Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells using the polymerase chain reaction.
We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light- and X-ray-induced hprt-deficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (11/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.
Xu
Z D
ZD
Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston 77550.
Yu
Y J
YJ
Hsie
A W
AW
Caskey
C T
CT
Rossiter
B
B
Gibbs
R A
RA
D000818:Animals; D002478:Cells, Cultured; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D005091:Exons; D005784:Gene Amplification; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D014466:Ultraviolet Rays; D014965:X-Rays
D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004259:DNA-Directed DNA Polymerase