应用套式PCR克隆ST13基因cDNA的5'端序列

Cloning the 5end fragment of ST13 cDNA by nested PCR

目的获得ＳＴ１３基因ｃＤＮＡ５′端的未知序列。方法应用套式ＰＣＲ方法直接从ｃＤＮＡ文库中扩增该基因的５′端序列，然后将扩增的片段克隆到ｐＧＥＭ－Ｔ．ｅａｓｙ载体，并进行序列分析。结果第１次ＰＣＲ后得到１条约５５０ｂｐ的片段，经套式ＰＣＲ后获得１条约４８０ｂｐ的片段，经序列分析证实该扩增的片段是ＳＴ１３基因ｃＤＮＡ的５′端未知序列。结论套式ＰＣＲ技术是克隆基因ｃＤＮＡ的５′端未知序列较简捷、有效的方法。

Objective To clone and sequence the 5end fragment of ST13 cDNA. Methods The 5end fragment of the ST13 cDNA was amplified directly from cDNA library by nested PCR method, and cloned into pGEMT.easy vector, then the sequencing of the inserted PCR product was performed. Results After primary and secondary PCR, two PCR products obtained with the size of about 550bp and 480bp, respectively, were both proved to be the 5end of ST13 cDNA by sequencing. Conclusion The method presented is very simple and effective for cloning and sequencing the 5end of a target gene.