Preelectrophoresis of agarose plugs containing bacterial chromosomal DNA prepared for analysis by pulsed field gel electrophoresis can improve the clarity of restriction patterns.
Pulsed field gel electrophoresis has indicated that chromosomal DNA isolated from stationary phase Pseudomonas fluorescens, Pseudomonas putida, and Escherichia coli cells immobilized in agarose can be fragmented during its release. For P. fluorescens it was demonstrated that the entire chromosome is affected and that there are no specifically fragile sites. The extent of the damage increased both during storage of the DNA and also when magnesium ions were provided, suggesting that nucleases may be involved. Chromosomal DNA isolated from exponentially growing cultures was subjected to significantly less damage during release and storage. Removal of damaged DNA by electrophoresis prior to digestion was shown to result in clearer restriction patterns of chromosomal DNA.
Whatling
C A
CA
School of Biological Sciences, University of Birmingham, Edgbaston, United Kingdom.
Thomas
C M
CM
D002876:Chromosomes, Bacterial / Q000737:chemistry; D004249:DNA Damage; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D003851:Deoxyribonucleases / Q000302:isolation & purification; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D011551:Pseudomonas fluorescens / Q000737:chemistry; D016958:Pseudomonas putida / Q000737:chemistry
D004269:DNA, Bacterial; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes