Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
Electrophoresis. 2013 Feb;34(4):590-7. doi: 10.1002/elps.201200462. Epub 2013 Jan 24.
We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated "sample-in/answer-out" devices with amplification, LDR, and detection all on one platform.
我们在这里展示了自由溶液共轭电泳（FSCE）作为一种无需筛分聚合物网络即可通过电泳分离 DNA 片段的方法的强大功能和灵活性。先前的工作介绍了将 FSCE 与连接酶检测反应（LDR）耦合，以检测点突变，即使与野生型 DNA 相比丰度较低也是如此。在这里，使用四个大的拖曳标签来实现 19 个 LDR 产物的自由溶液电泳分离，这些产物的大小范围为 42 到 66 个核苷酸，对应于 K-ras 癌基因中的突变。LDR-FSCE 通过 CE 在 13.5 分钟内（E = 310 V/cm）和通过微芯片电泳在 140 秒内（E = 350 V/cm）实现了这些 19 个 LDR-FSCE 产物的电泳分辨率。FSCE 的强大功能体现在自由溶液分离的独特特征中，无论电场强度如何，分离分辨率都是恒定的。通过微芯片电泳，电场被增加到电源的最大值（E = 700 V/cm），19 个 LDR-FSCE 产物在不到 70 秒的时间内分离，分辨率几乎与 E = 350 V/cm 时的分离相同。这些结果将有助于在具有扩增、LDR 和检测的集成“样品进/答案出”设备上筛选 K-ras 突变的目标，所有这些都在一个平台上。
