Tissue preservation and total DNA extraction from fish stored at ambient temperature using buffers containing high concentration of urea
Tissue preservation and total DNA extraction from fish stored at ambient temperature using buffers containing high concentration of urea
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DOI：
10.2331/fishsci.62.727
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发表时间：
1996-10-01
影响因子：
1.9
通讯作者：
Nakayama, I
中科院分区：
文献类型：
--
作者：
Asahida, T;Kobayashi, T;Nakayama, I
我们通过修改用于 DNA 分离的细胞裂解缓冲液，开发了一种用于 DNA 提取的高浓度尿素缓冲液（TNES-Urea：6 或 8 M 尿素；10 mM Tris-HCl，pH 7.5；125 mM NaCl；10 mM EDTA；1% SDS），我们发现该缓冲液适用于在环境温度下长期保存鱼类组织样本，以及从富含细胞的鱼类中提取 DNA。核酸内切酶。日本牙鲽牙鲆和大西洋鲱鱼 Clupea harengus 的组织样本在环境温度（10-36 摄氏度）下在 TNES-Urea 缓冲液中保存 1 个月至 3 年，而从瑞典运输到日本的大西洋鲱鱼的组织样本则保存在 TNES-Urea 缓冲液中。每条鱼的总DNA是从保存1个月至3年的肌肉或肝脏组织中提取的。 DNA产量为0.5-2.6μg总DNA/mg组织。所有来自保存组织的 DNA 都适合进行 DNA 分析，例如聚合酶链反应 (PCR) 技术、Southern blot 分析和随机扩增多态性 DNA (RAPD) 分析。 TNES-尿素缓冲液提供了一种方便的组织保存和 DNA 提取方法，并为以前需要在某些现场设置中受到限制的方案的方法提供了替代方案。
We have developed a high concentration urea containing buffer (TNES-Urea: 6 or 8 M urea; 10 mM Tris-HCl, pH 7.5; 125 mM NaCl; 10 mM 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 degrees 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 mu g 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.