Effectiveness of various methods of DNA isolation from bones and teeth of animals exposed to high temperature.

In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis.

Comparative?analysis?of?two?different?DNA?purification?methods?for?bones?and?teeth / 中国法医学杂志

Objective To compare the effect of silica-extraction method and Silico membrane based method in DNA purification from bones and teeth.Methods DNA samples were purified respectively with the silica-extraction method and MinElute PCR Purification kit from 6 bones and 8 teeth,then tested STR types by GlobalFiler? kits. And evaluated the two methods with the success rate and the peak height. Results Both of the two purification methods can successfully obtain the STR markers of the 14 samples. And there was no statistical difference between the two methods in the average peak height from bones and teeth. Conclusion The Silico membrane based method which have more advantages in operation is an efficient method to purify DNA from bones and teeth, and there is no significant difference compared with the silica-extraction method. But the cost is higher. It can be selectively used in forensic practice.

X-ray scattering evaluation of ultrastructural changes in human dental tissues with thermal treatment.

Micro- and ultrastructural analysis of burned skeletal remains is crucial for obtaining a reliable estimation of cremation temperature. Earlier studies mainly focused on heat-induced changes in bone tissue, while this study extends this research to human dental tissues using a novel quantitative analytical approach. Twelve tooth sections were burned at 400-900°C (30-min exposure, increments of 100°C). Subsequent combined small- and wide-angle X-ray scattering (SAXS/WAXS) experiments were performed at the Diamond Light Source synchrotron facility, where 28 scattering patterns were collected within each tooth section. In comparison with the control sample, an increase in mean crystal thickness was found in burned dentine (2.8-fold) and enamel (1.4-fold), however at a smaller rate than reported earlier for bone tissue (5-10.7-fold). The results provide a structural reference for traditional X-ray scattering methods and emphasize the need to investigate bone and dental tissues separately to obtain a reliable estimation of cremation temperature.