MOLECULAR BIOLOGY PROGRAM1,
DEPARTMENT OF COMPUTER SCIENCE
DEPARTMENT OF MATHEMATICS,
UNIVERSITY OF SOUTHERN CALIFORNIA,
LOS ANGELES, CA 90089-1340.
We propose a new experimental protocol, ExonPCR, which is able to identify exon-intron boundaries in a cDNA even in the absence of any genomic clones. ExonPCR can bypass the isolation, characterization and DNA sequencing of subclones to determine exon-intron boundaries: a major effort in the process of positional cloning. Given a cDNA sequence, ExonPCR uses a series of "adaptive" steps to analyze the PCR products from cDNA and genomic DNA thereby revealing the approximate positions of "hidden" exon boundaries in the cDNA. The nucleotide sequence of adjacent intronic regions is determined by ligation-mediated PCR. Primers adjacent to the "hidden" exon boundaries are used to amplify genomic DNA followed by limited DNA sequencing of the PCR product. The method was successfully tested on the 3 kb hMSH2 cDNA with 16 known exons and the 9 kb PRDII-BF1 cDNA with an unknown number of exons. We subsequently developed the ExonPCR algorithm and software to direct the experimental protocol using a strategy which is analogous to that used in the game "Twenty Questions". Using ExonPCR, the search for disease-causing mutations can be initiated almost immediately after cDNA clones in a genetically mapped region become available. This approach would be most valuable in gene discovery strategies that focus initially on cDNA isolation.