Amplification Refractory Mutation System Linear ExtensionA Novelcomma; Gelhyphen;Freecomma; Enzymehyphen;Linked Immunoassay Method for DNA Genotyping

摘要

A single synthesis cycle of the amplification refractory mutation system (ARMS) was applied to the analysis of K-rasalleles amplified by polymerase chain reaction and immobilized in streptavidin-coated microtiter plates. The ARMS 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-rasallele. Any digoxigenin-labeled deoxyuridine monophosphate substitution was then demonstrated by enzyme-linked immunoassay with a colorimetric endpoint. This method is capable of detecting underrepresented acquired mutations, and this has been shown by the unambiguous detection of specific K-rasmutations in cell line DNA/normal human genomic DNA admixtures. The characterization of K-rasmutations in frozen colorectal tumor samples and histologic material is also described.