AUTOMATED METHODS FOR SEQUENCING THE HUMAN C-ABL GENE

提出的研究的主要目标是继续我们正在进行的 努力开发新的和创新的DNA测序方法 这将导致更准确和更具成本效益的方法 大型DNA测序项目。在这项研究中，我们将开发， 改进和实施一系列DNA自动化程序 分离、DNA序列分析和数据采集。有了这些 技术，我们建议准确而快速地确定 大约50万个碱基的完整核苷酸序列 人类9号染色体的配对区域，包含人类c-abl基因 原癌基因。 建议的研究的具体目标是 以下是： 1.将我们为圣人开发的新协议自动化 双脱氧核苷酸DNA测序方法，从而获得更多 超过1000个核苷酸的准确、可重现的序列数据 从一个引爆点。 2.使单链M13的分离程序自动化 Sanger双脱氧核苷酸DNA所需的嵌合模板 测序，从而获得更均匀的DNA样本 测序。 3.开发新的算法，计算机程序 实现这些算法、分离技术和 扩展能力和能力所需的检测方法 自动荧光DNA测序仪的准确性。 4.开发必要的分离技术，以准确 确定超过1000个核苷酸的序列数据 单一引发点，从而提高了寡核苷酸的拆分率 荧光标记和放射性标记DNA测序凝胶的研究 以产生可以通过自动化更准确地分析的数据 荧光和放射自显影凝胶读数仪器。 在可预见的未来，发展出的途径和方法 在这个项目期间应该使我们不仅能够完成 人c-abl原癌基因的完整核苷酸序列，但 也是为了准确、快速地确定整个核苷酸 9号染色体远端的序列。通过直接定义 这个人类基因组区域的序列与慢性疾病有关 髓系白血病(CML)，我们可以更清楚地勾勒出基因 在9号染色体上，与远端相互易位 22号染色体末端产生异常的费城染色体 (PH1)。

The major goal of the research proposed is to continue our ongoing efforts to develop new and innovative approaches to DNA sequencing which will result in more accurate and cost effective methods for large DNA sequencing projects. In this research, we will develop, improve and implement a series of automated procedures for DNA isolation, DNA sequence analysis, and data acquisition. With these techniques, we propose to accurately and rapidly determine the complete nucleotide sequence of the approximately half-million base paired region of human chromosome 9 which contains the human c-abl proto-oncogene. The specific aims of the research proposed are the following: 1. To automate our newly developed protocol for the Sanger dideoxynucleotide DNA sequencing method and thereby obtain more accurate, reproducible sequence data in excess of 1000 nucleotides from a priming site. 2. To automate the isolation procedure for the single-stranded M13 chimeric templates necessary for Sanger dideoxynucleotide DNA sequencing, and thereby obtain more uniform DNA samples for sequencing. 3. To develop the new algorithms, the computer programs implementing these algorithms, the separation techniques, and the detection methods necessary for expanding both the capability and accuracy of automated fluorescent DNA sequencing instruments. 4. To develop the separation techniques necessary for accurately determining sequence data in excess of 1000 nucleotides from a single priming site, thereby improving oligonucleotide resolution on both fluorescent-labeled and radio-labeled DNA sequencing gels to yield data which can be more accurately analyzed via automated fluorescent and autoradiographic gel reading instruments. In the foreseeable future, the approaches and methods developed during this project should enable us not only to complete the entire nucleotide sequence of the human c-abl proto-oncogene but also to accurately and rapidly determine the entire nucleotide sequence of the distal end of chromosome 9. By directly defining the sequence of this human genomic region responsible for chronic myelogenous leukemia (CML), we can more clearly delineate the genes on chromosome 9 which are reciprocally translocated with the distal end of chromosome 22 to yield the abnormal Philadelphia chromosome (Ph1).