The DNA sequence analysis of 2.5 megabases of the human Xq28 chromosomal region will be performed. The Xq28 band spans approximately eight megabases of the long arm of the X chromosome and is rich in expressed sequences. Xq28 has at least 25 loci associated with inherited disease, making this one of the most 'disease rich' regions of the human genome. Some of the disorders are devastating and of high frequency, but most Xq28 disease genes have not yet been molecularly cloned. Genomic DNA sequence analysis is a possible strategy for the gene isolation, with the advantages of definitive characterization of the region and the generation of enormous amounts of data describing human genome architecture. In our hands fluorescent automated DNA sequencing methods are proven suitable for a project of several megabases. Overlapping clones spanning proximal markers within Xq28 will therefore be sequenced by a random/shotgun strategy using automated fluorescent methods. Several aspects of the sequencing process will be refined and developed within the project. Shotgun library construction, DNA image analysis in agarose gels, robot protocols, forward/reverse sequence strategies and sequence data management tools will each be improved and integrated into a streamlined sequencing process. Locally developed computer programs will be tested. A detailed cost analysis will be carried out in order to guide future 'genome scale' sequencing efforts. A study of sequence errors and natural variation in the sequenced region will be carried out. The finished sequence will be analyzed by computational and experimental methods to detect expressed sequence candidates. Final identification of the disease gene candidates will be carried out by collaborators or other members of the scientific community. Selected cDNA and murine genomic clones will also be sequenced. Data will be released from the sequencing laboratory at a maximum of six weeks from the initiation of the data analysis.
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