title: Evaluation of a new fluorimetric DNA-DNA hybridization method.
abstract: Standardized procedures must be followed when characterizing, officially describing, and validly naming novel bacteria. For species descriptions, DNA-DNA hybridization still is needed for whole-genome comparisons between close relatives, but many established hybridization methods have drawbacks, such as requiring labeled or large amounts of DNA. We evaluated a new technique based on the spectrophotometric method in which renaturation rates are used for calculating the degree of binding, which estimates relatedness. In this new approach, DNA is denatured and reassociated in a real-time PCR thermal cycler and the process monitored fluorimetrically using SYBR Green I dye that selectively binds to double-stranded DNA. We investigated the effects of different parameters on the renaturation rates, such as the quantities of DNA and SYBR Green I used. Then using this technique, we calculated the percent binding for pairs of selected bacterial species representing different taxonomic groups and compared our results with published values. We demonstrated that the SYBR Green I method is useful for describing new species and as a screening tool to quickly identify the relatedness of uncharacterized isolates with similar 16S rRNA gene sequences.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA. JXL26@psu.edu
authors.forename: Jennifer
authors.initials: J
authors.lastname: Loveland-Curtze
authors.forename: Vanya I
authors.initials: VI
authors.lastname: Miteva
authors.forename: Jean E
authors.initials: JE
authors.lastname: Brenchley
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics; D001482:Base Composition; D001483:Base Sequence; D052160:Benzothiazoles; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D003959:Diamines; D005470:Fluorometry / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I