Related Experiment Video
Updated: Aug 1, 2026
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations
1Molecular Oncology Laboratory, Huntington Medical Research Institutes, Pasadena, CA 91101, USA. eggerdfa@hmri.org
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.
Insights
This study introduces a novel method for detecting single nucleotide substitutions in ras oncogenes using multiplex PCR and probe ligation. This technique efficiently identifies common cancer-associated ras mutations in various cancer types.
Area of Science:
- Molecular Biology
- Oncology
- Genetic Diagnostics
Background:
- Ras oncogenes are frequently mutated in human cancers, driving tumor development.
- Accurate detection of single nucleotide substitutions in ras genes is crucial for cancer diagnosis and treatment.
Purpose of the Study:
- To develop and validate a sensitive method for detecting single nucleotide substitutions in H-, K-, and N-ras oncogenes.
- To apply this method for identifying ras mutations in diverse cancer patient samples.
Main Methods:
- Developed a multiplex PCR and multiplex allele-specific oligonucleotide probe ligation strategy.
- Utilized mutation-specific probes for codons 12, 13, and 61 of ras oncogenes.
- Analyzed ligation products using denaturing polyacrylamide gel electrophoresis and fluorescent DNA sequencing.
Main Results:
- The method successfully detects single-base, nonsilent mutations in ras oncogenes.
- Applied the technology to identify ras mutations in pancreatic cancer, lung cancer, myelodysplastic syndromes, and leukemias.
- Demonstrated the capability of competitive oligonucleotide probe ligation for simultaneous detection of normal and mutant genotypes.
Conclusions:
- The developed multiplex ligation assay is an effective tool for ras mutation detection.
- This technology facilitates the identification of genetic abnormalities in various hematologic and solid tumors.
- The strategy offers a robust approach for cancer genetic profiling and diagnostics.
