title: Differentiation of bacterial 16S rRNA genes and intergenic regions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage.
abstract: We describe here a new approach for analyzing nucleic acid sequences using a structure-specific endonuclease, Cleavase I. We have applied this technique to the detection and localization of mutations associated with isoniazid resistance in Mycobacterium tuberculosis and for differentiating bacterial genera, species and strains. The technique described here is based on the observation that single strands of DNAs can assume defined conformations, which can be detected and cleaved by structure-specific endonucleases such as Cleavase I. The patterns of fragments produced are characteristic of the sequences responsible for the structure, so that each DNA has its own structural fingerprint. Amplicons, containing either a single 5'-fluorescein or 5'-tetramethyl rhodamine label were generated from a 620-bp segment of the katG gene of isoniazid-resistant and -sensitive M. tuberculosis, the 5' 350 bp of the 16S rRNA genes of Escherichia coli O157:H7, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, Shigella dysenteriae, Campylobacter jejuni, staphylococcus, hominis, Staphylococcus warneri, and Staphylococcus aureus and an approximately 550-bp DNA segment comprising the intergenic region between the 16S and 23S rRNA genes of Salmonella typhimurium, Salmonella enteritidis, Salmonella arizonae, Shigella sonnei, and Shigella dysenteriae serotypes 1, 2, and 8. Changes in the structural fingerprints of DNA fragments derived from the katG genes of isoniazid-resistant M. tuberculosis isolates were clearly identified and could be mapped to the site of the actual mutation relative to the labeled end. Bland patterns which clearly differentiated bacteria to the level of genus and, in some cases, species were generated from the 16S genes. Cleavase I analysis of the intergenic regions of Salmonella and Shigella species differentiated genus, species, and serotypes. Structural fingerprinting by digestion with Cleavase I is a rapid, simple, and sensitive method for analyzing nucleic acid sequences and may find wide utility in microbial analysis.
authors.affiliation: Third Wave Technologies Inc., University of Wisconsin, Madison, USA. Mary_Ann_Brow@twt.com
authors.forename: M A
authors.initials: MA
authors.lastname: Brow
authors.forename: M C
authors.initials: MC
authors.lastname: Oldenburg
authors.forename: V
authors.initials: V
authors.lastname: Lyamichev
authors.forename: L M
authors.initials: LM
authors.lastname: Heisler
authors.forename: N
authors.initials: N
authors.lastname: Lyamicheva
authors.forename: J G
authors.initials: JG
authors.lastname: Hall
authors.forename: N J
authors.initials: NJ
authors.lastname: Eagan
authors.forename: D M
authors.initials: DM
authors.lastname: Olive
authors.forename: L M
authors.initials: LM
authors.lastname: Smith
authors.forename: L
authors.initials: L
authors.lastname: Fors
authors.forename: J E
authors.initials: JE
authors.lastname: Dahlberg
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics; D004720:Endonucleases; D005798:Genes, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D010544:Peroxidases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D010544:Peroxidases; C076136:catalase HPI; D004720:Endonucleases; D007538:Isoniazid