基于模板切换的mRNA5'末端全长扩增方法

本文介绍一种称为CapFinder的技术，可用于克隆基因mRNA序列的5'末端非翻译区全长。该技术是利用某些反转录酶在反转录达到mRNA的5'末端帽结构时表现出很高的加尾活性(主要添加dC)这一特点，在反转录体系中加入一种带GGG的寡核苷酸序列，当反转录反应到达mRNA模板的5'末端帽结构时，切换到以该寡核酸为模板继续进行反转录反应，即可合成完整的cDNA一链，且在其3'末端还带有一段额外的寡核苷酸序列。用GGG寡核苷酸序列为上游引物和基因特异性的下游引物进行PCR即可扩增得到mRNA5'末端非翻译区的全长。利用该技术克隆了棉铃虫幼虫中肠Bt毒素受体E-钙粘素基因的5'末端非翻译区序列。

Amplification of full-length mRNA 5'ends based on template-switching effects

"CapFinder" technology, which can be used to clone the full length of 5' UTR sequence of mRNA, was described. This technology used the terminal transferase activity of certain MMLV RT variants that added 3-5 residues (predominantly dC) to the 3'end of the first-strand cDNA exhibited when MMLV RT reached the 5'cap structure of mRNA. In the reverse reaction system containing GGG oligo, the terminal transferase activity was harnessed by the GGG oligo whose terminal stretch of dG residues can anneal to the dC-rich cDNA tail and serve as an extended template for RT. After RT switch templates from the mRNA template to the GGG oligo, a complete cDNA copy of the original RNA was synthesized with the additional GGG oligo sequences at the end. 5'UTR of mRNA can be amplified with GGG oligo as forward primer and a gene-specific reverse primer. 5'UTR of Bt toxin receptor E-Cadherin gene in midgut of cotton bollworm was cloned.