Preservation of bacterial DNA by human dentin.
BACKGROUND
The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-induced degradation was evaluated individually and by the incubation of DNA with nuclease-producing bacteria in a mixed culture.


METHODS
Extracted Fusobacterium nucleatum DNA was incubated with dentin shavings or collagen for 90 minutes. The DNA-bound substrates were incubated in different media (water, sera, and DNase I) for up to 3 months. Amplifiable DNA was released from dentin using EDTA,or from collagen using proteinase K, and evaluated by polymerase chain reaction (PCR). The stability of dentin-bound DNA was also assessed in a mixed culture (Parvimonas micra and Pseudoramibacter alactolyticus) containing a DNase-producing species, Prevotella intermedia. Samples were analyzed for amplifiable DNA.


RESULTS
In water, dentin-bound DNA was recoverable by PCR at 3 months compared with no detectable DNA after 4 weeks in controls (no dentin). DNA bound to collagen was detectable by PCR after 3 months of incubation in water. In 10% human sera, amplifiable DNA was detectable at 3 months when dentin bound and in controls (no dentin). In mixed bacterial culture, dentin-bound DNA was recoverable throughout the experimental period (3 months), compared with no recoverable F. nucleatum DNA within 24 hours in controls (no dentin).


CONCLUSIONS
There is a strong binding affinity between DNA and dentin, and between DNA and serum proteins or collagen. These substrates preserve DNA against natural decomposition and protect DNA from nuclease activity, factors that may confound molecular analysis of the endodontic microbiota yet favor paleomicrobiological studies of ancient DNA.
Department of Odontology/Endodontics, Faculty of Medicine, Umeå University, Umeå, Sweden. Electronic address: malin.brundin@odont.umu.se.
Malin
M
Brundin
Department of Microbiology, Monash University, Melbourne, Australia.
David
D
Figdor
Department of Odontology/Molecular Periodontology, Faculty of Medicine, Umeå University, Umeå, Sweden.
Anders
A
Johansson
Department of Odontology/Endodontics, Faculty of Medicine, Umeå University, Umeå, Sweden.
Ulf
U
Sjögren
D001769:Blood; D001798:Blood Proteins / Q000378:metabolism; D018920:Coculture Techniques; D003094:Collagen / Q000187:drug effects / Q000378:metabolism; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism*; D003804:Dentin / Q000187:drug effects / Q000382:microbiology*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D005051:Eubacterium / Q000235:genetics; D016967:Fusobacterium nucleatum / Q000235:genetics*; D006801:Humans; D006813:Humidity; D059013:Microbial Consortia; D010462:Peptostreptococcus / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D018713:Prevotella intermedia / Q000235:genetics; D011485:Protein Binding; D013696:Temperature; D013997:Time Factors; D014867:Water
D001798:Blood Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D014867:Water; D003094:Collagen; D004492:Edetic Acid; D003850:Deoxyribonuclease I; D019286:Endopeptidase K
Bacterial nucleases; DNA binding affinity; DNA decomposition; DNA preservation; dentin; polymerase chain reaction