DNA SEQUENCING OF MEGABASE SIZED REGIONS OF THE HUMAN GENOME

尽管对人类基因组全序列的阐明是一种 人类基因组计划的长期目标--测序效率 基因组DNA目前还不足以使这一目标成为 实现了跟踪这一原因，目前的两个目标是 HGP将对几个百万碱基大小DNA区域进行测序 生物兴趣和开发更有效的技术来 测序。在项目5中，我们建议确定约 在5年的资助期间，基因组的4个不同区域中有8个Mb 句号。我们计划使用转座子介导的(TM)来实现这一目标 测序策略，因为我们认为低水平的序列冗余和 它提供的序列组装的简单性将使这一过程变得高效 执行巨型数据库测序的方法。我们建议建立一个巨型数据库- 中心的测序组，并将测试 目前的TM测序程序，使用一个特征良好的粘粒克隆作为 一个模范系统。我们建议在这方面进行几项改进 取消一些限速措施的战略。具体来说，我们将 开发一种直接执行转座和测序步骤的方法 并开发一种有效的聚合酶链式反应方法来绘制宇宙粒子的位置图 这些宇宙体中的转座子。然后我们将使用此方法来确定 基因组的4个区域的DNA序列，主要在 根据它们的生物相关性，还因为许多所需的 试剂对我们来说是现成的。这些区域是：2Mb的区域 染色体21q22与唐氏综合征有关；一个富含基因的2Mb片段 性染色体的假常染色体区域；2 Mb区域 染色体4q25-4q26上的EGF基因与肝细胞癌 和Rieger综合征，以及染色体4Q11-4Q12上的2Mb区域 包括一组受体酪氨酸激酶基因。在结束时， 建议的5年资助期，我们不仅将提供生物 的几个感兴趣区域的相关序列信息 基因组，但也将测试的有效性和改进 作为主流猎枪的替代方案的排序策略 排序方法。我们认为，几种这样的方法应该是 试图改进测序技术，以便最终目标是 确定全基因组序列是可以实现的。

Although the elucidation of the entire sequence of the human genome is a long-term goal of the Human Genome Project, the efficiency of sequencing genomic DNA is not currently such that it would allow this goal to be realized keeping track of this reason, two of the current objectives of the HGP are to sequence several megabase-sized regions of DNA with high biological interest and to develop more efficient technologies for sequencing. In Project 5, we propose to determine the sequence of about eight Mb in 4 different regions of the genome during the 5-year funding period. We plan to achieve this goal using the transposon-mediated (TM) sequencing strategy as we believe the low levels of sequence redundancy and the ease of sequence assembly that it offers will make this an efficient way to perform megabase-sequencing. We propose to establish a megabase- sequencing group at the Center and will test the transferability of the current TM-sequencing procedure, using a well-characterized cosmid clone as a model system. We propose to develop several improvements in this strategy to eliminate some rate-limiting steps. Specifically, we will develop a method to perform the transposition and sequencing steps directly in cosmids and to develop an efficient PCR approach to map the positions of transposons in these cosmids. We will then use this method to determine the DNA sequence of 4 regions of the genome, selected primarily on the basis of their biological relevance, but also because many of the required reagents are readily available to us. These regions are: a 2 Mb region of chromosome 21q22 implicated in Down syndrome; a gene-rich 2 Mb segment in the pseudoautosomal region of the sex chromosomes; a 2 Mb region around the EGF gene on chromosome 4q25-4q26, implicated in hepatocellular carcinoma and Rieger's syndrome, and a 2 Mb region on chromosome 4q11-4q12 that includes a cluster of receptor tyrosine kinase genes. At the end of the proposed 5 year funding period, we not only will have provided biologically relevant sequence information for several interesting regions of the genome, but also will have tested the efficacy of and improved upon a sequencing strategy that is an alternative to the mainstream shotgun sequencing approach. We believe several such approaches should be attempted to improve sequencing technologies so that the ultimate goal of determining the sequence of the whole genome can be realized.