AUTOMATED METHODS FOR SEQUENCING THE HUMAN C-ABL GENE

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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).