<i>BRAF</i> mutation status determined by HRM using DNA treated with BSA or purified using the NucleoSpin^® Kit.

<p><i>BRAF</i> mutation status determined by HRM using DNA treated with BSA or purified using the NucleoSpin^® Kit.</p

Example of <i>BRAF</i> amplification in several tissue samples.

<p>(A) Image showing samples with a weak (+), moderate (++), or high (+++) level of melanin contamination after DNA extraction. (B) HRM <i>C</i>_<i>t</i>-values for samples harboring different levels of melanin contamination and treated with different pre-PCR procedures.</p

Removal of the inhibitory effects of melanin on PCR amplification.

<p>(A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of <i>Taq</i> polymerase. (D) NucleoSpin^® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.</p

Combinaison of the Nucleospin^® Kit and BSA treatment for routine detection of <i>BRAF</i> mutation in highly pigmented samples.

<p>Combinaison of the Nucleospin^® Kit and BSA treatment for routine detection of <i>BRAF</i> mutation in highly pigmented samples.</p

Patient and specimen characteristics.

<p>Patient and specimen characteristics.</p

Conclusive results obtained by HRM analysis according to the level of melanin contamination.

<p>Conclusive results obtained by HRM analysis according to the level of melanin contamination.</p

Reproducibility of the HRM <i>C</i>_<i>t</i>-values for 50 samples pre-treated with either BSA or the NucleoSpin^® Kit.

<p>Box-and-whisker plots represent the standard deviation of <i>C</i>_<i>t</i>-values obtained for samples with weak (+, n = 20), moderate (++, n = 21), or high (+++, n = 9) melanoma contaminations that were pre-treated with either BSA or the NucleoSpin^® Kit. Each sample was analyzed in duplicate.</p

Comparative Methods to Improve the Detection of <i>BRAF</i> V600 Mutations in Highly Pigmented Melanoma Specimens

<div><p>Genotyping <i>BRAF</i> 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 <i>BRAF</i> 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 <i>BRAF</i> 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.</p></div

Spatial distribution of genotypes of <i>M. bovis</i> isolated from multiple hosts.

<p>Spatial distribution of genotypes of <i>M. bovis</i> isolated from multiple hosts.</p

Evolution of the prevalence and the incidence rate of bTB (A), of the different strain profiles number (B) and yearly evolution of the strain collection (C) of M. bovis in France from 1978 to 2013.

<p>A: On the abscissa are represented the years, on the ordinate, the percentage of incidence (◼) and prevalence (●). Black line cutting the ordinate axe at 0.1% represents the limit under which an EU country is considered free of bTB. Below the graph, three lines (P1; P2; P3) represent the three periods studied. B: In black bars, spoligotypes, in white bars, MLVA profiles, in green bars, the association of both methods. C: Yearly evolution of the number of strains collected (Black) and spoligotype-MLVA identification (Pink) of the 4,654 <i>M</i>. <i>bovis</i> strains received in the National Reference laboratory between 1978 and 2013. For both graphs, lines represent the three studied periods (P1; P2; P3).</p