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Updated: Feb 19, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE
Claudia Huebner1, Remeny Weber1, Richard Lloydd1
1Department of Anatomical Pathology, Labtests Auckland Limited, New Zealand.
Abstract:
Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment.
