International Mammalian Genome Society

D15 Determination of Exon/Intron Boundaries by Direct Sequencing of BACs with Internal Primers

Lijun Feng, Edward, K. Novak, and Richard T. Swank. Department of Molecular and Cell Biology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263

Large insert genomic DNA clones like BACs, PACs or YACs are widely used in genetic studies. Direct sequencing of BACs/PACs has become routine. Most labs do end sequencing of BACs/PACs with vector primers to find STSs for contig building. Here we describe a novel application of BAC sequencing with internal primers to define exon/intron boundaries and the genomic structure of genes. One BAC that harbors part of the mouse Ap3b1 gene was sequenced with primers from b3A cDNA and several intron/exon boundaries were defined. From the intron sequences adjacent to each exon, primers were designed to amplify whole exons. The 3' UTR sequence was obtained and the genomic sequence beyond 3' UTR as well. Our data suggests novel application of BAC sequencing in many areas, like exon/intron determination, obtaining 3' or 5' UTR sequences and promoter sequences, thus generating primers for mutation analysis.