CDNA PROBES FOR EXON MAPPING OF THE HUMAN GENOME

We propose to participate in the overall effort that is directed towards the mapping and sequencing of the human genome by providing suitable cDNA probes that can be used for exon mapping. Our aim is to construct developmental and tissue-specific human cDNA libraries using novel vectors (lafmids) that we have constructed. Following cDNA cloning in lafmids, single-stranded versions of the libraries can be obtained that can be used directly for subtractive hybridization. Because of directional cloning, two libraries from the same tissue or developmental stage, but in opposite orientations, can be subtracted to yield an "equimolar" sub-library (i.e. the clones can be adjusted to equal abundances). Following subtraction of equimolar sub-libraries from different tissues or developmental stages, uniquely expressed sequences can be cataloged and sequenced to generate a collection of characterized probes. Such probes can then be used by other investigators, attempting to correlate genetic and physical macro-maps of the human genome, for "functional" exon mapping of the expressed portion of the genome. This approach will identify genomic regions that can then be sequenced selectively.