REAL-TIME IMAGING OF THE REORIENTATION MECHANISMS OF YOYO-LABELED DNA-MOLECULES DURING 90-DEGREES AND 120-DEGREES PULSED-FIELD GEL-ELECTROPHORESIS

Pulsed field gel electrophoresis (PFGE) techniques have been developed to overcome the limitations of conventional electrophoresis and to in crease the separation to DNA chromosomes of few megabase pairs in size , Despite of the large success of these techniques, the various separa tion protocols employed for PFGE experiments have been determined empi rically, However, a deep understanding of the molecular mechanisms of motion responsible for DNA separation becomes necessary for the ration al optimization of these techniques, This paper shows the first clear observations of individual molecules of DNA during the reorientation p rocess in 90 degrees PFGE and 120 degrees PFGE, Real-time visualizatio n of the DNA dynamics during PFGE was possible with the use of an epi- illumination fluorescence microscope specifically equipped to run thes e experiments and by staining the DNA with YOYO-1 ro-(benzo-1,3-oxazol e)-2-methyl-idene]-quinolinium tetraiodide). This dye forms a very sta ble, highly fluorescent complex with double-stranded DNA and dramatica lly improves the quality of the DNA images. The results of computer si mulations used to reproduce the molecular mechanisms of motion as well as the DNA separation features are also discussed.