Tissue Preservation and Total DNA Extraction form Fish Stored at Ambient Temperature Using Buffers Containing High Concentration of Urea

Abstract

We have developed a high concentration urea containing buffer (TNES-Urea: 6 or 8M urea; 10mM Tris-HCl, pH 7.5; 125mM NaCl; 10mM EDTA; 1% SDS) for DNA extraction by modifying the cell lysis buffer for DNA isolation, and we found this buffer is suitable for long-term preservation of tissue samples from fish at ambient temperatures and for DNA extraction from fish that are rich in cellular endonucleases. Tissue samples from the Japanese flounder Paralichthys olivaceus and Atlantic herring Clupea harengus were preserved for periods ranging from 1 month to 3 years and for the Atlantic herring transported from Sweden to Japan in TNES-Urea buffer at ambient temperature (10-36°C). The total DNA for each fish was extracted from the muscle or liver tissue which had been preserved for periods ranging from 1 month to 3 years. The DNA yield was 0.5-2.6 μg of total DNA/mg tissue. All DNA from preserved tissues was suitable for DNA analyses, e. g. Polymerase Chain Reaction (PCR) technique, Southern blot analysis and Random amplified polymorphic DNA (RAPD) analysis. The TNES-Urea buffer provides a convenient method of tissue preservation and DNA extraction and offers an alternative to previous methods which require protocols that are restrictive in some field settings.