AUTOMATED METHODS FOR SEQUENCING THE HUMAN C-ABL GENE

点击翻译按钮获取中文摘要

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.