An in vitro strategy for the selective isolation of anomalous DNA from prokaryotic genomes

In sequenced genomes of prokaryotes, anomalous DNA (aDNA) can be recognized, among others, by atypical clustering of dinucleotides. We hypothesized that atypical clustering of hexameric endonuclease recognition sites in aDNA allows the specific isolation of anomalous sequences in vitro. Clustering of endonuclease recognition sites in aDNA regions of eight published prokaryotic genome sequences was demonstrated. In silico digestion of the Neisseria meningitidis MC58 genome, using four selected endonucleases, revealed that out of 27 of the small fragments predicted (<5 kb), 21 were located in known genomic islands. Of the 24 calculated fragments (>300 bp and <5 kb), 22 met our criteria for aDNA, i.e. a high dinucleotide dissimilarity and/or aberrant GC content. The four enzymes also allowed the identification of aDNA fragments from the related Z2491 strain. Similarly, the sequenced genomes of three strains of Escherichia coli assessed by in silico digestion using XbaI yielded strain-specific sets of fragments of anomalous composition. In vitro applicability of the method was demonstrated by using adaptor-linked PCR, yielding the predicted fragments from the N.meningitidis MC58 genome. In conclusion, this strategy allows the selective isolation of aDNA from prokaryotic genomes by a simple restriction digest–amplification–cloning–sequencing scheme