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

该建议旨在生成经过验证的非冗余排列的全长cdna集，以便广泛用于全长测序程序。要实现这一目标，就必须执行一项利用互补资源和技术的双管齐下战略。我们提案中的湿实验室和信息学研究部分是完全整合的，它们对于成功实现我们的目标同样重要。简而言之，我们建议将5'端富集的随机引物文库与oligo-[dT]引物全长富集文库结合使用，生成混合选择文库，这些文库将大大富集全长cdna。从这些混合选择的文库中产生的5' EST数据的信息学分析将使cdna分为三组：全长、全编码和不完整。基于这些分析，全长cdna将被识别并重新排列。将产生三组这样的重新排列的cdna。第一个将包括从oligo-[dT]引物的全长富集文库中选择的15000个全长和全编码杂交cdna。第二组和第三组相对于第一组是非冗余的，每个包含7,500个cdna，来自一对oligo-[dT]引物的全长富集和5'端富集的随机引物文库。总之，后两组将允许另外15,000个mrna的全长表示。我们的核心策略是利用相同大小的分离细胞质mRNA构建全长富集和5'端富集文库。这对文库将分别为4个人类mRNA大小片段生成，跨度从2.37到9.4 kb。两种不同的方法将用于构建5'端富集随机引物文库：寡核苷酸capping技术和一种新方法-“末端拯救”-其发展构成本应用程序中提出的工作的一部分。从两种方法生成的文库中获得的5‘ EST数据的信息学分析将有助于确定最佳的5’端富集文库，用于我们提出的双管齐下的策略，以生成阵列的全长cdna集。

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.