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

Author(s)

Silva Almeida Vicentel, Anna Luiza ^[1] ; Bianchini, Raquel Alves ^[1] ; Laus, Ana Carolina ^[1] ; Macedo, Graziela ^[2] ; Reis, Rui Manuel ^{[3, 1, 4]} ; Vazquez, Vinicius de Lima ^{[1, 5]}

Affiliation

^[1] Barretos Canc Hosp, Mol Oncol Res Ctr, Antenor Duarte Villela 1331, BR-14784400 Sao Paulo - Brazil

^[2] Barretos Canc Hosp, Dept Pathol, Sao Paulo - Brazil

^[3] Univ Minho, Sch Hlth Sci, Life & Hlth Sci Res Inst ICVS, Braga - Portugal

^[4] ICVS 63Bs PT Govt Associate Lab, Braga - Portugal

^[5] Barretos Canc Hosp, Dept Surg Melanoma Sarcoma, Sao Paulo - Brazil

• Document type: Journal article
• Source: HISTOLOGY AND HISTOPATHOLOGY; v. 34, n. 9, p. 1089-1096, SEP 2019.
• Web of Science Citations: 0

Abstract

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 (R)-100, Chelex (R)-100 5%, centrifugation, OneStep (TM) PCR Inhibitor Removal Kit and centrifugation plus OneStep (TM) 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 (TM) 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. (AU)

• FAPESP's process: 17/09612-0 - The Role of Methylation in the Melanoma Initiation and Progression and the Epigenetic Impact in vitro
• Grantee: Anna Luiza Silva Almeida Vicente
• Support type: Scholarships abroad - Research Internship - Doctorate
• FAPESP's process: 12/04194-1 - The biologic and clinical characterization of the RAS-MAPK and PI3K-AKT pathways in cutaneous and mucosal melanomas among Brazilians and comparison with patients of the United States of America
• Grantee: Vinicius de Lima Vazquez
• Support type: Regular Research Grants
• FAPESP's process: 16/15941-3 - The Role of Methylation in the Melanoma Initiation and Progression and the Epigenetic Impact in vitro
• Grantee: Anna Luiza Silva Almeida Vicente
• Support type: Scholarships in Brazil - Doctorate