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DNA Binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA
Umeå University, Faculty of Medicine, Department of Odontology, Endodontics.
Umeå University, Faculty of Medicine, Department of Odontology, Endodontics.
Umeå University, Faculty of Medicine, Department of Odontology, Endodontics.
2013 (English)In: Journal of Endodontics, ISSN 0099-2399, Vol. 39, no 2, 211-216 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: Molecular methods are increasingly being deployed for analysis of the microbial flora in the root canal. Such methods are based on the assumption that recovered DNA is associated with the active endodontic infection, yet paleomicrobiology research is based on the recovery of ancient DNA from centuriesold tooth and bone samples, which points to considerable longevity of the DNA molecule in these tissues. The main component of dentin and bone is the mineral hydroxyapatite. This study assessed DNA binding to hydroxyapatite and whether thiS binding affinity stabilizes the DNA molecule in various media.

Methods: DNA was extracted from Fusobacterium nucleatum and added to ceramic hydroxyapatite for 90 minutes. The DNA-bound hydroxyapatite was incubated in different media (ie, water, sera, and DNase I) for up to 3 months. At predetermined intervals, the recovery of detectable DNA was assessed by releasing the DNA from the hydroxyapatite using EDTA and evaluating the presence of DNA by gel electrophoresis and polymerase chain reaction (PCR) amplification.

Results: When incubated with hydroxyapatite, nonamplified DNA was detectable after 3 months in water, sera, and DNase I. In contrast, DNA incubated in the same media (without hydroxyapatite) decomposed to levels below the detection level of PCR within 3 weeks, with the exception of DNA in sera in which PCR revealed a weak positive amplification product.

Conclusions: These results confirm a specific binding affinity of hydroxyapatite for DNA. Hydroxyapatite-bound DNA is more resistant to decay and less susceptible to degradation by serum and nucleases, which may account for the long-term persistence of DNA in bone and tooth.

Place, publisher, year, edition, pages
Elsevier, 2013. Vol. 39, no 2, 211-216 p.
Keyword [en]
Ancient DNA, DNA binding affinity, DNA decomposition, DNA preservation, hydroxyapatite, polymerase chain reaction
National Category
URN: urn:nbn:se:umu:diva-67045DOI: 10.1016/j.joen.2012.09.013ISI: 000314482500013OAI: diva2:612713
Available from: 2013-03-24 Created: 2013-03-12 Last updated: 2013-11-08Bibliographically approved
In thesis
1. Stability of bacterial DNA in relation to microbial detection in teeth
Open this publication in new window or tab >>Stability of bacterial DNA in relation to microbial detection in teeth
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA.

Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases.

The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 35 p.
Umeå University odontological dissertations, ISSN 0345-7532 ; 127
Cell-bound DNA, cell-death, dentine, DNA binding affinity, DNA decomposition, DNA preservation, extracellular DNA, hydroxyapatite, PCR, polymerase chain reaction
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Research subject
urn:nbn:se:umu:diva-82735 (URN)978-91-7459-743-1 (ISBN)
Public defence
2013-12-05, Sal B, tandläkarhögskolan 9 tr, Norrlands universitetssjukhus, Umeå, 09:00 (Swedish)
Available from: 2013-11-08 Created: 2013-11-07 Last updated: 2013-11-08Bibliographically approved

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Brundin, MalinSundqvist, GöranSjögren, Ulf
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