Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers.
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.
Perego
M
M
Department of Agricultural and Industrial Biotechnologies, University of Verona, Italy.
Gelfi
C
C
Stoyanov
A V
AV
Righetti
P G
PG
D000143:Acids; D001482:Base Composition; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D009841:Oligonucleotides / Q000302:isolation & purification*; D017186:Titrimetry
D000143:Acids; D002021:Buffers; D009841:Oligonucleotides