SBIR Phase II: High throughput screening for splice variant cDNAs

描述(申请人提供)：人类剪接变异体mRNAs的目录被认为远远不完整，许多未发现的异构体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文库来寻找剪接变异体的做法既耗时又昂贵。更高的生产能力和更具成本效益的筛查方法将有助于加快这一重要企业的发展。该项目的目的是创建一种有价值的研究工具，用于快速鉴定替代剪接异构体。本建议中描述的文库采样器代表了一组高质量的cDNA文库，这些文库组织在两个由384个低复杂性池组成的小组中，可用于从广泛的正常组织、肿瘤和癌细胞系中识别剪接变体、多态和突变。这项拟议工作的主要优点是允许一种简单的PCR方法有效地筛选由900多万个cDNA克隆代表的大量不同的人类来源。由于cDNA库的低复杂性，聚合酶链式反应产物可以直接测序并用于亚克隆实验，为研究人员提供了一组与感兴趣基因相对应的mRNA亚型的即时答案。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，以帮助科学家进行实验设计，并将从一系列选定的基因中确定一组新的剪接异构体，以展示这一新发现工具的价值。文库采样器将为研究人员群体提供一个强大的系统，以快速识别单个基因表达的所有变异转录本。对所有的mRNA异构体进行分类将是理解人类蛋白质组复杂性的基础，由于这些变异中的许多与遗传疾病有关，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将人类蛋白质编码基因的估计数量减少到只有20,000到25,000个基因，对于我们这个物种来说，这个数字令人惊讶地低，这表明基因调控远远比基因数量更重要。众所周知，我们的大多数基因通过其初级转录本的选择性剪接产生不止一种蛋白质，但对所有这些剪接变体的鉴定还远未完成。拟议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鉴定这些剪接异构体，由于许多剪接异构体与人类遗传病有关，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.