Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers

Marilena Perego, Cecilia Gelfi, Alexander V. Stoyanov, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

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, and G, 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.

Original languageEnglish
Pages (from-to)2915-2920
Number of pages6
JournalElectrophoresis
Volume18
Issue number15
DOIs
Publication statusPublished - Dec 1997

Keywords

  • Capillary electrophoresis
  • Conductivity
  • Isoelectric buffers
  • Oligonucleotidcs

ASJC Scopus subject areas

  • Clinical Biochemistry

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