Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.
Laboratory for Sensors, Department for Microsystem Engineering (IMTEK), Albert-Ludwigs-University Freiburg, Georges-Köhler-Allee 103, 79110 Freiburg, Germany.
Biosens Bioelectron. 2015 May 15;67:49-52. doi: 10.1016/j.bios.2014.06.003. Epub 2014 Jun 6.
With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.
采用快速简单的驱动方案，电泳核酸提取易于自动化，无需移动部件，易于小型化，并且具有尺寸相关的截止滤器，可通过水凝胶的孔径进行调节。然而，从细菌中进行电泳核酸提取迄今为止主要应用于短 RNA 靶标。原因之一是由于长度的原因，未片段化的基因组 DNA 链的电泳处理耗时较长。在这里，DNA 片段化会加速提取和分离。我们介绍了一种在芯片上进行的裂解和非酶 DNA 切割，然后直接进行纯化步骤，可在不到 25 分钟的时间内从细菌中获得可扩增的 DNA 片段。与限制性内切酶不同，芬顿反应已知可以无核苷酸特异性地切割 DNA。反应混合物包含铁(II)EDTA、抗坏血酸钠、过氧化氢和溶菌酶。通过试剂的浓度可以调节片段化的程度。该结果使得电泳提取方法能够在短时间内非特异性地处理长基因组 DNA，例如在微流控芯片上用于病原体检测。
