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An efficient method for genomic DNA extraction from different molluscs species.

ABSTRACT: The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

SUBMITTER: Pereira JC

PROVIDER: S-EPMC3233457 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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An efficient method for genomic DNA extraction from different molluscs species.

Pereira Jorge C JC Chaves Raquel R Bastos Estela E Leitão Alexandra A Guedes-Pinto Henrique H

International journal of molecular sciences 20111117 11

The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA ...[more]

PMID: 22174651

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Project description:BackgroundDNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S).ResultsOverall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did.ConclusionsMuseum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.

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An efficient method for genomic DNA extraction from different molluscs species.

Publications

An efficient method for genomic DNA extraction from different molluscs species.

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