Novel 5'-end tagging of mRNA for cDNA synthesis or RACE

描述（申请人的摘要）：在本申请中，我们将讨论 一种新的化学标记方法的开发原理 使用帽将合成的寡核苷酸连接到全长mRNA的5 '末端 结构（7-甲基鸟苷三磷酸，m7 Gppp）。有两大 全长cDNA文库构建的技术限制。一是 逆转录酶反应的效率降低， 限制是不能有效地仅选择全长cDNA。这 由于需要使用RNase H来产生RNA引物， 链合成，其偏向于分离全长cDNA。电流 RACE方法（cDNA末端快速扩增）和商业试剂盒分离 全长cDNA在技术上是困难的，充其量是可变的。在这 我们已经设计了一个程序来解决一些问题 这是当前协议所固有的。该技术可用作5 '-RACE 方法或全长cDNA文库构建方法。通过附加 核糖-寡核苷酸通过吗啉代-核苷残基至5 '-加帽的 在mRNA的末端，逆转录酶可以读取帽结构，并且 使用连接的核糖-寡核苷酸作为模板，继续合成 将第一链cDNA连接到该寡核苷酸的末端。的互补序列的连接 核糖寡核苷酸连接到cDNA的3 '末端，呈现一个 用于第二链合成以产生全长双链已知引发位点 该方法特别有利，因为它不需要 - RNase H步骤，以产生用于第二链合成的引物， 它本身对5 '端识别有偏见。 拟定商业应用： 最终的目标将是产生一种有效的方法，专门标记5 '-帽 全长cDNA的结构。 这项技术有望取代目前的 这些技术在技术上很困难，效率低下。高质量全长cDNA 文库和5 '-EST文库。 这些库将导致 鉴定新的5' -非翻译区和更长和更稀有的基因。

DESCRIPTION (Applicant's abstract): In this application we will discuss the rationale for the development of a novel method for chemically tagging a synthetic oligonucleotide to the 5'-end of full-length mRNAs using the cap structure (7-methyl guanosine triphosphate, m7Gppp). There are two major technical limitations in full-length cDNA library construction. The first is reduced efficiency of the reverse transcriptase reaction and the second limitation is the inability to efficiently select only full-length cDNA. This is compounded by the need to use RNase H to generate RNA primers for second strand synthesis, which biases against the isolation full-length cDNAs. Current RACE methods (Rapid Amplification of cDNA ends) and commercial kits to isolate full-length cDNAs are technically dificule whith variable at best. In this proposal we have designed a procedure which will address some of the problems inherent with current protocols. The technique can be used as either a 5'-RACE method or a full-length cDNA library construction method. By attaching a ribo-oligonucleotide through the morpholino-nucleoside residue to the 5'-capped end of mRNA, the reverse transcriptase can read through the cap structure, and using the attached ribo-oligonucleotide as a template, continuing systhesis of first strand cDNA to the end of this oligo. The complementary sequence to the ribooligonucleotide becomes attached to the 3'-end of the cDNA, presenting a known priming site for second strand synthesis to generate full-length double strand cDNA, This method is especially advantageous in that it does not require an RNase H step to generate primers for second strand synthesis, which in itself biases against 5'-end identification. PROPOSED COMMERCIAL APPLICATION: The ultimate goal will be to generate an efficient method for specifically tagging the 5'-cap structure of full-length cDNA. This technology will hopefully supercede the current technologies, which are technically difficult and inefficient. Quality full-length cDNA libraries and 5'-EST libraries will be developed. These libraries will result in the identification of novel 5' -untranslated regions and longer and rarer genes.