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

描述(申请人摘要)：在本申请中，我们将讨论 开发一种新的化学标记方法的基本原理 使用该帽合成全长mRNA5‘端的寡核苷酸 结构(7-甲基鸟苷三磷酸，m7Gppp)。有两个主要的 构建全长cdna文库的技术限制。第一个是 逆转录酶反应和第二次反应效率降低 限制是不能有效地只选择全长的cdna。这 更复杂的是需要使用RNaseH来为第二个 链合成，这与分离的全长cDNA有偏差。当前 RACE方法(cDNA末端快速扩增)和商业试剂盒分离 全长的DNA在技术上是可描述的，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。这项技术既可以作为5‘-比赛使用 方法或构建全长cDNA文库的方法。通过将一个 核糖核酸通过吗啉核苷残基到5‘端封端 在信使核糖核酸末端，逆转录酶可以读取帽子结构，并且 以所附的核糖核酸为模板，继续合成 该寡核苷酸末端的第一链cDNA.序列的互补序列 核糖核酸连接到cDNA3‘端，呈现一种 用于合成全长双链的第二链合成的已知引发点 链cDNA，这种方法特别有利，因为它不需要 一种RNaseH步骤，用于产生用于第二链合成的引物，其在 它本身就对5‘端识别有偏见。 建议的商业应用： 最终目标将是产生一种有效的方法来专门标记5‘-帽 全长c DNA的结构。这项技术有望取代目前的 技术，这在技术上是困难和低效的。高质量的全长cDNA 将开发文库和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.