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PLoS One. 2016 Jul 28;11(7):e0158698. doi: 10.1371/journal.pone.0158698. eCollection 2016.

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

Author information

1
CHU Poitiers, Department of Biopathology, Poitiers, France.
2
CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Pathology, Montpellier, France.
3
Institut du Cancer de Montpellier, Department of Biopathology, Montpellier, France.
4
CHU Montpellier, Arnaud de Villeneuve Hospital, Department of Bacteriology, Montpellier, France.
5
IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France.
6
INSERM, U1194, Montpellier, France.
7
Université de Montpellier, Montpellier, France.

Abstract

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.

PMID:
27466810
PMCID:
PMC4965116
DOI:
10.1371/journal.pone.0158698
[Indexed for MEDLINE]
Free PMC Article

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