Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma
| Autor: | Regine Schneider-Stock, Lucie Heinzerling, Lyudmila Lyubchenko, Sergei Kovalenko, Ekaterina Pisareva, Arndt Hartmann, Sarah Kuehnapfel, Nadezhda I. Gutkina, Vladimir A. Shamanin |
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| Rok vydání: | 2014 |
| Předmět: |
Adult
Male Proto-Oncogene Proteins B-raf Cancer Research Skin Neoplasms Adolescent Dermatology Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Sensitivity and Specificity Young Adult Sense (molecular biology) TaqMan medicine Humans Allele Codon Melanoma Alleles Aged Mutation Middle Aged medicine.disease Molecular biology Real-time polymerase chain reaction Oncology Cutaneous melanoma Female V600E |
| Zdroj: | Melanoma Research. 24:322-331 |
| ISSN: | 0960-8931 |
| Popis: | AQ1 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, V600DK601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allelespecific 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 BRAFV600 in cutaneous melanoma. Melanoma Res 00:000–000 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Melanoma Research 2014, 00:000–000 |
| Databáze: | OpenAIRE |
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