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.