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Updated: Aug 8, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Detection of the BRAF V600E mutation in melanocytic lesions using the ligase detection reaction
Daniel J Turner1, Monib A Zirvi, Francis Barany
1Department of Microbiology, Cornell University Graduate School of Medical Sciences, New York, NY, USA.
Background:
BRAF is a member of the RAF kinase family, and it promotes signaling through the RAS-MAP kinase signal transduction cascade. Research has shown that a majority of melanomas and nevi exhibit an activating V600E (T1799A) mutation in BRAF exon 15. Additional studies of BRAF have demonstrated that the T1799A mutation is absent in uveal melanomas and Spitz nevi.
Methods:
The ligase detection reaction (LDR) is a sensitive technique that can identify specific DNA mutations occurring at very low frequency within heterogeneous clinical samples, and it has previously been used to analyze mutations in paraffin-embedded tissue specimens.
Results:
The LDR can readily detect mutations in DNA extracted from non-microdissected paraffin-embedded sections of common nevi, dysplastic nevi, and melanomas. In addition, this method demonstrated the absence of the V600E (T1799A) mutation within Spitz nevi.
Conclusion:
The LDR is a highly sensitive and specific test for detecting mutations in formalin-fixed tissue. The LDR can be easily adapted to detect any mutation associated with a cutaneous disorder. The absence of the BRAF V600E mutation in Spitz's nevi may serve as a molecular signature to distinguish these lesions from common nevi, dysplastic nevi, and some types of malignant melanoma.
Insights
The ligase detection reaction (LDR) accurately detects BRAF V600E mutations in skin lesions. This method confirms the absence of this mutation in Spitz nevi, aiding in their distinction from other nevi and melanomas.
Area of Science:
- Oncology
- Genetics
- Dermatopathology
Background:
- BRAF kinase is crucial in the RAS-MAP kinase signaling cascade.
- Activating BRAF V600E mutations are common in melanomas and nevi.
- These mutations are notably absent in uveal melanomas and Spitz nevi.
Purpose of the Study:
- To evaluate the ligase detection reaction (LDR) for detecting BRAF mutations in various skin lesions.
- To determine the presence or absence of the BRAF V600E mutation in Spitz nevi.
Main Methods:
- Utilized ligase detection reaction (LDR), a sensitive DNA mutation detection technique.
- Analyzed DNA from paraffin-embedded tissue sections of common nevi, dysplastic nevi, melanomas, and Spitz nevi.
Main Results:
- LDR successfully detected BRAF mutations in common nevi, dysplastic nevi, and melanomas.
- The BRAF V600E (T1799A) mutation was confirmed to be absent in Spitz nevi.
Conclusions:
- LDR is a sensitive and specific method for mutation detection in formalin-fixed tissues.
- The absence of the BRAF V600E mutation can serve as a molecular marker to differentiate Spitz nevi from other skin lesions.
