NEW METHODS FOR PRODUCING CHROMOSOME SPECIFIC LIBRARIES

此次续签申请的重点是两个问题，即 是在人类基因组中寻找基因的核心。其中的第一个 关系到技术发展的精细化，以提高 CDNAa的选择和扩展。全长cDNA是 疾病基因定位克隆的宝贵工具，用于 注释基因组DNA序列和功能基因组学。 然而，目前还不存在从cDNA转移的快速路线 片段为全长的cDNA.我们建议发展 增强以增加直接选择的cDNA和 以使cDNA能够快速转换为全长。直接 CDNA选择已经成功地应用于整个人类 染色体。我们现在建议将这些技术应用于 使用整个人类基因组获得标准化的cDNA。这些 各种类型的cDNA文库将补充和加强现有的 归一化的cDNA源。第二个问题是，这 应用地址是开发和绘制新的 表达序列标签(EST)。人类基因目录 还远远没有完成，基因预测算法基于 基因组DNA序列并不完全可靠。我们建议 构建人类5号染色体上2,000个基因的详细图谱 它偏向于不在EST中的序列 数据库。这将通过我们开发的技术来促进 这是其他方法所缺少的丰富的cDNA。在我们的最终目标中 我们建议对这种方法进行有限的扩展，以派生出新的 染色体特异的cDNA文库和新的EST图谱 人类3号和8号染色体。

This renewal application is focused upon two issues that are central to finding genes in the human genome. The first of these relates to the development of technology refinements to improve the selection and extension of cDNAa. Full length cDNAs are valuable tools in the positional cloning of disease genes, for annotating genomic DNA sequences and for functional genomics. However, no quick routes currently exist for moving from a cDNA fragment to a full length cDNA. We propose developing enhancements to increase the length of direct selected cDNAs and to enable the rapid conversion of cDNAs to full length. Direct cDNAs selection has been successfully applied to entire human chromosomes. We now propose applying these techniques to derive normalized cDNAs using the whole human genome. These types of cDNA libraries would complement and enhance existing normalized cDNA resources. The second issue that this application addresses is the development and mapping of new Expressed Sequence Tags (ESTs). The catalogue of human genes is far from complete, and gene prediction algorithms based upon genomic DNA sequences are not fully reliable. We propose to build a detailed map of 2,000 genes on human chromosomes 5 which is biased towards sequences that are not in the EST database. This will be facilitated by techniques we have developed that enrich for cDNAs which other methods miss. In our final aim we propose a limited scale up of this approach to derive new chromosome-specific cDNA libraries and novel EST maps for human chromosomes 3 and 8.