COMPARISON OF RESOLUTION OF DOUBLE-STRANDED AND SINGLE-STRANDED-DNA IN CAPILLARY ELECTROPHORESIS

Capillary electrophoresis has become a powerful analytical tool for th e 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 migratio n times and enables pressure injection. When pressure injection is use d, rather than electrokinetic injection, it is assured that a represen tative part of the sample is introduced into the capillary. The possib le lower resolution, which is a side effect of the pressure injection, can be made up for by an increase of selectivity. Using fluorescent l abels, it is possible to detect DNA with a fluorescence detector under both native conditions in double-stranded (ds) form, and under denatu ring conditions in single-stranded (ss) form. When DNA is separated in its ss form, as is necessary for DNA sequencing, we observe an increa sed 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 achi eved with the same resolution in a shorter separation time compared to dsDNA. Therefore, it is advisable to separate DNA in the ss form if h igh resolution, size dependent separation is required. The enhanced re solution achieved with DNA migrating in the ss form enabled the separa tion of allelic ladders of short tandem repeats with a difference of 4 base pairs in the 200 base pair range, with separation times no longe r than 6 min.