We propose a new experimental protocol,ExonPCR,which is able to identify exon boundaries in a cDNA even in the absence of any genomic clones.ExonPCRcan bypass the isolation, characterization, and DNA sequencing of subclones of genomic DNA to determine exon boundaries: a major effort in the process of positional cloning. Given a cDNA sequence,ExonPCRuses 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 a previously unknown number of exons. We subsequently developed theExonPCRalgorithm and software to direct the experimental protocol using a strategy that is analogous to that used in the game Twenty Questions. Through the use ofExonPCR,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.
- Xu, G.; Sze, S. H.; Liu, C. P.; Pevzner, P. A. & Arnheim, N. (1998). Gene hunting without sequencing genomic clones: finding exon boundaries in cDNAs. Genomics, 47(2), 171-179.
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