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Proc. Natl. Acad. Sci. USA
Vol. 94, pp. 10373-10378, September 1997
Medical Sciences

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting

Ivan H. Still, Pauline Vince, and John K. Cowell

Department of Neurosciences NC30, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195

Communicated by George R. Stark, Cleveland Clinic Foundation, Cleveland, OH, July 10, 1997 (received for review May 30, 1997)

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.


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