DETECTION OF DELETION MUTATIONS EXTENDING BEYOND THE HPRT GENE BY MULTIPLEX PCR ANALYSIS

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determi ned. These cloned DNAs were derived from the ends of a set of overlapp ing yeast artificial chromosomes (YA C) defining a contig of 8 Mb at X q26 and including hprt. We used ''bubble'' PCR to isolate an additiona l YAC end-clone. Seven primer pairs were derived from DNA sequence ana lysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upst ream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstre am of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an in ternal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerate d at this hemizygous locus.