Detection of simple mutations and polymorphisms in large genomic regions

We have developed a novel technology that makes it possible to detect simpl e nucleotide polymorphisms directly within a sample of total genomic DNA. I t allows, in a single Southern blot experiment, the determination of sequen ce identity of genomic regions with a combined length of hundreds of kiloba ses. This technology does not require PCR amplification of the target DNA r egions, but exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endon uclease CEL I to cleave heteroduplex DNA with a very high specificity and s ensitivity. We have used this technique to detect various simple mutations directly in the genomic, DNA of isogenic pairs of recombinant Pseudomonas a eruginosa, Escherichia coli and Salmonella isolates. Also, by using a cosmi d DNA library and genomic fractions as hybridization probes, we have compar ed total genomic DNA of two clinical P.aeruginosa clones isolated from the same patient, but exhibiting divergent phenotypes. The mutation scan correc tly detected a GA insertion in the quorum-sensing regulator gene rhIR and, in addition identified a novel intragenomic polymorphism in rrn operons, in dicating very high stability of the bacterial genomes under natural non-mut ator conditions.