HIGH-RESOLUTION SEPARATION OF SINGLE-STRA NDED AND DOUBLE-STRANDED DNA USING CAPILLARY GEL-ELECTROPHORESIS

The effect of the gel composition on the resolution of single- and dou ble-stranded DNA was investigated in order to optimize the electrophor etic conditions in the separation of DNA using capillary gel electroph oresis. In this study we prepared gel-filled capillaries in which the gel was sometimes chemically bound to the capillary inner surface. A m ixture of single-stranded DNA fragments was separated by using capilla ries filled with linear polyacrylamide with differing polyacrylamide c oncentrations and a mixture of double-stranded DNA fragments by using capillaries filled with a crosslinked polyacrylamide gel with differin g gel compositions. A total of 250 bands of single-stranded DNA fragme nts was completely reserved within 60 min under the optimum electropho retic conditions. The base-line resolution of double-stranded DNA frag ments up to 12000 base pairs (bp) was performed under optimum separati on conditions. In addition, both the 500 bp PCR reaction product and t wo fragments of 506 and 517 bp, which differ from each other by only l ess than 10 bp and are usually not separated by slab gel electrophores is, have been base-line resolved. A plate number of(1 similar to 5) X 10(6) per meter was achieved. Some guidelines are presented based on t he experimental results in order to select the optimum gel composition in the separation of single- and double-stranded DNA.