Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations.
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.
Eggerding
F A
FA
Molecular Oncology Laboratory, Huntington Medical Research Institutes, Pasadena, CA 91101, USA. eggerdfa@hmri.org
D000483:Alleles; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D007938:Leukemia / Q000235:genetics*; D008025:Ligases / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics*; D009190:Myelodysplastic Syndromes / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000737:chemistry; D010190:Pancreatic Neoplasms / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D008025:Ligases