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Fabbri M., Venturi M., Boni S., Benedetti S., Pollicino R., Gaudio R. M., Avato F. M.
Laboratory of Forensic Genetics Section of Legal Medicine Department of Medical Sciences University of Ferrara, Ferrara, Italy
Aim: Present work involved the development of a new DNA extraction method, suggested for biological matrices represented by human skeletal remains.
Methods: Surface of the elements were cleaned by removing the residues of soft tissue eventually present, rinsed in sterile distilled water, dried and treated using UV light. A 2 x 5 cm portion was cut from each bone epiphyses and the surface of these portion were removed (at least 2-3 mm deep) in order to get rid of modern exogenous DNA contamination. Bone pellets were suspended in 7ml of digestion buffer containing 0.02 M Tris (pH 8.0), 0.1 M NaCl, 0.05 M EDTA (pH 7.5), 0.6M DTT and 200 μL of proteinase K (18 mg/mL). After digestion step, the tubes were placed at -20 °C for 24 hours. Subsequently, a purification was performed by the addition of an equivalent volume of a mixture of phenol-chloroform-isoamyl alcohol (25:24:1), and this step was repeated twice. In the final phase, DNA were concentrated by using Centricon YM-100.
Results: The new experimental protocol allowed to achieve results quantitatively higher, in terms of STR markers obtained after capillary electrophoresis. Of particular interesting were changes to the pH of the solution used in decalcification process and the concentrations of the compounds introduced during digestion. Further improvements were represented by the incubation temperature; it seems reasonable that this modify affect in a smaller fraction DNA structure already compromised by time and environmental. Finally, it was possible to observe how the skeletal elements offered the best results were represented by femurs.
Conclusion: The modifications introduced to the previous experimental protocol, identified by the evaluation of analytical parameters that affect the extraction process and verifying the influence on the entire analytical process were able to allow recovery DNA useful for PCR amplification and genotyping, in terms of quantity and quality.