Skip Navigation LinksHome > August 2014 - Volume 24 - Issue 4 > Sensitive allele-specific real-time PCR test for mutations i...
Melanoma Research:
doi: 10.1097/CMR.0000000000000090
ORIGINAL ARTICLES: Basic research

Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma

Pisareva, Ekaterinaa; Gutkina, Nadezhdaa; Kovalenko, Sergeia,b,d; Kuehnapfel, Sarahe; Hartmann, Arndte; Heinzerling, Lucief; Schneider-Stock, Reginee; Lyubchenko, Lyudmilac; Shamanin, Vladimir A.a,b

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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‚ÄČng 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.

© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins

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