Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE).
Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.
Bautsch
W
W
Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, Germany.
D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004269:DNA, Bacterial