运用重叠延伸PCR技术构建在甘丙肽全长cDNA嵌入第二内含子的重构分子

Construction of Recombinant Molecule of Galanin Full Length cDNA Including the Second Intron by OE-PCR

构建嵌入第二内含子的甘丙肽(Galanin,GAL)全长基因组cDNA的重构分子。通过RT-PCR扩增出cDNA编区的序列,分别从基因组中扩增出cDNA的5′和3′端部分非编码序列;使用重叠延伸PCR(overlap extention PCR,OE-PCR)方法将三个片段重叠获得全长cDNA序列;再将全长cDNA从第三外显子第15个碱基处分成两部分,分开的cDNA前半部分和后半部分以及第二内含子进行重叠延伸获得重构分子,含有第二内含子的甘丙肽(GAL)全长基因组cDNA;将重构分子连入pMDI9-Tsimple载体。电泳分析观察到清晰的重构分子片段;测序显示重构分子由所设计的序列组成,第二内含子插入的位置准确,且无移码。使用重叠延伸PCR能够成功在cDNA中插入内含子获得一段重构基因。

The study was design to construct a recombinant gene composed of galanin（GAL）full-length cDNA and the second intron of the galanin gene.Firstly,by RT-PCR the coding cDNA sequence was amplified,and the 5＇ and 3＇ noncoding sequences of Gal cDNA was amplified by PCR.Secondly,the full-length cDNA sequence was obtained by overlap extention PCR（OE-PCR）.Thirdly,the full-length cDNA was cuted into two part at the 15 bp of the third exon and amplified them respectively.Finally,Using the two part of the full-length cDNA and the second intron as a template by OE-PCR,and the second intron was inserted into the galanin full-length cDNA,and the recombinant molecule was obtained then cloned into pMD19-T simple vector.The electrophoresis analysis illustrated a clear band of recombinant molecule fragment,and the squencing result proved that the recombinant molecule was composed of the second intron and full-length cDNA of galanin.The insertion position of the second intron is accurate without base malposition.Therefore,it proved that the Gal recombinant gene was obtained successfully by OE-PCR.