The integrity of all genomic DNA samples isolated from several individuals was analyzed by agarose gel electrophoresis. In Figure 1 it is possible to observe eleven examples of genomic DNA isolated from several species (Crassostrea gigas, Ostrea stentina, Ostrea edulis, Ostrea chilensis, Chamelea gallina, Ruditapes decussatus, Venerupis pullastra, Venerupis aurea, Anomia ephippium, Cerastoderma edule and Hexaplex trunculus) with almost no DNA fragmentation and a high molecular weight band.

Purity, concentration and yield of genomic DNA samples were estimated with NanoDrop^® ND-1000 (NanoDrop Technologies) with the purpose of evaluating parameters such as quality and quantity. The DNA purity and concentration was directly measured by NanoDrop^® ND-1000 system, while, the DNA yield was estimated, for each sample, comparing the quantity of genomic DNA obtained with the quantity of tissue used (cf. Material and Methods). The DNA isolation can be influenced by several factors like species, tissue preservation method and extraction procedure. In molluscs this procedure is known to be challenging due to the high amount of mucopolysaccharides and polyphenolic proteins present in these animals tissues.

From Table 1 it is possible to analyze the concentration, yield and purity of genomic DNA extracted from several species (N = 100). One of the important features of this protocol is the possibility of its application to a great variety of mollusc species. The average concentration of the total extracted genomic DNA of all samples was 271.8 ± 64.5 ng·μL⁻¹ (mean ± SE), ranging from 200.7–370.3 ng·μL⁻¹ (min-max). Typical DNA yield ranges from 1000–5000 ng·mg⁻¹ in animal tissue [16]. For the mollusc analyzed in this study, the extraction method generated adequate DNA yield, ranging from 823.6–5053.8 ng·mg⁻¹ (mean ± SE). Indeed, it is not easy to compare our results with the ones achieved by other methods and that are already published for mollusc species, as most of them use different types of tissues such as gills, rectum and mantle. In fact, these tissues are easier for DNA isolation but in the end the DNA could be contaminated with alien DNA as for example from parasites. Nevertheless, in recent works published [15,16] with protocols for bivalves DNA extraction, the total DNA measured ranges between 0.5–250 μg, indicating similar values of high molecular weight DNA using this protocol, a mean of 27.2 ± 6.5 μg (mean ± SE).

There are several types of contaminations that can be acquired during the DNA extraction protocols, depending on the origin of the biological sample [12,13]. Phenolics and other secondary compounds cause damage of the DNA and/or inhibit enzymatic reactions. The quality of the samples evaluated in terms of RNA/protein and chaotropic salt contamination was respectively, 1.88 ± 0.04 and 1.83 ± 0.12 (mean ± SE) (Table 1), being all samples analyzed around the optimal value, for both quality standards. These minor levels of salt, protein or RNA contamination prevent interference in downstream molecular biology procedures and so, it is very important to maintain its levels to a minimum. Riemann et al. [17] suggested that quantity and quality of the isolated DNAs were slightly higher with manual extraction than automatic extraction methods. In our experience we notice that the automatic extraction is more efficient regarding the quality of genomic DNA, which is very important to downstream molecular procedures.

One of the main objectives of the protocol presented was the maximization of this process for small amounts of tissue, since one of the problems of genetic studies in molluscs is the scarcity of tissue. All the data was clustered in five different groups of weights and the mean of concentration, DNA yield and purity was quantified (Table 2). As can be observed in Table 2, the data demonstrates that the total genomic DNA yield is optimal in intervals of [0–5] and [5–10] mg, respectively 6887.30 ± 613.72 and 3577.16 ± 490.42 ng·mg⁻¹ (mean ± SE), meaning that with small quantities of tissue it was possible to obtain the highest yields of genomic DNA representing a prominent feature of this protocol. Moreover, the same company (i.e., the QuickGene 610) developed a new automatic DNA extraction system that allows a ten-fold starting amount (compared with the QuickGene 810), so correspondingly bigger amounts of DNA may be achieved. However, this system permits only the simultaneous handling of six samples.

The genomic DNA obtained with the presented methodology was of high quality regarding all standards employed. However, and as described by different authors, the quality and total DNA contents provided by NanoDrop do not accurately represents the quantity of DNA that is efficiently amplifiable by PCR [18,19]. In order to analyze the quality of amplifiable DNA, we PCR-amplified amplicons for the histone H3 gene in all samples. We also performed a random PCR that would ideally generate several DNA segments, like RAPDs, since this technique covers the entire genome.

PCR conditions were optimized in the genomic DNA samples obtained by the present method and the histone H3 gene was amplified with success in all species (Figure 2). This technique was also successfully applied in DNA extraction in population genetics studies using RAPDs already published [20,21] (Figure 3). Moreover, we also sequenced, with great success, specific genome fractions, major and minor ribosomal genes isolated from genomic DNA prepared with the methodology described here, being elucidative of the quality of the genomic DNA obtained. These sequences are available in GenBank sequence database with the following access numbers: JN797504, JN797505, JN797506 and JN797507.

Several species of bivalves, C. gallina (N = 10), V. aurea (N = 10), V. pullastra (N = 10), R. decussatus (N = 10) (Bivalvia: Veneridae), C. gigas (N = 10), O. stentina (N = 10), O. edulis (N = 10), O. chilensis (N = 10) (Bivalvia: Ostreidae), A. ephippium (N = 5) (Bivalvia: Anomiidae), C. edule (N = 10) (Bivalvia: Cardiidae) and one gastropod, H. trunculus (N = 5) (Gastropoda: Muricidae) (N = number of individuals), were collected from Ria Formosa populations, Algarve, Portugal. After two days of depuration, the samples were processed and placed in 70% ethanol at −20 °C, until further use.

Fresh adductor muscle tissue from different bivalves was used for DNA extraction, while for gastropod H. trunculus egg capsules were used. To extract the genomic DNA from all the animals we used the Automatic Nucleic Acid Isolation System (QuickGene 810, Fujifilm Life Science). This system uses a porous ultra thin membrane and an automatically pressurizing unit that promotes binding, washing and elution steps at low pressure. In order to apply this system to the isolation of genomic DNA from molluscs, several modifications were carried out: (a) the use of the “Pestle Pellet” and the adaption of 3–4 h of incubation ensured a more efficient digestion; (b) the increase of elution time in the automatic nucleic-acid isolation system QuickGene-810 was more efficient for the elution of genomic DNA, and consequently for the increasing of the DNA yield. Tissue samples preserved in 70% ethanol, were washed with 1× PBS and distilled water for 10 min each. A section of tissue (about 5–30 mg of tissue) was cut in small pieces followed by the addition of 180 μL of MDT (tissue lysis buffer) and 20 μL of EDT (buffer with Proteinase K) in a 2 mL eppendorf. The samples were homogenised with the aid of a “Pellet Pestle”, vortexed briefly and incubated at 55 °C between 3 and 4 h. The eppendorfs were removed from incubation and at this point if any debris are present at the lyses, is recommended to remove it by centrifugation (10,000 g, 3 min). The supernatant was carefully transferred to a new 2 mL eppendorf. A volume of 180 μL of LDT (buffer solution) was added and mixed thoroughly. This procedure must be performed in a vortex during 15 s and followed by a quick spin down. The solution was then incubated at 70 °C during 10 min and occasionally mixed with a vortex. At the end of the incubation step, a quick spin down was performed. An ethanol volume of 240 μL of 100% (v/v) was added and mixed very well. The lysate was then transferred to a cartridge of the automatic nucleic-acid isolation system QuickGene-810 and the “DNA tissue mode” was selected with a major modification in the elution time to maximum. A standardized final volume of 100 μL was used and the samples of genomic DNA were ready to be used immediately or stored at −20 °C for several months.

NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Inc.) was used to measure the absorbance. The values of absorbance (A) allowed estimating the purity, concentration and yield of the genomic DNA samples. Pure DNA exhibited an A₂₆₀/A₂₈₀ ratio (RNA/protein contamination) and an A₂₆₀/A₂₃₀ ratio (chaotropic salt contamination) in the range of 1.8–2.0. To compare the efficiency of DNA extraction on various tissue weights, the DNA yield (DNA_ng/Tissue weight_mg) was estimated.

To assess the DNA quality, several standard molecular laboratory procedures were performed: a PCR for amplification of the histone H3 gene and RAPDs according to Zhang et al. 2007 [22] and Pereira et al. 2010 [21], respectively. The integrity of the genomic DNA samples extracted as well as the PCR amplification samples were analyzed by electrophoresis on a 2% agarose gel with O’GeneRuler^TM DNA Ladder Mix (Fermentas, Glen Burnie, MD, USA). After electrophoresis run at 75 volts, for 1 h, the DNA bands were observed under UV light and the images were saved in a gel analyser (UVIDOC).