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Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers
Author(s) -
Perego Marilena,
Gelfi Cecilia,
Stoyanov Alexander V.,
Righetti Pier Giorgio
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181532
Subject(s) - chemistry , capillary electrophoresis , chromatography , oligonucleotide , electrophoresis , isoelectric focusing , isoelectric point , titration , base (topology) , urea , iminodiacetic acid , analytical chemistry (journal) , biochemistry , inorganic chemistry , dna , chelation , mathematical analysis , mathematics , enzyme
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 T 9 and G 9 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 p I of 2.23 (for a 100 m M solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.