Abstract

During the last few years, an important element in the improvement of the molecular biology techniques has been the necessity for availability of high quality and functionality DNA. Several DNA extraction procedures with different results in both performance and quality, have been proposed. In this study our objective was to determine the most reliable extraction method that balances DNA quantity, and to assess the sample quantification of the fluorometric DNA quantification methods. For this, blood extracted by venopunction from 20 healthy volunteers was used to obtain DNA from buffy coat, and 4 commercial DNA extraction kits were assessed as well as two fluorometric DNA quantification methods with protocols of different complexity. Results suggest that manual methods achieve higher quality and larger yields of DNA. DNA purity obtained with the 4 extraction kits evaluated through the 260/280 and 260/230 ratio showed that the Qiacube kit fulfilled the criteria established in this work, followed very close by the Flexigene kit. On the other hand, the fluorometric DNA methods used in the samples quantification showed a higher variability when using QuantiFlour method, obtaining better results probably due to the simplicity of this protocol.

摘要

在过去的几年中，分子生物学技术改进的一个重要因素是获得高质量和功能性 DNA 的必要性。已经提出了几种在性能和质量方面具有不同结果的 DNA 提取程序。在本研究中，我们的目标是确定平衡 DNA 数量的最可靠的提取方法，并评估荧光 DNA 定量方法的样品定量。为此，使用静脉穿刺从 20 名健康志愿者身上提取的血液来从血沉棕黄层中获取 DNA，并评估了 4 种商业 DNA 提取试剂盒以及两种具有不同复杂性方案的荧光 DNA 定量方法。结果表明，手动方法可以获得更高的 DNA 质量和产量。通过 260/280 和 260/230 比率评估的 4 个提取试剂盒获得的 DNA 纯度表明，Qiacube 试剂盒满足本工作中建立的标准，其次是 Flexigene 试剂盒。另一方面，使用 QuantiFlour 方法时，样品定量中使用的荧光 DNA 方法显示出更高的变异性，可能由于该方案的简单性而获得了更好的结果。