Abstract
Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps.
Highlights
Advances in DNA analysis of human skeletal remains are providing high-resolution insights into the origin [1], migrations [2], health [3], biogeographic ancestry [4,5], phenotype [6,7] and identification [8,9,10] of deceased individuals and populations for evolutionary, archaeological, medical and forensic studies. Much of this progress has resulted from post-DNA-extraction advances in polymerase chain reaction (PCR) sensitivity [11], the design and optimization of short-amplicon DNA typing technologies [4], and next-generation sequencing [1,2] that focus on the small amounts of highly degraded DNA recovered from skeletal remains
Resolving these issues is critical to future improvements in DNA analysis of skeletal remains and could clarify intra- and inter-individual variation in DNA content of skeletal tissues leading to better predictive models for sample selection
Our results demonstrate differential distribution and variable postmortem preservation of nuclear and mitochondrial DNA across within individual tissues of human teeth suggesting that targeted sampling of specific tooth tissues will improve downstream analyses benefiting in doi:10.1371/journal.pone.0126935.g006
Summary
Advances in DNA analysis of human skeletal remains are providing high-resolution insights into the origin [1], migrations [2], health [3], biogeographic ancestry [4,5], phenotype [6,7] and identification [8,9,10] of deceased individuals and populations for evolutionary, archaeological, medical and forensic studies. The precise selection of tissue for post-mortem sampling is crucial to maximise recovery of endogenous DNA and to minimise destructive sampling, the potential for contamination, the co-extraction of inhibitors and the need to remove large amounts of inorganic (hydroxyapatite) and organic (collagen) fractions These non-DNA components of bone and teeth are primarily responsible for the large volume/low throughput/high cost nature of DNA extractions from skeletal remains [16,23], and represent potent PCR inhibitors, if not completely removed during the extraction process [24]. Informed targeted sampling would allow for smaller sample sizes to be processed facilitating the use of medium- to high-throughput techniques and standard laboratory equipment, leading to considerable cost and time saving
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