Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.
Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device.
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DOI：
10.1002/elps.200800197
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发表时间：
2008-12
期刊：
影响因子：
2.9
通讯作者：
Soper, Steven A.
中科院分区：
文献类型：
--
作者：
Sinville, Rondedrick;Coyne, Jennifer;Meayher, Robert J.;Cheng, Yu-Wei;Barany, Francis;Barron, Annelise;Soper, Steven A.
关键词：
我们开发了一种新的分析基因组DNA中低丰度点突变的方法，使用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）的组合来产生和分析基因产物。FSCE通过将化学合成的聚酰胺“拖拽标签”缀合到LDR引物上而消除了对聚合物筛分基质的需要。电荷中性拖曳标签的额外拖曳打破了DNA的电荷摩擦比的线性缩放，并且使得能够使用电泳在没有筛分基质的情况下在游离溶液中基于尺寸分离DNA。我们成功地证明了聚酰胺拖动标签到一组四LDR引物的共轭设计，以探测K-ras癌基因的突变高度相关的结直肠癌，荧光标记的LDR/拖动标签（LDR-dt）产品的同时生成在一个多路复用，单管格式与突变：野生型的比例低至1：100，分别和所有四个LDR-dt产品的单碱基，高分辨率分离。在没有聚合物网络的自由溶液中进行分离，使用商业毛细管阵列电泳（CAE）系统和聚（甲基丙烯酸甲酯），PMMA，通过热压印复制的微芯片，其中仅使用含有抑制电渗流（electroosmotic flow）的添加剂的基于Tris的运行缓冲液。使用CAE系统，LDR-dt缀合物的典型分析时间为11分钟，PMMA微芯片的分析时间低至85秒。与传统的基于凝胶的CAE的分辨率相当，FSCE沿着微芯片电泳将分离时间减少了40倍以上。
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-tagged (LDR-dt) products in a multiplexed, single-tube format with mutant:wild-type 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 poly(methylmethacrylate), PMMA, microchip replicated via hot-embossing with only a Tris-based running buffer containing additives to suppress the electroosmotic flow (EOF). Typical analysis times for LDR-dt conjugates 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.
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影响因子：
2.8
作者：
Hupert, Mateusz L.;Guy, W. Jason;Soper, Steven A.
通讯作者：
Soper, Steven A.
DOI：
10.1073/pnas.0507904102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Diehl, F;Li, M;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
5.6
作者：
Gerry, NP;Witowski, NE;Barany, F
通讯作者：
Barany, F
影响因子：
14.9
作者：
Luo, JY;Bergstrom, DE;Barany, F
通讯作者：
Barany, F
影响因子：
2.2
作者：
Rothschild, CB;Brewer, CS;Triscott, MX
通讯作者：
Triscott, MX