ONLINE MELTING OF DOUBLE-STRANDED DNA FOR ANALYSIS OF SINGLE-STRANDED-DNA USING CAPILLARY ELECTROPHORESIS

Capillary electrophoresis (CE) is a convenient, fast and non-radioacti ve method with possibilities for automatization. To analyse single-str anded DNA molecules in a more automated way, we developed a heating de vice to melt double-stranded DNA fragments in the capillary during ele ctrophoresis. In this study we used this device to obtain single-stran ded DNA, necessary for the detection of point mutations in DNA using t he single-strand conformation polymorphism technique. Results show tha t double-stranded DNA molecules can be melted on-line into single-stra nded DNA molecules, although not for 100%. In an attempt to find unive rsal electrophoretic conditions for the analysis of single-stranded DN A, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-str anded DNA peaks. We demonstrate that this heating device is a technica l adjustment of CE which contributes to more automated analyses of DNA fragments.