Sequence-based separation of single-stranded DNA at high salt concentrations in capillary zone electrophoresis.

Abstract

DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between single or hybridized strands or differences in thermal or chemical stability of hybridized strands. Previously, it was shown that four single-stranded DNA (ssDNA) 76-mers that differ by only a few A-G substitutions could be separated based solely on sequence by adding guanosine-5'-monophosphate to the running buffer in capillary zone electrophoresis (CZE). The separation was attributed to interactions of the ssDNA with self-assembled guanine-tetrad structures; however, subsequent studies of an expanded set of ten 76-mers showed that the separation was a more general phenomenon that occurred at high salt concentrations. With the long-term goal of using experimental and computational methods to provide insight into the basis of the separation, a set of ssDNA 15-mers was designed including a poly(dT) 15-mer and nine variants. Separations were performed using fluorescent-labeled ssDNA in CZE with laser-induced fluorescence detection. Results show that separation improves with increasing buffer concentration and decreasing temperature, due at least in part to longer separation times. Migration times increase with increasing purine content, with A having a much larger effect that G. Circular dichroism spectra of the mixtures of the strands suggest that the separation is not due to changes in conformation of the ssDNA at high salt concentrations.