Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma
Pisareva E, Gutkina N, Kovalenko S, Kuehnapfel S, Hartmann A, Heinzerling L, Schneider-Stock R, Lyubchenko L, Shamanin VA (2014)
Publication Type: Journal article
Publication year: 2014
Journal
Publisher: Lippincott, Williams & Wilkins
Book Volume: 24
Pages Range: 322-31
Journal Issue: 4
DOI: 10.1097/CMR.0000000000000090
Abstract
Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.
Authors with CRIS profile
Arndt Hartmann
Lehrstuhl für Allgemeine Pathologie und Pathologische Anatomie
Lucie Heinzerling
Department of Dermatology
Regine Schneider-Stock
Professur für Experimentelle Tumorpathologie
Involved external institutions
Russian Federation (RU)How to cite
APA:
Pisareva, E., Gutkina, N., Kovalenko, S., Kuehnapfel, S., Hartmann, A., Heinzerling, L.,... Shamanin, V.A. (2014). Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma. Melanoma Research, 24(4), 322-31. https://doi.org/10.1097/CMR.0000000000000090
MLA:
Pisareva, Ekaterina, et al. "Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma." Melanoma Research 24.4 (2014): 322-31.
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