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 (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.
Haque
K
K
Zeneca Diagnostics, Northwich, Cheshire, U.K.
Hehir
J
J
Fox
J C
JC
Newton
C R
CR
Little
S
S
D000483:Alleles; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000032:analysis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D011905:Genes, ras / Q000235:genetics; D005838:Genotype; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured