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.
Highlights
Reliability, feasibility and reproducibility of molecular genetics studies are often limited by the preliminary step of DNA isolation
DNA damage may occur during the extraction procedure due to oxidation and enzymatic hydrolysis problems, associated with extraction buffers formulation [1] and excessive mechanical shearing [2]
The Automatic Nucleic Acid Isolation System (QuickGene 810, Fujifilm Life Science) associated with the modifications in QuickGene DNA Tissue kit were applied with great success to different and large numbers of mollusc species
Summary
Reliability, feasibility and reproducibility of molecular genetics studies are often limited by the preliminary step of DNA isolation. The obtainment of great amounts of high quality DNA from small quantities of tissue is often a laborious task. DNA extraction methods should ideally be straightforward, quick, efficient, and reproducible while minimizing the potential for cross-contamination. It should be suitable for extracting multiple samples and generate minimal risk for the operator. DNA quality is a critical issue for most amplification-based analysis, since the DNA amplification is influenced by the presence of co-purifying inhibitors from matrix or extraction reagents, which can reduce subsequent PCR efficiency. DNA damage may occur during the extraction procedure due to oxidation and enzymatic hydrolysis problems, associated with extraction buffers formulation [1] and excessive mechanical shearing [2]
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