In situations of mass disaster (1), which include airline crashes (2), terrorist attacks (2), large fires (3), and mass homicide (4), the human remains are often damaged beyond recognition (5). In these cases, bones and teeth are potentially the only acceptable source of deoxyribonucleic acid, or DNA (6). Previous studies have evaluated a plethora of techniques to purify DNA from hard tissue, but there is no consensus on the optimal process by which to extract and purify DNA from these samples. Not only are hard tissue samples difficult to process, in many cases the samples are subjected to extreme environmental conditions, such as high temperature. Thus, there is interest in obtaining information on DNA quality from samples exposed to high temperatures (7). This work hopes to fill the gap by: 1) optimizing a DNA extraction protocol from hard tissue; and 2) measuring the degree to which the DNA is degraded in an effort to link the quantity and quality of the DNA recovered to the outer appearance of the tooth.
To accomplish this, individual teeth were burned in a furnace at 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, and 600 °C for 10, 20, and 30 minutes. The optimal extraction procedure utilized Amicon® Ultra-4 Centrifugal 30K filter devices and the QIAGEN MinElute Polymerase Chain Reaction (PCR) Purification Kit. Samples were quantified using the Quantifiler® Trio quantification kit to obtain the quantity and quality metrics.
After heat exposure, each tooth was photographed and subsequently given a color designation or value: light yellow to beige teeth were assigned a value of 1; dark yellow to orange were assigned a value of 2; brown was assigned a value of 3; shiny black was assigned a value of 4; and black to light gray teeth were assigned a value of 5. Both carbonization and the early stages of calcination were observed. The mass of DNA per mass of tooth was determined by examining quantitative PCR (qPCR) results for both a large and small autosomal fragment. The degradation index, or DI, was also calculated from qPCR measurements.
The results demonstrate a strong correlation between the quantity of DNA recovered, the quality of the DNA obtained, and the designated color value. The highest recovery rates were obtained from teeth assigned a color value of 1 (unaltered beige) or 2 (yellow to orange). These teeth were exposed to either room temperature, 100 °C or 200 °C. At temperatures exceeding 300 °C, the amount of DNA recovered drastically decreased and was inconsistent. Some of the samples subjected to temperatures at and above 300 °C resulted in no quantifiable DNA. In contrast, the DI results suggested that when the teeth were subjected to temperatures ≤ 100 °C, the quality of the DNA was good, wherein the DI value was approximately 1. At 200 °C, the temperature began to impact the DI value, which increased with time to the point where a DI was no longer able to be calculated because the large autosomal fragment could not be detected.
In conclusion, the current work compares five different methods of DNA extraction to establish a best practice extraction procedure for these difficult samples. Furthermore, this work suggests that examination of the tooth’s appearance can be used to deduce whether successful DNA recovery is likely. In summary, the results demonstrate that when the tooth sample was assigned a color value of 1, the quantity and quality of the DNA obtained was high. Once the color value of the sample rose to 2, the quantity and quality of DNA varied greatly and the probative value of the sample was diminished. Samples that exhibited large color changes or had begun the calcination process resulted in no recoverable DNA.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/26625 |
| Date | 01 November 2017 |
| Creators | Federchook, Taylor Joan |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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