Effectiveness of various methods of DNA isolation from bones and teeth of animals exposed to high temperature. (February 2021)

• Record Type: Journal Article
• Authors: Grela, Małgorzata; Jakubczak, Andrzej; Kowalczyk, Marek; Listos, Piotr; Gryzińska, Magdalena
• Is Part Of: Journal of forensic and legal medicine. Volume 78(2021)
• Publication Date: 2021-02
• Subjects: Forensic genetics -- DNA extraction methods -- Burnt bones and teeth -- Degraded DNA -- Real-time PCR -- Species identification; Medical jurisprudence -- Periodicals; Forensic sciences -- Periodicals; Forensic Medicine -- Periodicals; Médecine légale -- Périodiques; Electronic journals; 614.1
• DOI: 10.1016/j.jflm.2021.102131
• Languages: English
• ISSNs: 1752-928X

Abstract

Abstract: 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. Highlights: Four methods of DNA isolation from burnt remains were compared. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation. More DNA was recovered from bone than from teeth. Mitochondrial DNA constituted a larger part of the isolated genetic material. The duration of high temperature negatively affects DNA stability in hard tissues.