Purification and staining of intact yeast DNA chromosomes and real-time observation of their migration during gel electrophoresis.
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.
Gurrieri
S
S
Dipartimento di Scienze Chimiche, Università di Catania, Italy.
Bustamante
C
C
D001482:Base Composition; D001583:Benzoxazoles; D015825:Chromosomes, Fungal / Q000737:chemistry*; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000295:instrumentation / Q000379:methods; D004996:Ethidium; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D013194:Staining and Labeling
D001583:Benzoxazoles; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004996:Ethidium