Application of an Alkaline and Silica Membrane DNA Extraction Method to Detect Mitochondrial DNA in Foods
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Application of an Alkaline and Silica Membrane DNA Extraction Method to Detect Mitochondrial DNA in Foods Naoki Harikai & Kazufusa Shinomiya
Received: 21 February 2014 / Accepted: 15 September 2014 # Springer Science+Business Media New York 2014
Abstract To improve the efficiency of mitochondrial DNA (mtDNA) extraction from food, a simple method, PrepM, was developed by effectively combining alkaline sodium dodecyl sulfate lysis and DNA binding to a silica membrane. PrepM was evaluated using fresh, cold, and autoclaved rat liver samples and compared with commercial total DNA extraction kits. When PrepM was applied to fresh and cold samples, the DNAyield was low, but the OD260/280 ratio was close to 1.8. The cytochrome C oxidase subunit I (COI) copy number, which is the number of mtDNA copies, per DNAweight and the ratio of COI to β-actin, which is the ratio of mitochondrial to genomic DNA, were higher with PrepM than with total DNA extraction kits. When PrepM was applied to autoclaved samples, the DNA purity and yield and the COI copy number per DNAweight were relatively high, but the COI to β-actin ratio was low. PrepM was then applied to shrimp samples, a known allergenic food. Data obtained from cold and autoclaved shrimp samples were similar to data obtained from rat liver samples. When PrepM was applied to food samples spiked with shrimp, the DNA purity was high, but the DNA yield and the mtDNA levels were almost same as those obtained with total DNA extraction kits. We concluded that PrepM has a comparative advantage for sensitive polymerase chain reaction detection of mtDNA sequences in relatively fresh samples, and it has almost the same performance as general total DNA extraction kits with autoclaved and/or complex mixture samples.
N. Harikai School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11-68 Kyuban-cho, Koshien, Nishinomiya, Hyogo 663-8179, Japan N. Harikai (*) : K. Shinomiya School of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan e-mail: [email protected]
Keywords Alkaline sodium dodecyl sulfate lysis . Mitochondrial DNA extraction . β-Actin . Cytochrome C oxidase subunit I
Introduction Mitochondrial DNA (mtDNA) is highly conserved and present in all species with distinguishable variability (Bellis et al. 2003). The number of mtDNA copies per cell is much larger than that of genomic DNA (gDNA) (Bodenhagen and Clayton 1974). Accordingly, mtDNA has been used as a specific and sensitive detection tool for species identification. Polymerase chain reaction (PCR) is useful for the detection of mtDNA in foods such as meat and fish (Haider et al. 2012; Michelini et al. 2007; Taguchi et al. 2011). Furthermore, DNA extraction methods that enrich mtDNA are applied to meat and fish samples to identify species, and they allow the sensitive detection of mtDNA sequences using PCR (Tanabe et al. 2007; Burgener and Hubner 1998). Recently, a DNA sample after mtDNA enrichment from total DNA was able to allow direct DNA sequence analysis witho
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