利用GSP逆转录结合巢式PCR技术鉴定剪接变体的新方法及其在小鼠TrkC基因变体发现中的应用  

A new method for screening splice variants using gene-specific primer for reverse transcription followed by nested PCR approaches

作　　者：张艳春[1] 严瑞芬[1] 吴永红[1] 张成岗[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,蛋白质组学国家重点实验室,北京100850

出　　处：《军事医学》2011年第8期582-586,共5页Military Medical Sciences

基　　金：国家科技重大专项课题(2009ZX09503-002,2009ZX09301-002,2009ZX09103-616);国家自然科学基金课题(30900830,30900862);蛋白质组学国家重点实验室自主研究课题(SKLP-K201004);蛋白质组学国家重点实验室开放课题(SKLP-O201002)

摘　　要：目的利用基因特异性引物(gene specific primer,GSP)逆转录结合巢式PCR(nPCR)技术,建立鉴定剪接变体的新方法。方法以小鼠TrkC基因为例,参考其已知变体1和变体2的序列分别设计GSP和nPCR引物,以逆转录产物为模板进行nPCR反应,扩增产物采用1.5%琼脂糖凝胶分离并回收具有递减趋势的DNA片段,利用T/A克隆技术亚克隆到载体pMD19-T进行直接测序,并与小鼠RefSeq数据库进行比对。结果利用GSP逆转录结合nPCR技术不仅可鉴定出TrkC基因的已知剪接变体,而且还能够筛选到TrkC基因的新剪接变体并命名为变体3,与变体1相比属于盒式外显子剪接模式,缺失了变体1的520～2188位共1669 bp。结论利用GSP结合nPCR技术建立了一种鉴定剪接变体的方法,为新剪接变体的挖掘与功能研究以及理解异常剪接相关疾病的机制研究提供了重要参考。Objective To develop a new method for screening splice variants using gene-specific reverse primer （GSP） to specifically reverse transcribe （RT） the target genes followed by nested PCR （nPCR） approaches. Me,otis The reverse primer for transcription and nPCR primers of the known splicing variant No. 1 and variant No. 2 of the mouse TrkC gene were used to obtain the GSP-transcribed cDNA products ,followed by two or three rounds of nPCR. The PCR products were separated and extracted by 1.5% agarase gel electrophoresis and subcloned into the pMD19-T vector for direct sequencing and similarity analysis with the known cDNA sequence of the TrkC gene in the RefSeq database. Results Not only was the known variant （ splicing variant No. 1 ） of the TrkC gene obtained, but the new splicing variant was found. The 1669 bp from 520 to 2188 of the sequence of splicing variant No. 1 of the TrkC gene was spliced in the newly identified variant No. 3, indicating a splicing pattern of cassette exon. Conclusion This method of screening gene splice variants using RT-GSPs and nPCR is a simple and valuable approach to screening novel splice variants, which is important for the function studies of eukaryotic genes and for understanding the mechanism of aberrant splicing-related diseases.

关 键 词：基因特异性引物 巢式PCR TrkC基因 新剪接变体 神经营养因子3 基因组 DNA 互补 

分 类 号：Q78[生物学—分子生物学]