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MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES
Citation:
Fuscoe, J.C., L. Zimmerman, K. Harrington-Brock, AND M. Moore. MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-94/411 (NTIS PB95125357), 1994.
Description:
A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes for deletions. he hprt clonal assay was used to isolate in-vivo-arising hprt-deficient T-cells form six healthy males. utant frequencies ranged from 9-27 X 10-6. imple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. ixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. he relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-y genes. ighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR-y pattern. ne-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone suggesting that this region of the hprt gene is prone to deletion. he small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. he ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.
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MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES