A HIGHLY SENSITIVE ASSAY FOR MUTANT RAS GENES AND ITS APPLICATION TO THE STUDY OF PRESENTATION AND RELAPSE GENOTYPES IN ACUTE-LEUKEMIA

Most studies of ras oncogene activation use assays for ras mutations b ased on the polymerase chain reaction (PCR) of DNA segments containing ras exons 1 and 2, followed by allele-specific oligonucleotide (ASO) hybridization or direct sequencing, which require that to be detectabl e, a mutation must be present in at least 3-25% of ras alleles. Thus, studies of tissues in which only a fraction of cells contains a ras mu tation risk false negative results. To minimize this risk, we have dev eloped a highly sensitive, non-radioactive assay for uas mutations. Ra s genes were PCR-amplified using mismatched primers, to introduce rest riction sites into products derived from normal alleles. Repeated rest riction digestion and PCR enriched for mutant alleles, visualized by a garose gel electrophoresis. Serially diluted DNA samples containing I as mutations demonstrated detection of 1 mutant/10(6) normal alleles ( four orders of magnitude more sensitive than PCR/ASO hybridization). T his assay was applied to DNA from four patients with relapsed acute le ukemia in whom ras mutations present at diagnosis were not detectable by PCR/ASO hybridization at relapse. In one case, the mutation present at diagnosis was demonstrated at relapse. In the others, loss of the mutation was confirmed, at a greatly increased sensitivity. This metho d is widely applicable to detection of mutant ras alleles admired with larger numbers of normal alleles.