Perego M, Gelfi C, Stoyanov A V, Righetti P G
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Electrophoresis. 1997 Dec;18(15):2915-20. doi: 10.1002/elps.1150181532.
A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18-mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3-4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co-migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.
报道了一种基于在相当酸性pH值下的自由区毛细管电泳（CZE）分析相同长度但带有单个碱基替换的寡核苷酸的新方法。为此，合成了一组四个碱基组成相当随机的18聚体，在第9个核苷酸处带有以下碱基：T、C、G或A。基于单个游离核苷酸滴定曲线的理论预测使我们能够预测，在pH 3-4范围内有可能同时分离所有四种寡核苷酸的混合物。事实上，在pH 5.7下进行电泳得到一个单一的不对称峰，而在pH 4.8下的CZE可以分离出四种中的三种（T9和G9寡核苷酸共迁移到一个单一区域）。在含有两性物质（等电亚氨基二乙酸，IDA）和7 M尿素的缓冲液中，在pH 3.3时可获得独特的分离能力。尽管IDA的pI为2.23（对于100 mM溶液），但加入7 M尿素（使寡核苷酸变性所必需）将溶液的表观pH提高到3.3。
