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Nucleic Acids Research 2004 32(14):e114; doi:10.1093/nar/gnh115
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Published online 10 August 2004

Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved

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

M. W. J. van Passel, A. Bart, R. J. A. Waaijer1, A. C. M. Luyf1, A. H. C. van Kampen1 and A. van der Ende*

Department of Medical Microbiology and 1 Bioinformatics Laboratory, Academic Medical Center, Amsterdam, the Netherlands

* To whom correspondence should be addressed. Tel: +31 20 5664862; Fax: +31 20 6979271; Email: a.vanderende{at}amc.uva.nl

Received May 19, 2004; Revised and Accepted July 28, 2004

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.


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