Capillary electrophoresis of DNA in the 20–500 bp range: recent developments

Abstract

The present mini-review summarizes recent developments in the field of DNA separations by capillary zone electrophoresis (CZE), as developed by our group. Separation of antisense oligonucleotides in sieving liquid polymers in isoelectric buffers is first discussed. It is shown that the use of isoelectric buffers (notably His) permits very high voltage gradients (up to 1000 V/cm) with much reduced transit times and increased resolution of all truncated and failed sequences. Oligonucleotides can also be analyzed by zone electrophoresis against a stationary pH gradient (typically a pH 6.5–10 range): if injected at the alkaline end, the sample components experience stacking and zone sharpening due to modulation of charge as the oligonucleotides move along the pH gradient. Oligonucleotides having the same length, but differing by one single nucleotide in the chain, can be separated in free solution (i.e., in the absence of a sieving matrix) at strongly acidic pH values (pH 3.0–3.3) where charge differences due to base protonation are maximized. By working in free solution, it has also been possible to measure accurately the free mobility of DNAs, shown to reach a constant value of 3.75±0.04·10−4 cm2 V−1 s−1 at 25°C and in Tris–acetate–EDTA buffer, pH 8.3, above a critical length of ca. 400 bp. However, when double-stranded, rather than single-stranded, DNA is analyzed in isoelectric His buffer, some peculiar phenomena are observed: improved resolution for smaller DNA fragments (up to ca. 150 bp) and a rapid deterioration of resolution above this critical length. Direct binding of His to the DNA helix is hypothesized, via a bidentate salt bridge of the two charged amino groups of His on the negatively charged oxygen of the phosphate group. Upon extensive binding, occupying every available phosphate site, π–π interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.