International Iberian Nanotechnology Laboratory (INL), Avenida Mestre José Veiga 4715-310, Braga, Portugal.
Department of Analytical Chemistry, Faculty of Veterinary Science, University of Santiago de Compostela, 27002 Lugo, Spain.
Anal Chim Acta. 2018 Aug 22;1020:30-40. doi: 10.1016/j.aca.2018.02.079. Epub 2018 Mar 13.
Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of "bench-top" based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.
从含有微量 DNA 的复杂基质中进行样品制备，可以通过固相萃取 (SPE) 基于设备的小型化而极大地受益于增加的表面积与体积比。然而，将基于“台式”的 DNA 纯化协议适用于小型化设备并不像看起来那么简单，需要考虑几个问题。需要仔细评估 DNA 提取和纯化协议，考虑到这些样品的复杂性，并为了便于与后续步骤（通常是 DNA 扩增）集成。在这项工作中，开发了一种基于微尺度固相萃取 (μSPE) 的可清洗和可重复使用的微型 DNA 纯化设备，其中包含商用一次性硅胶膜作为 DNA 捕获的固相。首先通过测试一组不同的条件来优化 DNA 纯化协议，包括分析过程中三个步骤中的缓冲液组成以及洗脱步骤中的孵育时间，目的是增加 DNA 产量并促进在小型化设置中的集成。然后，使用橄榄油样品测试了该协议，包括本工作中还开发和优化的预处理步骤。橄榄油样品的 DNA 分析对于检测与其他种子油的欺诈性掺假以及品种鉴定具有很高的兴趣。通过估计 DNA 产量和评估吸光度比 A260/280 和 A260/230，将结果与商用 NucleoSpin Food 试剂盒的效率和纯度进行了比较。微型 DNA 纯化设备的性能优于测试的商用试剂盒，这使得该方法成为橄榄油和其他微量 DNA 含量样品的非常有前途的样品制备方法。
