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Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions

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dc.contributor.authorSilva Almeida Vicente, Anna Luiza
dc.contributor.authorAlves Bianchini, Raquel
dc.contributor.authorLaus, Ana Carolina
dc.contributor.authorMacedo, Graziela
dc.contributor.authorReis, Rui Manuel
dc.contributor.authorVazquez, Vinicius de Lima
dc.date.accessioned2022-07-11T08:20:49Z
dc.date.available2022-07-11T08:20:49Z
dc.date.issued2019
dc.description.abstractMelanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffinembedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.es
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dc.format.extent8es
dc.identifier.citationHistology and Histopathology, Vol.34, nº9, (2019)
dc.identifier.doiDOI: 10.14670/HH-18-112
dc.identifier.issn1699-5848
dc.identifier.issn0213-3911
dc.identifier.urihttp://hdl.handle.net/10201/122283
dc.languageenges
dc.publisherUniversidad de Murcia. Departamento de Biología Celular e Histologíaes
dc.relationSin financiación externa a la Universidades
dc.rightsinfo:eu-repo/semantics/openAccesses
dc.rightsAttribution-NonCommercial-NoDerivatives 4.0 Internacional*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/*
dc.subjectMelanines
dc.subjectPCR inhibitores
dc.subjectPigmented melanomases
dc.subjectPurificationes
dc.subjectPolymerase inhibitiones
dc.subject.otherCDU::6 - Ciencias aplicadas::61 - Medicina::616 - Patología. Medicina clínica. Oncologíaes
dc.titleComparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesionses
dc.typeinfo:eu-repo/semantics/articlees
dspace.entity.typePublicationes
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Histology and histopathology Vol.34, nº9 (2019)