Comparison of resolution of double-stranded and single-stranded DNA in capillary electrophoresis

Abstract

Capillary electrophoresis has become a powerful analytical tool for the analysis of DNA restriction fragments and polymerase chain reaction (PCR) products. The use of replaceable polymer solutions increases the lifetime of the capillary, improves the repeatability of the migration times and enables pressure injection. When pressure injection is used, rather than electrokinetic injection, it is assured that a representative part of the sample is introduced into the capillary. The possible lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent labels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denaturing conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increased selectivity compared to separation of that sample in its ds form. In this work, we exploited this increased selectivity for the analysis of denatured PCR products. It was found that DNA separated in the ss form yields superior separation; that is, a given analysis can be achieved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if high resolution, size dependent separation is required. The enhanced resolution achieved with DNA migrating in the ss form enabled the separation of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longer than 6 min.