The goal of the international human genome effort is to identify all of the genes encoded by the genome and map them to specific regions of individual chromosomes. The advent of efficient methods to construct physical maps of the genome and the successful completion of physical maps for chromosomes 21 and Y underscore the need to accelerate efforts to isolate and map coding sequences. In this project of the program, we propose to isolate cDNAs corresponding to genes encoded by chromosome 12. The first step of this program is to construct a highly representative, short fragment cDNA library which is normalized so that each member of the library is represented equally. Highly purified poly(A)+ RNA from a large number of adult and developing human tissues will be used as substrates for preparation of short fragment cDNA libraries using random oligomers as primers. Individual size selected libraries will be cloned into phage vectors and each of the libraries will be normalized by hybridization procedures which we have developed. Different libraries will be mixed and renormalized to generate a comprehensive normalized library. We expect this library to represent at least 75-80% of the genes encoded by the human genome. DNA from individual or small groups of non-chimeric YACs which have been physically mapped in Projects 1 and 2 will be immobilized on nylon membranes and hybridized with PCR amplified short fragment cDNA library. The cDNAs which hybridize to the YACs will be isolated by PCR and cloned. This procedure is expected to enable us to isolate cDNAs corresponding to one megabase of chromosome 12 DNA at a time. Several members of each of these cDNA packets will be sequenced by the cycle sequencing procedure to examine the quality and integrity of the cDNA packets isolated. In addition, these sequences can yield a low resolution expressed sequence tag (EST) map of chromosome 12. The availability of cDNA packets corresponding to specific, highly defined regions of the chromosome would be a starting point to isolate individual genes from those regions or to initiate a larger scale sequencing effort to define all of the genes encoded by chromosome 12.
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