Characterization of the human and other animal genomes requires the ability to establish the functional significance of DNA sequences being cloned and organized during the intensive genetic and physical mapping studies which are ongoing. One approach is to identify the structural genes contained within this complex collection of information. The development of exon amplification allows for rapid and efficient identification and cloning of coding sequences from complex sources of mammalian genomic DNA. These sequences are isolated by virtue of selection for both functional 5' and 3' splice sites in the genomic sequence being analyzed. The experimental approach outlined in this proposal is aimed at a complete characterization of the exon amplification method. Preliminary results indicate that exon amplification will facilitate rapid isolation of coding sequences from complex genomic DNA sources such as cosmid (35-40 kbp) clones. To establish the overall efficacy of this system, the upper limit of genomic DNA complexity which can be scanned for exons in a single assay, and the frequency of occurrence and nature of any false positives which occur, will be determined. Also, whether all possible coding exons are identifiable in a given genomic fragment using exon amplification, will be determined. Modifications to increase the efficacy of the method, and eliminate any identified false positives, will also be tested. These include: modifications to pSPL1, the in vivo splicing plasmid used in exon amplification; and modifications to aspects of the exon amplification protocol. The efficacy of these modifications will be compared to that of the original approach. From this, an optimized method will be established and used for the generation of exon libraries, which will be extremely useful for identifying all coding sequences in the genomic region being analyzed, due to the relatively equal representation of multi-exon genes recovered. These libraries should enhance fine genome mapping strategies and facilitate efficient identification of genes which are targets for genetic disease.
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