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Molecular Analysis of Mutations in the Human HPRT Gene

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 291))

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Abstract

The HPRT assay uses incorporation of toxic nucleotide analogs to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosporibosyltransferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.

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Authors and Affiliations

  1. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

    Phouthone Keohavong & Stephen G. Grant

  2. Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA

    Liqiang Xi

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  1. Phouthone Keohavong
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  2. Liqiang Xi
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  3. Stephen G. Grant
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  1. Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

    Phouthone Keohavong  & Stephen G. Grant  & 

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© 2005 Humana Press Inc.

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Keohavong, P., Xi, L., Grant, S.G. (2005). Molecular Analysis of Mutations in the Human HPRT Gene. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:161

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  • DOI: https://doi.org/10.1385/1-59259-840-4:161

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-084-7

  • Online ISBN: 978-1-59259-840-3

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