HISTOLOGY AND HISTOPATHOLOGY

From Cell Biology to Tissue Engineering

 

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

Anna Luiza Silva Almeida Vicente1, Raquel Alves Bianchini1, Ana Carolina Laus1, Graziela Macedo2, Rui Manuel Reis1,3,4 and Vinicius de Lima Vazquez1,5

1Molecular Oncology Research Center, 2Department of Pathology, Barretos Cancer Hospital, Barretos, São Paulo, Brazil , 3Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, 4ICVS/63B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal and 5Department of Surgery Melanoma/Sarcoma, Barretos Cancer Hospital, Barretos, São Paulo, Brazil

Offprint requests to: Vinicius de Lima Vazquez, MD, PhD, , Molecular Oncology Research Center - Barretos Cancer Hospital, Antenor Duarte Villela, 1331, Zip Code: 14784 400, Barretos, São Paulo, Brazil. e-mail: viniciusvazquez@gmail.com


Summary. Melanin 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 paraffin-embedded (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. Histol Histopathol 34, 1089-1096 (2019)

Key words: Melanin, PCR inhibitor, Pigmented melanomas, Purification, Polymerase inhibition

DOI: 10.14670/HH-18-112