Cordier Y, Roch O, Cordier P, Bischoff R
Transgène SA, Strasbourg, France.
J Chromatogr A. 1994 Oct 7;680(2):479-89. doi: 10.1016/0021-9673(94)85146-8.
Chemically synthesized oligodeoxyribonucleotides were subjected to capillary gel electrophoresis on three different polyacrylamide-based matrices. Analysis of about 1000 samples over a 1-year period showed that the gel matrix evolved with time resulting in shifting migration times, making it essential to use an internal standard. Cross-linked polyacrylamide matrices had the highest stability, allowing an average of 100 injections on the same capillary. Computer-aided prediction of migration times was subsequently evaluated to confirm the size and base composition of oligonucleotides more accurately. A number of problems were noted when using this approach on a routine basis, such as insufficient stability of the gel matrices, effects of secondary structure on migration and insufficient differences in migration times for oligonucleotides containing > 50 bases. Capillary gel electrophoresis at pH 3.5 in replaceable gels showed that migration was mainly dependent on the charge per base ratio resulting in separations of significantly altered selectivity which complemented analyses under the commonly used basic pH conditions.
对化学合成的寡脱氧核糖核苷酸在三种不同的基于聚丙烯酰胺的基质上进行毛细管凝胶电泳。在一年的时间里对约1000个样品进行分析表明，凝胶基质随时间演变，导致迁移时间发生变化，因此使用内标至关重要。交联聚丙烯酰胺基质具有最高的稳定性，在同一根毛细管上平均可进行100次进样。随后对迁移时间的计算机辅助预测进行了评估，以更准确地确认寡核苷酸的大小和碱基组成。在常规使用这种方法时发现了一些问题，例如凝胶基质稳定性不足、二级结构对迁移的影响以及含>50个碱基的寡核苷酸迁移时间差异不足。在可替换凝胶中于pH 3.5条件下进行的毛细管凝胶电泳表明，迁移主要取决于每碱基电荷比，从而导致选择性发生显著改变的分离，这补充了常用碱性pH条件下的分析。
