Du Ming, Flanagan James H, Lin Bingcheng, Ma Yinfa
Department of Chemistry, University of Missouri-Rolla, Rolla, MO 65409, USA.
Electrophoresis. 2003 Sep;24(18):3147-53. doi: 10.1002/elps.200305572.
The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.
DNA 中点突变及其他简单突变的检测对于癌症研究、诊断及其他生物学研究至关重要。毛细管电泳已成功用于分离 DNA 片段。然而，尚未见有用于 DNA 分离的具有在线涂层的低粘度聚合物筛分缓冲液的报道。本文展示了一种采用毛细管电泳结合在线涂层和激光诱导荧光检测（CE-LIF）来筛选 DNA 点突变或简单突变的新方法。该方法采用在线动态涂层技术，可延长毛细管使用寿命并提高分析重现性，还使用了低粘度聚合物溶液，这使得用户能够快速冲洗毛细管并轻松重新填充聚合物溶液。实验证明，用于在线毛细管涂层的分离缓冲液中的添加剂不会影响运行缓冲液的分离效率，也不会干扰硼酸、甘露醇和羟丙基甲基纤维素聚合物之间氢键网络的形成。对动态涂层毛细管的稳定性进行了定量研究；与永久涂层的 CE 柱相比，毛细管使用寿命提高了 6 至 7 倍。在含突变的 DNA 片段经 CEL-I 核酸内切酶切割后，该系统成功分离并检测了大小为 209、219 和 338 个碱基对的含突变标准 DNA 片段。该技术对低范围、中范围和高范围 DNA 片段的大小分离非常灵敏。将结果与 Transgenomic 公司开发的 HPLC 方法进行比较，结果吻合良好。该方法应适用于所有相关生物学和临床研究中的突变检测。本文还讨论了影响分离及动态毛细管涂层稳定性的因素。
