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

• Published in: Diagnostic molecular pathology Vol. 7; no. 5; p. 248
• Main Authors: Haque, K, Hehir, J, Fox, J C, Newton, C R, Little, S
• Format: Journal Article
• Language: English
• Published: United States 01-10-1998
• Subjects: Alleles; Colorectal Neoplasms - chemistry; Colorectal Neoplasms - genetics; Colorectal Neoplasms - pathology; DNA - analysis; DNA - genetics; DNA Primers - chemistry; DNA, Neoplasm - analysis; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay - methods; Genes, ras - genetics; Genotype; Humans; Point Mutation; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Tumor Cells, Cultured
• ISSN: 1052-9551
• DOI: 10.1097/00019606-199810000-00003

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