In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs.

Development of a Simple and Cost-Effective Bead-Milling Method for DNA Extraction from Fish Muscles

ARMANI, ANDREA;CASTIGLIEGO, LORENZO;GIANFALDONI, DANIELA;GUIDI, ALESSANDRA
2014

Abstract

In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs.
D'Amico, P.; Armani, Andrea; Castigliego, Lorenzo; Sheng, G.; Gianfaldoni, Daniela; Guidi, Alessandra
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/494870
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