|Institution:||University of Otago|
|Keywords:||Oral forensics; microbiology; dental calculus; ancient DNA; demineralization; Next generation sequencing; NGS; aDNA|
|Full text PDF:||http://hdl.handle.net/10523/4960|
The study of ancient DNA is a relatively new field and the methods and standards involved are evolving. DNA isolated from ancient specimens is defined as aDNA. As a DNA source, teeth are the hardest structures of the body and are resistant to harsh conditions such as incineration, mutilation, immersion and trauma. Calculus is a feature of archaeological teeth. The DNA from bacteria, viruses and fungi are more likely to be preserved in amorphous calcifications, making dental calculus a potential source of microbial aDNA. In this study, 16S rRNA and rpoB genes served as the molecular markers of microbial DNA from calculus. The three specific aims of this project involved (i) amplification of bacterial DNA from calculus and assessing the feasibility of preparing 454 sequencing template directly from bacterial DNA within calculus, (ii) comparing the effects of three buffer systems, namely Tris, Citrate and EDTA, at varying pH and concentrations, on the demineralization of calculus (mimicked by HAP beads) and on DNA integrity, and (iii) application of 454 high-throughput sequencing of dental bacteria as a method of tracking human domestic interactions. The results indicate that direct amplification of bacterial sequences from calculus was possible and established a protocol for generating bacterial amplicons for sequence analysis. In two of the three buffer systems (Citrate and EDTA), DNA degradation occurred regardless of the presence of HAP beads, with increased degradation of DNA in the presence of HAP beads, suggesting their lack of suitability for aDNA recovery. Analysis of the molecular markers used in this study, showed unrelated individuals sharing the same household did not share strains of oral streptococci, hence suggesting microbial DNA is unable to track human domestic interactions in this scenario.