A review of the current understanding of burned bone as a source of DNA for human identification.

Identification of incinerated human remains may rely on genetic analysis of burned bone which can prove far more challenging than fresh tissues. Severe thermal insult results in the destruction or denaturation of DNA in soft tissues, however genetic material may be preserved in the skeletal tissues. Considerations for DNA retrieval from these samples include low levels of exogenous DNA, the dense, mineralised nature of bone, and the presence of contamination, and qPCR inhibitors. This review collates current knowledge in three areas relating to optimising DNA recovery from burned bone: 1) impact of burning on bone and subsequent effects on sample collection, 2) difficulties of preparing burned samples for DNA extraction, and 3) protocols for bone decalcification and DNA extraction. Bone decalcification and various DNA extraction protocols have been tested and optimised for ancient bone, suggesting that prolonged EDTA (Ethylenediaminetetraacetic acid) demineralisation followed by solid-phased silica-based extraction techniques provide the greatest DNA yield. However, there is significantly less literature exploring the optimal protocol for incinerated bones. Although burned bone, like ancient and diagenetic bone, can be considered "low-copy", the taphonomic processes occurring are likely different. As techniques developed for ancient samples are tailored to deal with bone that has been altered in a particular way, it is important to understand if burned bone undergoes similar or different changes. Currently the effects of burning on bone and the DNA within it is not fully understood. Future research should focus on increasing our understanding of the effects of heat on bone and on comparing the outcome of various DNA extraction protocols for these tissues.

Comparison of bone demineralisation procedures for DNA recovery from burned remains.

Recovering DNA from modern incinerated bones can be challenging and may require alteration of routine DNA extraction protocols. It has been postulated that incinerated bones share some similarities with ancient bones, including fragmented DNA, surface contamination and highly mineralised structure, all of which can inhibit the successful recovery of genetic material. For this reason, ancient DNA extraction protocols are often used for incinerated modern samples; however, their effectiveness is still somewhat unclear. Much of this uncertainty exists around the demineralisation step of extraction, specifically the length of incubation and retention or removal of supernatant. As obtaining human samples for forensic research can be challenging, porcine models (Sus scrofa domesticus) are often used as substitutes. This study developed real time PCR assays for porcine nuclear DNA in order to investigate the effects of modified demineralisation protocols on DNA yield from femurs exposed to either short (60 min) or prolonged (120 min) burning. Gradient PCR results indicated 56 °C was the ideal amplification temperature for targeted amplicons, with melt curve analysis showing short and long amplicons corresponded to 80.3 °C and 83 °C peaks respectively. Results of altered extraction protocol showed a trend towards higher DNA yields from longer demineralisation periods however this was not significant. By comparison, retaining supernatant post-demineralisation resulted in significantly greater DNA yields compared to discarding it (P < 0.009). Although DNA content yield decreased with burn duration, the demineralisation treatment variations appeared to have the same effect for all burn lengths. These results suggest that for incinerated modern bone retaining the supernatant following demineralisation can dramatically increase DNA yield.

A comparison of crystal structure in fresh, burned and archaic bone - Implications for forensic sampling.

Standard protocols for extracting DNA from bone are variable and are largely dependent on the state of preservation. In archaic samples, endogenous DNA is believed to be tightly bound to crystal aggregates in the Hydroxyapatite (HAp) matrix requiring prolonged demineralisation to allow its release. By comparison, fresh bone contains abundant cellular material, discounting the need for demineralisation. Recommendations for incinerated bone, specifically how viable sampling sites should be selected and the ideal techniques for DNA recovery are unclear, and the protocol used is often selected based on macroscopic sample appearance. It has been postulated that like archaic bone, burned bone is 'highly degraded' and therefore aDNA techniques may present better results for DNA recovery than using fresh protocols. However, little research has been undertaken comparing the crystal structure of burnt, fresh and archaic bone. This study uses a combination of XRPD and SEM analysis to compare the crystalline profile and microscopic appearance of burned bone subjected to temperatures ranging from 100-1000°C, with archaic and fresh samples. Although macroscopically visually different, fresh samples and samples heated up to 500°C showed no microscopic differences or significant changes in crystallinity. By comparison, samples heated above 500°C became significantly more crystalline, with HAp crystal size increasing dramatically. Archaic samples were different again, more closely resembling the amorphous fresh samples than the highly crystalline incinerated samples. These results suggests that, potentially, samples burned at 500°C or lower can be treated as fresh samples, whilst samples exposed to higher temperatures may require adapted protocols. Whether or not these highly burned samples require demineralisation needs to be investigated.

Growth Patterns and Individual Variation in Mid-sagittal Facial Soft Tissue Depth from Childhood to Adulthood.

Previous studies have used longitudinal samples to investigate growth of the skeletal aspects of the face, although far less has been done on facial soft tissue. This study uses a larger sample than previous studies on the same data (Denver and Fels growth series) to explore covariation of bony and soft facial dimensions comprehensively from childhood to adulthood. A total of 1036 lateral cephalometric radiographs were digitized from 60 individuals, and distances between facial landmarks were measured using ImageJ. Multivariate analysis of variance (MANOVA) showed all bony facial landmark distances were significantly different (p > 0.05) between age groups; however, only half were significantly different between sexes. Further analysis showed the effect of age (calculated as eta-squared) explained a greater percentage of total variation (20%) than sex (15%). Overall, soft tissue changes between 0 and 19 years of age were small (<3 mm) and only some were correlated to underlying facial skeleton dimensions.