Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations.

Abstract

A mutation detection strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was developed to detect single nucleotide substitutions in ras oncogenes, a common genetic abnormality in many human cancers. Mutation-specific probes are synthesized for each possible single-base, nonsilent mutation in codons 12, 13, and 61 of H-, K-, and N-ras oncogenes. Mutations are identified by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) are needed for analysis of each mutation. Probes hybridized to target ras oncogene DNA are joined by a thermostable ligase if there are no mismatches at their junctions; temperature cycling results in a linear increase in product. Common probes are labeled with fluorochromes, and allelic probes each have different lengths. Ligation products are analyzed by denaturing polyacrylamide gel electrophoresis on a fluorescent DNA sequencer. We have applied this technology to identify ras mutations in pancreatic cancers and lung cancers and in patients with myelodysplastic syndromes and leukemias.