ISOLATION OF CHROMOSOME-SPECIFIC ESTS BY MICRODISSECTION-MEDIATED CDNA CAPTURE

Despite dramatic advances in the identification of human expressed seq uence tags (ESTs), techniques that facilitate isolation of chromosome or chromosome band-specific ESTs would be of considerable value. This report demonstrates the feasibility of identifying chromosome-specific ESTs following microdissection of a single-copy chromosome region. Fo r this study, a reduced complexity cDNA library was linkered and chrom osome 6 (6q) was microdissected. Following PCR amplification using lin ker-specific primers, captured cDNAs were subcloned and 187 individual clones picked at random. These 187 clones were then sorted by filter cross-hybridization into 34 unique groups. Of these 34 groups, 19 (56% ) mapped to chromosome 6 by Southern blot. We identified three previou sly known genes, human cytovillin (ezrin) mapped previously to 6q25-26 , human cardiac gap junction protein (connexin 43) mapped previously t o 6q21-23.2 and prolyloligopeptidase, which had not been mapped previo usly. BLASTN identified three clone groups with homology to known ESTs and 12 representing novel cDNA sequences. Six of the groups were subl ocalized to specific band regions of 6q using a chromosome 6 hybrid ma pping panel, five representative clones were tested on Northern analys is to verify their expression, and Finally, nine clones were mapped ag ainst the Gene bridge 4 reduction hybrid panel to confirm their geneti c map location on 6q. These results demonstrate that microdissection o f single-copy sequences has sufficient specificity for isolation of ch romosome-specific cDNAs.