SBIR Phase II: High throughput screening for splice variant cDNAs

描述（由申请人提供）：人类剪接变异mrna的目录被认为远未完成，许多未发现的亚型可能在健康和疾病中发挥重要作用。目前通过一次筛选一个cDNA文库来寻找剪接变异体的做法既耗时又昂贵。更高的通量和更具成本效益的筛选方法将有助于加快这一重要事业的发展。该项目的目的是创建一个有价值的研究工具，用于快速鉴定备选剪接异构体。本提案中描述的文库采样器代表了高质量cDNA文库的集合，这些文库组织在384个低复杂性池的两个面板中，可用于识别来自广泛的正常组织，肿瘤和癌细胞系的剪接变体，多态性和突变。这项工作的主要优点是允许一种简单的PCR方法有效地筛选由超过900万个cDNA克隆所代表的大量人类来源。由于cDNA池的复杂性较低，PCR产物可以直接测序并用于亚克隆实验，从而使研究人员能够立即得到与感兴趣基因对应的一组mRNA同种异构体的答案。将使用一个全面的真核剪接变体数据库开发一个图形网络界面，以协助科学家进行实验设计，并从选定的基因列表中识别一组新的剪接同种异构体，以证明这一新发现工具的价值。Library Sampler将为研究人员提供一个强大的系统来快速识别单个基因表达的所有变体转录本。对所有mRNA亚型进行编录将是理解人类蛋白质组复杂性的基础，而且由于许多这些变异与遗传疾病有关，它们的发现将为药物开发和分子诊断提供新的潜力。2004年10月，国际人类基因组测序联盟（International Human Genome Sequencing Consortium）将人类蛋白质编码基因的估计数量减少到只有2万到2.5万个，这对人类来说是一个惊人的低数字，这表明基因调控远比基因数量重要。众所周知，我们的大多数基因通过其初级转录物的选择性剪接产生不止一种蛋白质，但所有这些剪接变体形式的鉴定还远未完成。所提出的文库采样器提供了一个强大的研究工具，可以从广泛的正常组织、肿瘤和癌细胞系中快速识别这些剪接异构体，并且由于许多剪接异构体与人类遗传疾病有关，它们的发现将为药物开发和分子诊断提供新的潜力。

DESCRIPTION (provided by applicant): The catalog of human splice variant mRNAs is believed to be far from complete with many undiscovered isoforms likely to play an important role in health and disease. The current practice of hunting for splice variants by screening cDNA libraries one at a time is both time consuming and expensive. Higher throughput and more cost-effective methods of screening would be helpful in accelerating this important enterprise. The aim of this project is to create a valuable research tool for the rapid identification of alternative splice isoforms. The Library Sampler described in this proposal represents a collection of high-quality cDNA libraries organized in two panels of 384 low-complexity pools that can be used to identify splice variants, polymorphisms and mutants from a broad range of normal tissues, tumors and cancer cell lines. The main advantage of this proposed work is to allow a simple PCR approach to efficiently screen a large diversity of human sources represented by over 9 million cDNA clones. Due to the low complexity of the cDNA pools the PCR products can be sequenced directly and used in sub-cloning experiments, giving the researcher an immediate answer on a set of mRNA isoforms corresponding to the gene of interest. A graphical web interface will be developed using a comprehensive eukaryotic splice variant database to assist scientists with their experimental design, and a set of novel splice isoforms from a list of selected genes will be identified to demonstrate the value of this new discovery tool. The Library Sampler will provide the researcher community with a powerful system to quickly identify all variant transcripts expressed from a single gene. Cataloging all the mRNA isoforms will be fundamental for understanding the complexity of the human proteome, and since many of these variants have been associated with genetic diseases, their discovery will offer new potentials for drug development and molecular diagnostics. In October 2004 the International Human Genome Sequencing Consortium reduced the estimated number of human protein-coding genes to only 20,000 to 25,000 genes, a surprisingly low number for our species, suggesting that gene regulation is far more important than gene number. It is well known that most of our genes produce more than one protein by alternative splicing of their primary transcript, but the identification of all these splice variant forms is far from complete. The proposed Library Sampler provides a powerful research tool to rapidly identify these splice isoforms from a broad range of normal tissues, tumors and cancer cell lines, and because many splice isoforms have been associated with human genetic diseases, their discovery will offer new potentials for drug development and molecular diagnostics.