BRAFV600E detection in melanoma is highly improved by COLD-PCR.
作者： Pamela Pinzani , Claudio Santucci , Irene Mancini , Lisa Simi , Francesca Salvianti
DOI: 10.1016/J.CCA.2011.01.014
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摘要: Abstract Background The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation shown to be associated with clinico pathological features of primary invasive melanomas. As may attractive therapeutic target, it is crucial have a sensitive method for detecting mutated DNA biological samples. Our aim was investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) new approach pre-analytical enrichment BRAFV600E variant formalin fixed paraffin embedded (FFPE) melanoma tissues. Methods used selectively amplify minority alleles from mixtures wild-type sequences, shows higher specificity than other conventional PCR-based methods somatic mutations. Results We increase theoretical sensitivity three different post-PCR methods: sequencing, pyrosequencing HRMA. gain seems more evident HRMA, which allows detection 3.1% alleles. More 20% patients initially classified negative were found positive after COLD-PCR. Conclusions confirmed suitable alleles, particularly samples percentage tumor cells very low.
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