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 restric tion site. The alleles of interest are amplified by nested PCR and lab eled in a second round of PCR. The product is digested with a restrict ion enzyme specific for that site, resolved on a non-denaturing gel an d quantified by phosphor imaging techniques. Cell types with known num bers of wild-type and mutant alleles of c-Harvey-ras are used to valid ate the assay The method is then ap plied to a cell line, homogygous f or the mutation, to determine the gene copy number The applicability o f the method to other genes is shown using the matrix metalloproteinas e gene, matrilysin. A cell line transfected with a plasmid carrying a mutant, auto-activated form of the gene is compared to its pai-ent cel l line. Advantages of this technique compared with Southern analysis a re ease of screening a large number of clones or foci and accuracy of quantitation.of quantitation.