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Updated: May 12, 2026
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Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based
Alexander Skorokhod1, Peter Helmbold, Benedikt Brors
1Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany. Alexander.Skorokhod@med.uni-heidelberg.de
Plos One
|April 5, 2013
Summary
A new universal pyrosequencing assay, U-BRAF(V600), accurately detects BRAF mutations in melanoma. This automated tool aids in identifying patients eligible for targeted BRAF inhibitor therapy.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Malignant melanoma is an aggressive cancer with high metastatic potential.
- BRAF mutations are key therapeutic targets in metastatic melanoma.
- Existing BRAF detection methods have limitations.
Purpose of the Study:
- To develop and validate a universal pyrosequencing assay for BRAF mutation detection.
- To enable automated, quantitative identification of BRAF V600 mutations.
- To establish a diagnostic tool for BRAF status assessment in melanoma patients.
Main Methods:
- Development of the U-BRAF(V600) pyrosequencing assay.
- Analysis of 75 formalin-fixed paraffin-embedded (FFPE) melanoma metastasis samples.
- Validation using ultra-deep sequencing (approx. 60,000-fold coverage).
Main Results:
- Identified 5 different BRAF mutations in a single assay.
- Detected BRAF mutations in 21 out of 29 patient metastases.
- U-BRAF(V600) assay quantitatively detected all mutant variants and excluded false wild-type results.
Conclusions:
- The U-BRAF(V600) assay is a powerful, automated sequencing-based diagnostic tool.
- It can identify at least 36 previously published BRAF mutations.
- Suitable for large-scale clinical sample analysis for targeted therapy selection.
