A TWO-PRONGED STRATEGY TO GENERATE FULL-LENGTH CDNA'S

生成全长 CDNA 的双管齐下策略

• 批准号: 6377589
• 负责人: Marcelo Bento Soares
• 金额: $ 66.51万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377589
• 关键词: DNA primers; animal genetic material tag; animal tissue; bioengineering /biomedical engineering; complementary DNA; genetic library; human genetic material tag; human tissue; messenger RNA; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; polymerase chain reaction; technology /technique development

This proposal is aimed at the generation of verified non-redundant arrayed sets of full-length cDNAs to be made widely available for full-length sequencing programs. The achievement of this goal relies on the implementation of a two-pronged strategy that takes advantage of complementary resources and technologies. The wet laboratory and informatics research components of our proposal are totally integrated and they are both equally important for the successful achievement of our goals. Briefly, we propose to use 5' end-enriched random primed libraries in conjunction with oligo-[dT] primed full-length- enriched libraries to generate hybrid-selected libraries that will be greatly enriched for full-length cDNAs. Informatics analysis of 5' EST data generated from these hybrid-selected libraries will enable classification of cDNAs into three groups: full-length, full-coding and incomplete. Based on these analyses, full-length cDNAs will be identified and re-arrayed. Three such re-arrayed sets of cDNAs will be produced. The first will comprise a collection of 15,000 full-length and full-coding hybrid selected cDNAs from the oligo-[dT] primed full-length enriched libraries. The second and third sets, which will be non-redundant respective to the first set, will each contain 7,500 cDNAs, derived from a pair of oligo-[dT] primed full-length enriched and 5' end-enriched random primed libraries. Together, the latter two sets will allow for full-length representation of an additional 15,000 mRNAs. Central to our strategy is the utilization of the same size fractionated cytoplasmic mRNA for construction of both full-length-enriched and 5' end-enriched libraries. This pair of libraries will be generated for each of four human mRNA size fractions, spanning from 2.37 to 9.4 kb. Two different approaches will be used for construction of 5' end-enriched random primed libraries: the oligo-capping technology, and a novel method - "end rescuing" - whose development constitutes part of the work proposed in this application. Informatics analysis of 5' EST data obtained from libraries generated by both methods will be instrumental for identification of the best 5' end-enriched libraries to be selected for use in our proposed two-pronged strategy for generation of arrayed sets of full-length cDNAs.

该建议旨在产生经验证的非冗余的全长cDNA阵列集，以广泛用于全长测序程序。 要实现这一目标，就必须执行一项双管齐下的战略，利用互补的资源和技术。 我们建议的湿实验室和信息学研究部分是完全集成的，它们对于成功实现我们的目标同样重要。 简而言之，我们建议使用5'端富集的随机引物文库与寡聚[dT]引物全长富集文库结合，以产生杂交选择文库，其将大大富集全长cDNA。 从这些杂交选择文库中产生的5' EST数据的信息学分析将使cDNA分成三组：全长、全编码和不完整。 基于这些分析，将鉴定全长cDNA并重新排列。将产生三组这样的重新排列的cDNA。 第一个将包括来自寡聚-[dT]引发的全长富集文库的15，000个全长和全编码杂合选择的cDNA的集合。 第二组和第三组相对于第一组是非冗余的，将各自含有7，500个cDNA，其衍生自一对寡聚-[dT]引发的全长富集和5'末端富集的随机引发文库。 后两个集合一起将允许另外15，000个mRNA的全长表示。 我们策略的核心是利用相同大小的分级细胞质mRNA来构建全长富集和5'末端富集文库。 这对文库将针对跨越2.37至9.4 kb的四种人mRNA大小级分中的每一种生成。两种不同的方法将用于构建5'末端富集的随机引发库：寡聚帽技术和一种新方法--“末端拯救”--其开发构成本申请中提出的工作的一部分。 从这两种方法产生的文库中获得的5' EST数据的信息学分析将有助于鉴定最好的5'末端富集文库，以用于我们提出的用于产生全长cDNA阵列集的双管齐下的策略。