Department of Animal Production, College of Agriculture, Al-Qasim Green University, Babil, Iraq.
Mol Biol Rep. 2023 Apr;50(4):3963-3968. doi: 10.1007/s11033-023-08317-4. Epub 2023 Feb 24.
DNA extraction is an essential step for many genetic techniques like PCR and other molecular analyses. Based on the method of extraction and type of tissue used, the quality of extracted DNA for genetic studies varies. An appropriate extraction method is evaluated by the high concentration and purity of DNA. Thus, this study aimed to find a more efficient and effective method of DNA extraction from fish tissues and compare it to commercially available kits.
A total of 200 fish tissue samples were extracted using each method and then validated with restriction enzymes and PCR amplification. The result revealed that the mean quantity of the isolated genomic DNA, when measured by Nanodrop for grass and common carp, was estimated at (624.41 ± 34.51) µg/ml and (651.27 ± 46.31) µg/ml, respectively, and the purity of this DNA was about (1.83 ± 0.04) and (1.88 ± 0.03) respectively, as compared to commercial extraction kits. Furthermore, gel electrophoresis was performed on the PCR-ready DNA, and the results were confirmed with restriction enzymes and PCR amplification. Based on results obtained from restriction enzymes and PCR analysis, it was determined that no significant inhibitors existed for the enzymes that were used in molecular biology reactions.
As a result, this technique provides an efficient and versatile alternative to the traditional method for obtaining bulk amounts of highly qualified DNA from fish tissue and can be easily used for subsequent analyses such as PCR and several molecular experiments on other fish species.
DNA 提取是许多遗传技术(如 PCR 和其他分子分析)的重要步骤。基于提取方法和组织类型的不同,用于遗传研究的提取 DNA 的质量也有所不同。适当的提取方法可以通过 DNA 的高浓度和纯度来评估。因此,本研究旨在寻找一种更有效和高效的从鱼类组织中提取 DNA 的方法,并将其与市售试剂盒进行比较。
使用每种方法从总共 200 个鱼类组织样本中提取 DNA,然后用限制性内切酶和 PCR 扩增进行验证。结果表明,当用 Nanodrop 测量草和鲤鱼时,分离的基因组 DNA 的平均数量估计分别为(624.41±34.51)μg/ml 和(651.27±46.31)μg/ml,并且 DNA 的纯度分别约为(1.83±0.04)和(1.88±0.03)。与商业提取试剂盒相比。此外,对 PCR 准备好的 DNA 进行凝胶电泳,并用限制性内切酶和 PCR 扩增进行验证。基于从限制性内切酶和 PCR 分析获得的结果,确定不存在对用于分子生物学反应的酶有显著抑制作用的抑制剂。
因此,该技术为从鱼类组织中获得大量高质量 DNA 提供了一种高效且通用的替代传统方法,并且可以轻松用于后续分析,如 PCR 和其他鱼类物种的几种分子实验。