Amplification refractory mutation system linear extension: A novel, gel-free, enzyme-linked immunoassay method for DNA genotyping

A single synthesis cycle of the amplification refractory mutation system (A RMS) was applied to the analysis of K-ras alleles amplified by polymerase c hain reaction and immobilized in streptavidin-coated microtiter plates. The ARE;IS cycle provided the specificity and molecular switch characteristics of a conventional ARMS assay. This allowed linear extension from an allele -specific primer and the incorporation of digoxigenin-labeled deoxyuridine monophosphate from digoxigenin-11-deoxyuridine triphosphate in the presence of the appropriate K-ras allele. Any digoxigenin-labeled deoxyuridine mono phosphate substitution was then demonstrated by enzyme-linked immunoassay w ith a colorimetric endpoint. This method is capable of detecting underrepre sented acquired mutations, and this has been shown by the unambiguous detec tion of specific K-ras mutations in cell line DNA/normal human genomic DNA admixtures. The characterization of K-ras mutations in frozen colorectal tu mor samples and histologic material is also described.