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Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis

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Somatic Cell and Molecular Genetics

Abstract

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 thehprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used “bubble” PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal 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 tolerated at this hemizygous locus.

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Author notes

  1. James C. Fuscoe

    Present address: Integrated Laboratory Systems, P.O. Box 13501, 27709, Research Triangle Park, North Carolina

  2. Anita Jeffers Nelsen

    Present address: MacroNex, Inc., 120, Southecenter Ct., 27560, Morrisville, North Carolina

Authors and Affiliations

  1. Environmental Health Research and Testing, Inc., Research Triangle Park, North Carolina

    James C. Fuscoe & Anita Jeffers Nelsen

  2. Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri

    Giuseppe Pilia

Authors
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  2. Anita Jeffers Nelsen
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  3. Giuseppe Pilia
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Fuscoe, J.C., Nelsen, A.J. & Pilia, G. Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis. Somat Cell Mol Genet 20, 39–46 (1994). https://doi.org/10.1007/BF02257484

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  • DOI: https://doi.org/10.1007/BF02257484

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