Purification and staining of intact yeast DNA chromosomes and real-time observation of their migration during gel electrophoresis

Abstract

In the past few years, fluorescence microscopy has been used successfully to characterize the motion of intermediate-size DNA molecules (50–500 kbp) during steady- and pulsed-field gel electrophoresis. However, experimental difficulties had prevented the application of this technique to the direct observation of longer DNA chromosomes (1–2 Mbp). In the present study a particular procedure was followed for the purification and staining of chromosomal yeast DNA to protect it from shear forces. Also, a new highly fluorescent DNA-labelling dye, YOYO-1, was employed to improve brightness and contrast. Finally, the motion of such long DNA molecules (1–2 Mbp) was characterized under steady-field electrophoresis conditions. An accurate description of the molecular mechanisms of motion of such long molecules should provide the basis for a detailed analysis of the mechanisms responsible for DNA trapping.