Analysis of large deletions in the HPRT gene of primary human fibroblasts using the polymerase chain reaction.
Spontaneous and X-ray-induced mutants of the HPRT gene were isolated from two primary human fibroblast lines. The limited life-span of the mutants restricted the use of methods requiring large quantities of DNA, and the polymerase chain reaction (PCR) was used in particular to check for the presence of multiple genomic sites in mutant analysis. Robust PCR primers were designed to amplify sites of up to 1 kb, mostly with 1-kb spacings between sites, over the entire 56-kb HPRT gene region. Using PCR, large deletions were found in 43% of independent X-ray-induced mutants, and their breakpoints were localized where these fell within the gene. Anonymous DNA sites in the Xq26 chromosomal region containing HPRT (covering > or = 1.5 Mb) were also amplified by PCR to assess codeletion with HPRT; sites up to 1 Mb distal to the gene (DXS86, DXS10) were codeleted in some mutants, but no mutant was found with loss of a proximal site (DXS79).
Morris
T
T
MRC Radiobiology Unit, Didcot, Oxon, England.
Masson
W
W
Singleton
B
B
Thacker
J
J
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000201:enzymology / Q000528:radiation effects; D017353:Gene Deletion; D008040:Genetic Linkage; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014960:X Chromosome
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase