A comparison of conservative DNA extraction methods from fins and scales of freshwater fish: A useful tool for conservation genetics.

Non-destructive protocols for DNA isolation from fresh or preserved specimens are fundamental to study endangered or elusive species, breeders or samples sibship and specimens derived from public or private collections. Because of the low yield and poor quality DNA, particular laboratory care and standardizations were needed to allow reproducible results. We compared six DNA extraction procedures from conservative samples: three are commercial kits and three are largely employed methodologies. For each method, the standard and several variations were tested varying sample storage and pre-lysis conditions, homogenization procedures, buffer solutions and concentrations, incubations and resuspension times and temperatures. Our results indicate that the quality and quantity of DNA extracted from fin clips and scales varied according to extraction methods and storage conditions. Alcohol preservation at -20°C is fundamental for fin specimens, although dried scales conservation at room temperature allowed good DNA extraction. One percent agarose electrophoresis revealed that WGDPK extracted good DNA from pike and trout fin and scales; Chelex provided scarce DNA and WMDPF, NSF, C-TAB and Trizol protocols were inadequate because no clear band or just a smearing could be visible. In conclusion, the modified procedures allowed a standardization of results, using a commercial kit (WGDPK) which gave stable, easily reproduced and good quality DNA extraction from conservative samples, avoiding costs and time wasting. The use of dried scales, stored for 1 week up to 34 years, allows the analysis of ancient public or private collections, particularly widespread for Salmonids (Nielsen et al., 1999). DNA suitability was successfully tested through two molecular markers currently used for conservation genetics purposes. The solidity of the results obtained was tested through the genotyping errors analysis, fundamental for conservative approaches (Hoffman & Amos, 2005; Roon et al., 2005). Detailed experimental procedures can be requested to the authors at (livia@unipg.it)