AUTOMATED METHODS FOR SEQUENCING THE HUMAN C-ABL GENE

我们研究的主要目标是继续开发新的， DNA测序的创新方法，这将导致更多的 准确、快速和经济的大规模DNA测序方法 项目，并通过系统地排序来实施这些方法 人类22号染色体。 在过去的两年里，我们修改了 测序反应，开发了改进嵌套片段的方法， 在ABI 373 A上设置分辨率，并编写了一系列DNA分析 我们现在可以从一个基因组中读取超过750个核苷酸， 从一张血管造影图中找到一个致敏部位。 通过这些 方法，正如这里所描述的，在我们最近的手稿，我们将有 获得了几个人c-abl粘粒克隆的大部分序列 在第一个资助期结束前由我们的合作者提供。 在即将到来的赠款期间，我们的具体目标是 1.制定、改进和实施DNA自动化程序 分离、DNA序列分析、数据采集和数据分析 在上一个资助期开始的项目， 2.为了完成大约250 kb的整个核苷酸序列， 来自人9号染色体人c-abl原癌基因， 3.为了对22号染色体上约100 kb的人BCR基因进行测序， 目前也在进行中， 4.为了对P1克隆进行测序， 来自人类22号染色体的感兴趣区域。 因为相关的人类个体染色体图谱绘制项目仍在继续 为了生成所选染色体的更详细的描述， 显然，更多的实验室必须更多地参与长期的研究， 长期测序项目侧重于收集额外的染色体水平 序列数据 我们最初的目标是生产超过100万台 独特的22号染色体特异性碱基序列/年份/荧光 测序仪（3倍覆盖率）通过实施我们改进的策略， 用于在目前可获得的商业上进行DNA序列分析的方法 DNA测序仪 在5年的拟议资助期内，我们的目标是 引入测序化学、硬件和软件的改进 至少能让我们的生产力提高三倍 在此期间，我们 将继续培养下一代科学家， 发展新的测序方法所需的理论和方法， 解释数据，从而继续为整体 测序整个人类基因组的目标。 与附加 设备，训练有素的人员和技术建议，我们将序列 人类22号染色体的近一半在5年内， 了解选定的22号染色体特异性 基因组区域和发现其他未知的 人类未知区域的结构/功能关系 基因组

The major goal of our research is to continue to develop new and innovative approaches to DNA sequencing, which will result in more accurate, rapid and cost effective methods for large DNA sequencing projects, and to implement these methods by systematically sequencing human chromosome 22. During the past two years we have modified the sequencing reactions, developed methods to improve the nested fragment set resolution on the ABI 373A, and written a series of DNA analysis programs with which we now can read in excess of 750 nucleotides from a single priming site from a single electrophoretogram. Through these methods, as described here and in our recent manuscripts, we will have obtained most of the sequence of several human c-abl cosmid clones supplied by our collaborators by the end of this first funding period. In the upcoming grant period our Specific Aims are as 1. To develop, improve and implement the automated procedures for DNA isolation, DNA sequence analysis, data acquisition, and data analysis that were begun during the previous funding period, 2. To complete the entire nucleotide sequence of the approximately 250 kb human c-abl proto-oncogene from human chromosome 9, 3. To sequence the approximately 100 kb human BCR gene on chromosome 22, also presently underway, and 4. To sequence P1 clones representing mapped and partially characterized regions of interest from human chromosome 22. Because the related individual human chromosome mapping projects continue to generate more detailed descriptions of selected chromosomes, it is apparent that additional laboratories must become more involved in long term sequencing projects focused on gathering additional chromosome-level sequence data. Our initial goal is to produce in excess of 1 million unique chromosome 22-specific bases of sequence/year/fluorescent sequencer (3-fold coverage) by implementing our improved strategies and methods for DNA sequence analysis on the presently available commercial DNA sequencers. Over the 5 year proposed funding period, our goal is to introduce improvements to the sequencing chemistry, hardware and software that will at least triple our productivity. Throughout this period we will continue to train the next generation of scientists in the basic theories and methods needed to evolve new approaches to sequencing and to interpreting the data, thereby continuing to contribute to the overall goal of sequencing the entire human genome. With the additional equipment, trained personnel and techniques proposed, we will sequence almost half of human chromosome 22 within 5 years within the context of understanding the biological relevance of selected chromosome 22-specific genomic regions and discovering additional as yet unknown structure/function relationships in uncharted regions of the human genome.