Isolation of Gene Sequences from the BRCA1 Region of Chromosome 17q21 by Exon Amplification

Abstract

A variety of techniques now exist for the identification and isolation of gene sequences from cloned genomic DNA. We report the use of exon amplification to isolate candidate exons of genes in the chromosome 17q21 region associated with familial breast and ovarian cancer. We have used the second generation splicing vector pSPL3, which provides greater flexibility for cloning genomic fragments and which reduces the frequency of the major classes of false positive clones. In two experiments, exon amplification was performed using DNAs of approximately 170 cosmids spanning 1–2 Mb of this region. Cosmid DNAs were pooled in groups of 6–10 each. More than 2000 candidate exon clones from these experiments have been arrayed in microtiter dishes. The average size determined for nearly 400 cloned inserts was approximately 200 base pairs. Ongoing efforts to identify and eliminate clone redundancy have thus far yielded more than 100 unique exon clones. Less than 10% of the clones were found to be repetitive or to be artifacts resulting from cryptic splicing involving sequences present in the splicing vector. Thus the great majority of clones were found to be single copy and to derive from the correct chromosomal location. These exons have been used as hybridization probes to isolate cDNA clones derived from normal breast tissue. The cloned exons and corresponding cDNAs are being localized within developing cosmid contigs in order to assemble a transcription map of the region, and to position transcribed sequences with respect to critical recombinants in breast/ovarian cancer families. While database searches suggest that many of the exon sequences are unique, these searches have also identified several genes which were either mapped previously to proximal 17q or which appear to be homologs of genes in other species. Exon amplification represents a rapid and efficient means for isolating candidate gene sequences from genomic clones, facilitating efforts to identify specific genes associated with disease using positional cloning strategies. Utilization of this technique to survey large genomic regions will also assist in efforts to construct transcription maps of chromosomes.