Williams P E, Marino M A, Del Rio S A, Turni L A, Devaney J M
Armed Forces DNA Technology Development Branch, Armed Forces Institute of Pathology, Gaithersburg, MD 20877.
J Chromatogr A. 1994 Oct 7;680(2):525-40. doi: 10.1016/0021-9673(94)85152-2.
Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.
毛细管电泳（CE）是一种用于分析DNA限制性片段和通过聚合酶链反应（PCR）扩增的DNA的新型高分辨率工具。通过将传统平板凝胶方法的许多原理整合到毛细管形式中，可以对人和植物的PCR扩增产物以及DNA限制性片段进行分子大小测定。通过动态筛分电泳（DSE）和毛细管凝胶电泳（CGE）对DNA限制性片段和PCR产物进行分析。作为本研究的一部分，对样品制备程序、进样模式和分子量标记物的使用进行了评估。使用uPage-3（3%T，3%C）CGE柱在260nm处进行紫外检测实现了最佳分离。膜透析和超滤/离心被证明是几乎等效的样品制备方法。重现性研究表明，平端、非磷酸化标记物（特别是等位基因生成标记物）为PCR产物分析提供了最准确的校准。本研究表明，与传统方法相比，CE提供了一种用于准确确定分子大小和/或等位基因类型的高速、高分辨率分析方法。
