Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites

A method for generating limited representations of total bacterial DNA, wit hout prior knowledge of the DNA sequence, has been developed. This method c onsists of three steps: digestion with two restriction enzymes, ligation of two oligonucleotide adapters corresponding to the restriction sites, and s elective PCR amplification of the ligation products. The method relies on t he use of two restriction enzymes with considerable differences in cleavage frequency of the investigated DNA and the ligation of two different oligon ucleotides, each corresponding to one of the two cohesive ends of DNA fragm ents. Three subsets of DNA fragments are generated during digestion and sub sequent ligation: terminated with the same oligonucleotide on both 5' ends of DNA fragments (two subsets) and terminated with two different oligonucle otides. Suppression PCR allows only the third subset of DNA fragments to be amplified exponentially. The method allows bacterial species strain differ entiation on the basis of the different DNA band patterns obtained after el ectrophoresis in polyacrylamide gels stained with ethidium bromide and visu alized in UV light.