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Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis
Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic aci...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Impact Journals LLC
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581030/ https://www.ncbi.nlm.nih.gov/pubmed/28881731 http://dx.doi.org/10.18632/oncotarget.17014 |
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author | Emelyanova, Marina Ghukasyan, Lilit Abramov, Ivan Ryabaya, Oxana Stepanova, Evgenia Kudryavtseva, Anna Sadritdinova, Asiya Dzhumakova, Cholpon Belysheva, Tatiana Surzhikov, Sergey Lyubchenko, Lyudmila Zasedatelev, Alexander Nasedkina, Tatiana |
author_facet | Emelyanova, Marina Ghukasyan, Lilit Abramov, Ivan Ryabaya, Oxana Stepanova, Evgenia Kudryavtseva, Anna Sadritdinova, Asiya Dzhumakova, Cholpon Belysheva, Tatiana Surzhikov, Sergey Lyubchenko, Lyudmila Zasedatelev, Alexander Nasedkina, Tatiana |
author_sort | Emelyanova, Marina |
collection | PubMed |
description | Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy. |
format | Online Article Text |
id | pubmed-5581030 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Impact Journals LLC |
record_format | MEDLINE/PubMed |
spelling | pubmed-55810302017-09-06 Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis Emelyanova, Marina Ghukasyan, Lilit Abramov, Ivan Ryabaya, Oxana Stepanova, Evgenia Kudryavtseva, Anna Sadritdinova, Asiya Dzhumakova, Cholpon Belysheva, Tatiana Surzhikov, Sergey Lyubchenko, Lyudmila Zasedatelev, Alexander Nasedkina, Tatiana Oncotarget Research Paper Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy. Impact Journals LLC 2017-04-10 /pmc/articles/PMC5581030/ /pubmed/28881731 http://dx.doi.org/10.18632/oncotarget.17014 Text en Copyright: © 2017 Emelyanova et al. http://creativecommons.org/licenses/by/3.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Research Paper Emelyanova, Marina Ghukasyan, Lilit Abramov, Ivan Ryabaya, Oxana Stepanova, Evgenia Kudryavtseva, Anna Sadritdinova, Asiya Dzhumakova, Cholpon Belysheva, Tatiana Surzhikov, Sergey Lyubchenko, Lyudmila Zasedatelev, Alexander Nasedkina, Tatiana Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title | Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title_full | Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title_fullStr | Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title_full_unstemmed | Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title_short | Detection of BRAF, NRAS, KIT, GNAQ, GNA11 and MAP2K1/2 mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis |
title_sort | detection of braf, nras, kit, gnaq, gna11 and map2k1/2 mutations in russian melanoma patients using lna pcr clamp and biochip analysis |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581030/ https://www.ncbi.nlm.nih.gov/pubmed/28881731 http://dx.doi.org/10.18632/oncotarget.17014 |
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