PCR/RFLP assay for copy number of mutant and wild-type alleles.
A PCR method for quantitating the copy number of mutant vs. wild-type alleles in DNA from cell lines is described. The assay can be used to detect a point mutation in any gene that creates or destroys a restriction site. The alleles of interest are amplified by nested PCR and labeled in a second round of PCR. The product is digested with a restriction enzyme specific for that site, resolved on a non-denaturing gel and quantified by phosphor imaging techniques. Cell types with known numbers of wild-type and mutant alleles of c-Harvey-ras are used to validate the assay. The method is then applied to a cell line, homogygous for the mutation, to determine the gene copy number. The applicability of the method to other genes is shown using the matrix metalloproteinase gene, matrilysin. A cell line transfected with a plasmid carrying a mutant, auto-activated form of the gene is compared to its parent cell line. Advantages of this technique compared with Southern analysis are ease of screening a large number of clones or foci and accuracy of quantitation.
Finch
J S
JS
Dept. of Radiation Oncology, University of Arizona, Tucson 85724, USA.
Bowden
G T
GT
D000483:Alleles; D000818:Animals; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification; D018628:Gene Dosage; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured / Q000737:chemistry
D017931:DNA Primers; D004273:DNA, Neoplasm