MboII endonuclease heat inactivation before agarose gel electrophoresis to prevent artifactual bands in restriction patterns.

MboII restriction enzyme belongs to class-IIS endonucleases group (3–5). Like all of the enzymes belonging to this class, MboII cleaves DNA at a specific distance from its recognition sequence and still binds to its recognition sequence after DNA has been cleaved, because binding and cleaving domains have separate functions (5). In our laboratory, we apply amplified ribosomal DNA restriction analysis (ARDRA) to bacterial identification following the technique of Garcìa-Arata et al. (2). MboII was retained, among other enzymes, because of appropriate frequency of the endonuclease recognition sequence on the amplified ribosomal RNA operon (rrn) of Escherichia coli. Since E. coli K-12 MG1655 strain has now been entirely sequenced (1), we could retrieve from the European Molecular Biology Laboratory (EMBL) database (GenBank Accession No. U00096) the sequences corresponding to the amplified fragment in the seven rrn copies (A, B, C, D, E, G and H). Virtual MboII in silico restrictions could be performed on each of the seven sequences with the Geneman software (DNASTAR, Madison, WI, USA) on a Macintosh (Apple Computer, Cupertino, CA, USA). From the combination of all virtual restriction fragments from the seven operons, an in silico restriction pattern for E. coli K-12 MG1655 strain was obtained and could be compared to experimental patterns. Because the seven rrn operons in the E. coli chromosome differ in length and sequence, some of the fragments are contributed to by only one or a few operons. The amount of DNA contained in a single band will be proportional to the number of operons producing the band. Moreover, because the number of ethidium bromide molecules bound to double-stranded DNA fragments is proportional to fragment length, longer fragments will stain better than smaller ones even if they are produced by the same number of operons. Staining ability (S) of a band was calculated as S = L × N, where L is the fragment length, and N is the number of operons contributing to the band. S values were considered as strong (above 1500), intermediate (600–1499) and weak (below 600). Surprisingly, experimental patterns never entirely corresponded to the in silico one and, moreover, they seemed to vary according to enzyme concentration (Figure 1). MboII endonuclease is heat-sensitive, and enzyme inactivation can be obtained after a 15-min incubation at 50°C (3). To verify enzyme-inactivaBenchmarks