A rapid, nondestructive, reproducible and cheap DNA extraction method from body mucus and buccal cells of northern pike and brown trout is described. Buccal cells and body mucus were sampled on FTA Cards; the captured DNA was used directly for microsatellite and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses. A complete concordance with control DNA was found. The genotyping error rate for microsatellite ranged from 1.9% to 3.3% for the northern pike and brown trout, respectively. This methodology, using for the first time these materials as a fish DNA source, combines speed of sampling and processing, with a twofold to a threefold time and costs saving.

No abstract

No abstract

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 (Nielsen et al. 1999;Wasko et al. 2003;Hansen and Jensen 2005;Lucentini et al. 2006). 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 [Wizard Genomic DNA Purification Kit (WGDPK) (Promega); Wizard Magnetic DNA Purification System for Food (WMDPF) (Promega); NucleoSpin Food (NSF) (Macheray-Nagel)] and three are largely employed methodologies [Trizol (Life Technologies); Chelex (Sigma-Aldrich); C-TAB]. For each method, the standard protocol (reported on data sheets or the first published one) and several variations were tested varying sample storage and pre-lysis conditions, homogenization procedures, buffer solutions and concentrations, incubations and resuspension times and temperatures. DNA was extracted from 200 northern pikes (Esox lucius L.) and 100 brown trout (Salmo trutta fario L.) (Table 1) from small (£10 mg) fin pieces and 5-10 scales stored dried or in absolute alcohol both at )20°C and at room temperature. DNA extraction was carried out on fresh materials and repeated within a period of three years (Table 1), and from 34 years old dried scales culled from a museum collection. Furthermore, DNA was extracted from liver and muscle of both species, from individuals that had naturally died and used as controls. Spectrophotometric readings (260 and 280 nm) and densitometric evaluations (Image J) of DNAs obtained through different methodologies and protocols (conservative versus control DNA) were analyzed by means of ANOVA and t-test. DNA suitability was assayed through PCR-RFLP and microsatellite analyses (Lucentini et al. (2006) (Table 2). Microsatellites were run on ABI377 DNA sequencer whereas PCR-RFLPs were assayed on 2.5% agarose electrophoresis for two mtDNA NADH coding regions (ND-1 ND5/ 6) (Cronin et al. 1993). DNA stability and amplificability was controlled every three months, for three years, in parallel with control DNA. To evaluate genotyping errors, null alleles presence and allelic dropout, all the experiments were replicated and statistically treated as suggested (Hoffman and Amos 2005; Roon et al. 2005) through MICRO-CHECKER 2.2.3 (Van oosterhout et al. 2004). Deviations from the Hardy-Weinberg equilibrium were tested by Arlequin 2000 and Genepop.Our results indicate that the quality and quantity of DNA extracted from fin clips and scales varied according to extraction methods (Table 2) 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

No abstract