RT PCR分别扩增跨外显子 9的cDNA片段和缺失外显子 9的cDNA片段 ,并克隆到pMD1 8 T载体上 ;其次 ,PCR扩增外显子 9片段 ,限制性内切酶StuI酶切缺失外显子9cDNA片段的重组质粒 ,二者以一定的比例混合 ,互为模板 ,在pfuDNA聚合酶的作用下进行延伸 ,从而产生一个双链的DNA分子 .最后 ,以延伸产物为模板 ,用COX 1cDNA两端的引物进行PCR扩增 ,产生包含外显子 9的COX 1基因的全长cDNA .这种限制性内切酶介导的重叠延伸方法 ,对于克隆mRNA剪接水平上受调控的基因尤为有用 。

The overlap extension mediated by restriction endonuclease to obtain the full length gene was established. Two partial fragments were isolated separately. A proper restriction site was cleavaged by the corresponding restriction endonuclease, and the two fragments were overlapped, used as template for each other and extended into a full length gene under catalysis of DNA polymerase. The exon 9 of cyclooxygenase 1 (COX 1) gene was inserted into COX 1 cDNA fragment without exon 9, and the full length cDNA of COX 1 gene was obtained. The method includes three steps. Firstly, the partial COX 1 cDNA fragment containing exon 9 and COX 1 cDNA without exon 9 were isolated separately by RT PCR, and cloned into pMD18 T vector. Secondly, the partial COX 1 cDNA containing exon 9 was amplified by PCR, and was mixed with combinative plasmid without exon 9 cleaved by Stu Ⅰ. The two fragments used as template for each other, and overlap extended into a double strand DNA molecule under catalysis of pfu DNA polymerase. Finally, the full length cDNA of COX 1 gene containing exon 9 was obtained by PCR using the terminal primer of COX 1 cDNA. The overlap extension mediated by restriction endonuclease was especially applied for cloning genes regulated on mRNA splicing level,and also expected to be a new method for gene recombination and modification.