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

The effect of the gel composition on the resolution of single- and double-stranded DNA was investigated in order to optimize the electrophoretic conditions in the separation of DNA using capillary gel electrophoresis. In this study we prepared gel-filled capillaries in which the gel was sometimes chemically bound to the capillary inner surface. A mixture of single-stranded DNA fragments was separated by using capillaries filled with linear polyacrylamide with differing polyacrylamide concentrations and a mixture of double-stranded DNA fragments by using capillaries filled with a crosslinked polyacrylamide gel with differing gel compositions. A total of 250 bands of single-stranded DNA fragments was completely reserved within 60 min under the optimum electrophoretic conditions. The base-line resolution of double-stranded DNA fragments up to 12000 base pairs (bp) was performed under optimum separation conditions. In addition, both the 500 bp PCR reaction product and two fragments of 506 and 517 bp, which differ from each other by only less than 10 bp and are usually not separated by slab gel electrophoresis, 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 the experimental results in order to select the optimum gel composition in the separation of single- and double-stranded DNA.