Efficient DNA Extraction from Nail Clippings Using the Protease Solution from Cucumis melo

Abstract

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR-RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.

DNA from Nails for Genetic Analyses in Large-Scale Epidemiologic Studies

“…Amplification of 911-and 969-bp PCR products in fingernail DNA samples was unsuccessful. In the majority of fingernail samples they tested, YoshidaYamamoto and colleagues (10) were able to amplify a 286-bp fragment of the ESRX gene. In conclusion, our study and other studies have shown that toenail DNA is considerably fragmented to sizes generally <200 bp, but larger fragments are present and can be amplified in part of the samples, probably depending on the protocols used to isolate the DNA from the nails and the specific genomic regions of interest to be amplified.…”

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

“…In a previous study, Li [3] evaluated 59 clostridiopeptidase and trypsin to develop an improved method 60 for recovering DNA from bone. We evaluated DNA recovery using 61 proteinase K, bromelain, and papain due to their success in a study 62 of DNA recovery from fingernail clippings; bromelain exhibited a 63 1.78-fold higher keratinolytic activity than proteinase K [4]. Both 64 bromelain and papain outperformed proteinase K [4].…”

“…We evaluated DNA recovery using 61 proteinase K, bromelain, and papain due to their success in a study 62 of DNA recovery from fingernail clippings; bromelain exhibited a 63 1.78-fold higher keratinolytic activity than proteinase K [4]. Both 64 bromelain and papain outperformed proteinase K [4]. 65 Proteinase K (EC 3.5.21.64), a serine protease enzyme of the 66 fungus Engyodontium album (Tritirachium album Limber), has a 67 molecular weight of 18 kDa, has an optimal pH of 7.5 to 9.0 (active 68 between pH 4.0 and 12.0), and is active between 37 and 60°C 69 (<65°C) [5,6].…”

“…Serine proteases share mechanistic features 75 with the cysteine and threonine proteases [7]. ing plants from invasive herbivorous insects [4,8] and have been 83 used in DNA extraction methods [4]. In this study, pineapple stem 84 bromelain (24 kDa), with an optimal pH of 4.5 to 5.5 (active 85 between pH 4.5 and 9.8) and a temperature range of 40 to 60°C 86 (<65°C), was employed [4,9].…”

“…ing plants from invasive herbivorous insects [4,8] and have been 83 used in DNA extraction methods [4]. In this study, pineapple stem 84 bromelain (24 kDa), with an optimal pH of 4.5 to 5.5 (active 85 between pH 4.5 and 9.8) and a temperature range of 40 to 60°C 86 (<65°C), was employed [4,9]. Bromelain will hydrolyze esters 87 and amides and will cleave amino acids that are leucine or glycine, 88 are basic, or exhibit large hydrophobic side chains at the P2 position [10].…”