TY - JOUR
T1 - Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers
AU - Perego, Marilena
AU - Gelfi, Cecilia
AU - Stoyanov, Alexander V.
AU - Righetti, Pier Giorgio
PY - 1997/12
Y1 - 1997/12
N2 - 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.
AB - 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.
KW - Capillary electrophoresis
KW - Conductivity
KW - Isoelectric buffers
KW - Oligonucleotidcs
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U2 - 10.1002/elps.1150181532
DO - 10.1002/elps.1150181532
M3 - Article
C2 - 9504830
AN - SCOPUS:0031473069
VL - 18
SP - 2915
EP - 2920
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 15
ER -