选择性剪接变异体的RT-PCR检测及结果分析  

Detection and Analysis of Alternative Splicing by RT-PCR

作　　者：勾文峰[1] 杨雪[1] 徐小燕[1] 王建平[1] 郑华川[1] 

机构地区：[1]中国医科大学基础医学院病理与病理生理研究所,生物化学与分子生物学教研室,沈阳110001

出　　处：《中国医科大学学报》2012年第6期490-493,共4页Journal of China Medical University

基　　金：国家自然科学基金资助项目(81172371;81001093)

摘　　要：目的利用RT-PCR方法检测并分析mRNA选择性剪接变异体。方法针对糖原合成酶激酶(GSK)3β和B细胞转位基因(BTG)3基因变异体差别设计引物,利用RT-PCR扩增后进行电泳和克隆DNA测序。结果 GSK3β和BTG3的RT-PCR产物电泳均出现3条带,经过电泳和克隆DNA测序发现GSK-3β中上面2条带为同一产物。对BTG3上(位于319 bp)、下(位于70 bp)2条带进一步PCR扩增后测序,结果发现3个条带均为同一产物。结论 RT-PCR法检测选择性剪接产物操作方便,对Western蛋白印迹结果评价具有指导意义,但是产生的非特异条带可能是由于某种特殊原因造成,如选择性剪接部位的特异核酸序列,需DNA测序做进一步确定。Objective To detect and analyze the alternative splicing of mRNA by RT-PCR. Methods The specific primers were designed targeting the differential DNA sequence of glycogen synthase kinase （GSK） 313 and B-cell translocation gene （BTG） 3 variants,amplified by RT-PCR and analyzed by electrophoresis and DNA cloning sequencing. Results Three bands appeared during the electrophoresis of GSK3β and BTG3 RT-PCR products respectively. On two top bands of GSK3β, the same product was found by electmphoresis and DNA cloning sequencing. On the ttuee bands of BTG3,the same produce was found after the top （319 bp） and lower （70 bp） bands of BTG3 were amplified by RT-PCR. Conclusion RT-PCR is a simple method to detect the alternative splicing, which is helpful for the evaluation of Western blot results. However,the non-specific bands of PCR might result from the specific nucleic acid sequence of alternative splicing and should be confirmed by DNA sequencing.

关 键 词：选择性剪接 RT-PCR 

分 类 号：Q354[生物学—遗传学]