Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format
Evaluation of silica resins for direct and efficient extraction of DNA from complex biological matrices in a miniaturized format
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DOI：
10.1006/abio.2000.4577
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发表时间：
2000-08-01
影响因子：
2.9
通讯作者：
Landers, JP
中科院分区：
文献类型：
--
作者：
Tian, HJ;Hühmer, AFR;Landers, JP
为了将DNA纯化功能集成到微芯片中进行高通量DNA分析，必须开发一种小型化的纯化方法，该方法可以很容易地适应微芯片格式。在这项研究中，我们评估了各种硅树脂的有效性，小型化的DNA纯化和衡量芯片上的固相萃取的潜在用途。一个微固相萃取（亩SPE）装置只含有纳克的二氧化硅树脂被证明是有效的吸附和解吸的DNA在程序纳克质量范围内。荧光光谱法以及毛细管电泳与激光诱导荧光检测用于分析从固相树脂回收的DNA，而聚合酶链反应（PCR）是用来评估洗脱的DNA的可重复性。我们证明，DNA可以直接从白色细胞中回收，效率约为70%，而大于80%的蛋白质被去除，500-nl床体积mu SPE过程，需要不到10分钟。容量在10-30 ng/mg硅胶树脂范围内，我们表明，从白色细胞，培养的癌细胞，甚至低微升规模的全血也适用于直接PCR扩增。DNA提取的小型化格式以及快速时间框架与微制造芯片上的快速电泳兼容。(C)北京大学出版社.
For DNA purification to be functionally integrated into the microchip for high-throughput DNA analysis, a miniaturized purification process must be developed that can be easily adapted to the microchip format. In this study, we evaluate the effectiveness of a variety of silica resins for miniaturized DNA purification and gauge the potential usefulness for on-chip solid-phase extraction. A micro-solid-phase extraction (mu SPE) device containing only nanograms of silica resin is shown to be effective for the adsorption and desorption of DNA in the program-nanogram mass range. Fluorescence spectroscopy as well as capillary electrophoresis with laser-induced fluorescence detection is employed for the analysis of DNA recovered from solid-phase resins, while the polymerase chain reaction (PCR) is used to evaluate the amplifiable nature of the eluted DNA. We demonstrate that DNA can be directly recovered from white blood cells with an efficiency of roughly 70%, while greater than 80% of the protein is removed with a 500-nl bed volume mu SPE process that takes less than 10 min. With a capacity in the range of 10-30 ng/mg of silica resin, we show that the DNA extracted from white blood cells, cultured cancer cells, and even whole blood on the low microliter scale is suitable for direct PCR amplification. The miniaturized format as well as rapid time frame for DNA extraction is compatible with the fast electrophoresis on microfabricated chips. (C) 2000 Academic Press.