Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

Genotyping BRAF in melanoma samples is often challenging. The presence of melanin greatly interferes with thermostable DNA polymerases and/or nucleic acids in traditional polymerase chain reaction (PCR)-based methods. In the present work, we evaluated three easy-to-use strategies to improve the dete...

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Main Authors:	Frouin, Eric, Maudelonde, Thierry, Senal, Romain, Larrieux, Marion, Costes, Valérie, Godreuil, Sylvain, Vendrell, Julie A., Solassol, Jérôme
Format:	Online
Language:	English
Published:	Public Library of Science 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965116/

id	pubmed-4965116
recordtype	oai_dc
spelling	pubmed-49651162016-08-18 Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens Frouin, Eric Maudelonde, Thierry Senal, Romain Larrieux, Marion Costes, Valérie Godreuil, Sylvain Vendrell, Julie A. Solassol, Jérôme Research Article Genotyping BRAF in melanoma samples is often challenging. The presence of melanin greatly interferes with thermostable DNA polymerases and/or nucleic acids in traditional polymerase chain reaction (PCR)-based methods. In the present work, we evaluated three easy-to-use strategies to improve the detection of pigmented DNA refractory to PCR amplification. These pre-PCR processing methods include the addition of bovine serum albumin (BSA), the dilution of DNA, and the purification of DNA using the NucleoSpin® gDNA Clean-up XS Kit. We found that BRAF genotyping in weakly and moderately pigmented samples was more efficient when the sample was processed with BSA or purified with a NucleoSpin® gDNA Clean-up XS Kit prior to PCR amplification. In addition, the combination of both methods resulted in successful detection of BRAF mutation in pigmented specimens, including highly pigmented samples, thereby increasing the chance of patients being elicited for anti-BRAF treatment. These solutions to overcome melanin-induced PCR inhibition are of tremendous value and provide a simple solution for clinical chemistry and routine laboratory medicine. Public Library of Science 2016-07-28 /pmc/articles/PMC4965116/ /pubmed/27466810 http://dx.doi.org/10.1371/journal.pone.0158698 Text en © 2016 Frouin et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Frouin, Eric Maudelonde, Thierry Senal, Romain Larrieux, Marion Costes, Valérie Godreuil, Sylvain Vendrell, Julie A. Solassol, Jérôme
spellingShingle	Frouin, Eric Maudelonde, Thierry Senal, Romain Larrieux, Marion Costes, Valérie Godreuil, Sylvain Vendrell, Julie A. Solassol, Jérôme Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
author_facet	Frouin, Eric Maudelonde, Thierry Senal, Romain Larrieux, Marion Costes, Valérie Godreuil, Sylvain Vendrell, Julie A. Solassol, Jérôme
author_sort	Frouin, Eric
title	Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
title_short	Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
title_full	Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
title_fullStr	Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
title_full_unstemmed	Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens
title_sort	comparative methods to improve the detection of braf v600 mutations in highly pigmented melanoma specimens
description	Genotyping BRAF in melanoma samples is often challenging. The presence of melanin greatly interferes with thermostable DNA polymerases and/or nucleic acids in traditional polymerase chain reaction (PCR)-based methods. In the present work, we evaluated three easy-to-use strategies to improve the detection of pigmented DNA refractory to PCR amplification. These pre-PCR processing methods include the addition of bovine serum albumin (BSA), the dilution of DNA, and the purification of DNA using the NucleoSpin® gDNA Clean-up XS Kit. We found that BRAF genotyping in weakly and moderately pigmented samples was more efficient when the sample was processed with BSA or purified with a NucleoSpin® gDNA Clean-up XS Kit prior to PCR amplification. In addition, the combination of both methods resulted in successful detection of BRAF mutation in pigmented specimens, including highly pigmented samples, thereby increasing the chance of patients being elicited for anti-BRAF treatment. These solutions to overcome melanin-induced PCR inhibition are of tremendous value and provide a simple solution for clinical chemistry and routine laboratory medicine.
publisher	Public Library of Science
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965116/
_version_	1613617172428357632

Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

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