Chapter title |
Molecular Analysis of Mutations in the Human HPRT Gene.
|
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Chapter number | 23 |
Book title |
Molecular Toxicology Protocols
|
Published in |
Methods in molecular biology, March 2014
|
DOI | 10.1007/978-1-62703-739-6_23 |
Pubmed ID | |
Book ISBNs |
978-1-62703-738-9, 978-1-62703-739-6
|
Authors |
Keohavong P, Xi L, Grant SG, Phouthone Keohavong, Liqiang Xi, Stephen G. Grant |
Editors |
Phouthone Keohavong, Stephen G. Grant |
Abstract |
The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. 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 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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