Department of Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, 421 Washington Ave. SE, Minneapolis, MN 55455, USA.
Lab Chip. 2019 Jan 15;19(2):281-290. doi: 10.1039/c8lc01163j.
A number of outstanding problems in genomics, such as identifying structural variations and sequencing through centromeres and telomeres, stand poised to benefit tremendously from emerging long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels. However, optimal application of these new genomics technologies requires facile methods for extracting long DNA from cells. These sample preparation tools should be amenable to automation and minimize fragmentation of the long DNA molecules by shear. We present one such approach in a poly(dimethylsiloxane) device, where gel-based high molecular weight DNA extraction and continuous flow purification in a 3D cell culture-inspired geometry is followed by electrophoretic extraction of the long DNA from the miniaturized gel. Molecular combing reveals that the device produces molecules that are typically in excess of 100 kilobase pairs in size, with the longest molecule extending up to 4 megabase pairs. The microfluidic format reduces the standard day-long and labor-intensive DNA extraction process to 4 hours, making it a promising prototype platform for routine long DNA sample preparation.
基因组学中有许多突出问题，例如识别结构变异和通过着丝粒和端粒进行测序，这些问题都将极大地受益于新兴的长读长基因组学技术，如纳米孔测序和纳米通道基因组图谱。然而，这些新技术的最佳应用需要从细胞中提取长 DNA 的简便方法。这些样品制备工具应易于自动化，并通过剪切最小化长 DNA 分子的片段化。我们在聚二甲基硅氧烷器件中展示了一种这样的方法，其中基于凝胶的高分子量 DNA 提取和在 3D 细胞培养启发的几何形状中的连续流纯化，随后通过从微型化凝胶中电泳提取长 DNA。分子梳理表明，该器件产生的分子通常超过 100 千碱基对，最长的分子可延伸至 400 万个碱基对。微流控格式将标准的耗时一整天且劳动强度大的 DNA 提取过程缩短至 4 小时，使其成为常规长 DNA 样品制备的有前途的原型平台。
