Sinville Rondedrick, Coyne Jennifer, Meagher Robert J, Cheng Yu-Wei, Barany Francis, Barron Annelise, Soper Steven A
Center for BioModular Multi-Scale Microsystems, Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Electrophoresis. 2008 Dec;29(23):4751-60. doi: 10.1002/elps.200800197.
We have developed a new method for the analysis of low abundant point mutations in genomic DNA using a combination of an allele-specific ligase detection reaction (LDR) with free-solution conjugate electrophoresis (FSCE) to generate and analyze the genetic products. FSCE eliminates the need for a polymer sieving matrix by conjugating chemically synthesized polyamide "drag-tags" onto the LDR primers. The additional drag of the charge-neutral drag-tag breaks the linear scaling of the charge-to-friction ratio of DNA and enables size-based separations of DNA in free solution using electrophoresis with no sieving matrix. We successfully demonstrate the conjugation of polyamide drag-tags onto a set of four LDR primers designed to probe the K-ras oncogene for mutations highly associated with colorectal cancer, the simultaneous generation of fluorescently labeled LDR/drag-tag conjugate (LDR-dt) products in a multiplexed, single-tube format with mutant:WT ratios as low as 1:100, respectively, and the single-base, high-resolution separation of all four LDR-dt products. Separations were conducted in free solution with no polymer network using both a commercial capillary array electrophoresis (CAE) system and a PMMA microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the EOF. Typical analysis times for LDR-dt were 11 min using the CAE system and as low as 85 s for the PMMA microchips. With resolution comparable to traditional gel-based CAE, FSCE along with microchip electrophoresis decreased the separation time by more than a factor of 40.
我们开发了一种新方法，通过将等位基因特异性连接酶检测反应（LDR）与自由溶液共轭电泳（FSCE）相结合来分析基因组DNA中的低丰度点突变，以生成和分析遗传产物。FSCE通过将化学合成的聚酰胺“拖尾标签”与LDR引物共轭，消除了对聚合物筛分基质的需求。电荷中性拖尾标签的额外拖曳打破了DNA电荷与摩擦比的线性比例关系，并能够在无筛分基质的自由溶液中使用电泳按大小分离DNA。我们成功地证明了将聚酰胺拖尾标签共轭到一组四个LDR引物上，这些引物旨在探测与结直肠癌高度相关的K-ras癌基因中的突变；以低至1:100的突变体：野生型比例在多重单管格式中同时生成荧光标记的LDR/拖尾标签共轭物（LDR-dt）产物；以及对所有四个LDR-dt产物进行单碱基高分辨率分离。使用商业毛细管阵列电泳（CAE）系统和通过热压复制的PMMA微芯片在无聚合物网络的自由溶液中进行分离，仅使用含有添加剂以抑制电渗流的基于Tris的运行缓冲液。使用CAE系统时，LDR-dt的典型分析时间为11分钟，而PMMA微芯片的分析时间低至85秒。与传统的基于凝胶的CAE分辨率相当，FSCE与微芯片电泳一起将分离时间缩短了40多倍。
