EFFICIENCY OF HUMAN DNA ISOLATION AND STR PROFILING FROM BURNT TEETH

Sara C. Zapico, PhD1*, Joe Adserias Garriga, PhD, DDS2*, Douglas H. Ubelaker, PhD11Department of Anthropology, NMNH, Smithsonian Institution, Washington, DC, USA

2University Rey Juan Carlos, Madrid, Spain

Identification of a deceased individual is important from the viewpoint of the relatives, and is a vital factor in facilitating criminal investigations, inquests and other tribunals. Mass disasters, aircraft or car accidents and accidental deaths frequently involve the presence of fire. The identification of human remains subjected to incineration depends on the degree of destruction of the remains, which is affected by the intensity and duration of the fire. One of the approaches for identification of burned remains is genetic analysis. The genetic approach is usually unproblematic in cases of fire victims with conserved internal organs. However, extremely charred bodies frequently render highly degraded DNA, hampering STR analysis. Also, few studies focus on the possibility of amplifying authentic DNA from burnt remains. No consensus has been reached regarding the degree of cremation at which bone will still yield authentic DNA signals. Although previous studies have used similar temperature ranges (between 100ºC to 1000ºC), the duration of fire exposure differs among them. In the studies developed on whole teeth and pulp DNA, the same problem is encountered: the temperatures were similar to those used in the bone studies and the durations of exposure varied. Therefore more research is needed to evaluate the possibility of extracting DNA from burned remains.

INTRODUCTION OBJECTIVES Teeth are the hardest tissue of human body and one of the most abundant types of biological remains available in forensic cases, the present study is focused on the evaluation of the efficiency of DNA isolation from burnt teeth and the achievement of obtaining a DNA profile at different conditions of temperature and time exposure.

MATERIALS -  Teeth: 28 healthy erupted third molars, aged 20-70, collected from dental

clinics. -  DNA isolation: Dneasy Blood and Tissue Kit (Qiagen, Hilden, Germany). -  DNA quatification: Qubit system (Life Technologies, CA, USA) -  Conventional PCR: Biolase DNA Polymerase (Bioline, MA, USA) -  Gel electrophoresis: GelRed (Biotium, CA, USA); GelDoc 2000 (Bio-Rad

Laboratories, CA, USA). -  QPCR: Perfecta SYBR Green FastMix Low Rox (Quanta Biosciences, CA,

USA); Applied Biosystems ViiA7 (Applied Biosystems, CA, USA)

METHODS -  Sample preparation. Teeth were divided into seven groups treated at different temperatures: 100, 200, 300, 400, 500, 600, 700ºC. Each group

was treated at these temperatures for 1 minute, 5 minutes, 10 minutes and 15 minutes, removing one tooth after each time period. Two non-burnt teeth were used as controls.

-  DNA extraction. Control and burnt teeth were mechanically ground with agate mortar and pestle. DNA was extracted using Dneasy Blood and Tissue Kit according to the manufacturer´s protocol.

-  DNA quantification. DNA was quantified using the Qubit system, according to the manufacturer´s protocol. -  Conventional PCR. DNA was amplified looking for AMEL gen. This technique was performed using Biolase DNA Polymerase in 50 ml reactions

that contained PCR Buffer 10X, Mg2Cl2 1.5 mM, dNTPs 1 mM, 0.5 U Biolase enzyme and 0.5 mM specific primers. Thermocycling conditions were as follows: 1 cycle of 95ºC 10 minutes; 35 cycles with these conditions: 95ºC 45 seconds; 48ºC 45 seconds; 72ºC 1 minute; and 1 cycle of 72ºC 10 minutes.

-  Visualization of PCR product. Amplified PCR products were checked via gel electrophoresis in a 1.5 percent agarose gel containing GelRed conducted for one hour at 100 volts. After electrophoresis, amplicon bands were visualized under UV light and save the images digitally with Gel Doc 2000.

-  QPCR. The efficiency of STR profiling was estimated using real-time PCR by analysis of different STRs: D7S820, D13S317, D5S818, CSFO, TPOX, THO1, VWA, D16S539, FES/FPS. To perform this study, Perfecta SYBR Green FastMix, Low Rox was used in an Applied Biosystems ViiA 7. STRs primers were obtained from the general identification database. As a housekeeping gene, GAPDH was used. The amplification reaction of both genes was performed in a total volume of 20 µl with 2 µl DNA, 10 µl SYBR Green Mix and 0.3 µM of each primer. The PCR conditions were developed in a Fast 2-Step Cycling: 95ºC, 30 s; 40 cycles at 95ºC for 3s and 60ºC, 20 s. After the real-time PCR, melting curve was performed. Each sample was tested in duplicate. The analysis of relative gene expression data was calculated using the 2ΔCT method.

RESULTS AND DISCUSSION

2. DNA quantificationIt was possible to extract DNA from almost all samples. Only 400ºC at 10 and 15 min; 500ºC at 15 min; 600ºC at 5 and 15 min; 700ºC at 5, 10 and 15 min gave DNA values lower than the detection limit of the quantification assay. The first two treated samples (100ºC 1 and 2 min) showed similar quantification results than control teeth. It was found values below 1mg/ml in the rest of temperatures and times. The study of Williams et al treated deciduous teeth with temperatures between 100ºC to 500ºC for 15 min finding that DNA quantification was unreliable for the majority of the samples. This research used the same DNA extraction kit as used in the present study, however DNA was extracted from the pulp. In the present study, the whole tooth was grounded and submitted for DNA extraction, letting the rest of the tissues like dentin, cementum and enamel increase the DNA yield.

Table 1. DNA extraction efficiency at different temperatures and times. DNA concentration was measured in ug/ml.

3. STRs amplificationThe results were similar for all STRs tested (Fig 5,4,7). In the first temperatures and times (100ºC and 200ºC 1 and 5 min), it was possible to get amplification similar to the controls, sometimes better, like D5S818. However, in the majority of STRs the amplification was very low from 300ºC and 1 or 5 minutes. There are not so many studies in the literature that analyze the amplification profiles efficiency in teeth after temperature exposure. Particularly the study of Álvarez-García et al. analyzed the amplification of HLA DQA1, D1S80, HUMTH01, HUMFES/FPS treated at temperatures ranging from 75ºC to 500ºC for 1 and 2 min, finding that from temperature of 400ºC the amplification efficiency of specific STRs (HUMTH01 and HUMFES/FPS) was lower than previous temperatures. In the present study, TH01 amplification was almost undetectable from 300ºC 1 min. The amplification of Amelogenin gene by conventional PCR (Fig 7) pointed out to similar results, although it was possible to see slight bands until temperature 300ºC treated 15 min. These results, particularly related to identification of the sex are in agreement with previous studies. The study of Williams et al demonstrated the successful identification of human biological sex from deciduous teeth exposed to incineration temperatures of 200ºC or below, as it was possible to determine in our study. Respect to permanent teeth, the study of Álvarez García et al reported success in sex determination at 200ºC for 10 min and 300ºC, 400ºC and 500ºC for 2 min. In the present research, higher temperatures (400ºC and 500ºC) did not show Amelogenin bands, although as the study of Urbani et al, at 300ºC for 15 min was possible to see slightly band. Another important issue is the difference on amplification between STRs, the ones located on p arm and in an intron to specific gene (like vWA, TH01 and TPOX, Fig 6) showed almost undetectable amplification from 200ºC 15 min. In spite of these results with STRs, the analysis of amplification of the housekeeping gene used for the QPCR quantification, GAPDH, showed that DNA for this region was amplified in all combinations of temperatures and times (Fig 7), finding highest Ct (meaning the least amplification) with the increase in temperatures and times. This indicates that even in high-temperature burnt teeth it is possible to amplify DNA, at least housekeeping DNA. However, a STR profile would be difficult to obtain probably due to the small size of these regions, which makes them more prone to degradation.

Figure 7. Relative Amplification of CSFO STR. Representative GAPDH Amplification, showing Ct. Amel gen conventional PCR results.

Figure 6. Relative Amplification of FES/FPS, vWA, TH01 and TPOX. Temperature in Celsius degrees and time in minutes.

Figure 5. Relative Amplification of D7S820, D13S317, D5S818 and D16S539. Temperature in Celsius degrees and time in minutes.

CONCLUSIONS

1.   It is possible to get a full STR profilefrom burnt teeth until 300ºC 5 min.

2.   STRs located in “p” arm showed lessamplification efficiency.

3.   Housekeeping genes, like GAPDH,s h o w e d a g o o d a m p l i f i c a t i o n efficiency.

4.   The findings from this researchprovide a quantitative study for theachievement of obtaining DNA profilefrom burnt teeth

REFERENCES 1. Williams D, Lewis M, Franzen T, Lissett V, Adams C, Whittaker D, Tysoe C, Butler R(2004). Sex determination by PCR analysis of DNA extracted from incinerated deciduous teeth. Science & Justice. 44: 89-94. 2. Álvarez-García A, Munoz I, Pestoni C, Lareu MV, Rodríguez-Calvo MS, Carracedo A (1996). Effect of environmental factors on PCR-DNA analysis from dental pulp. International Journal of Legal Medicine 109:125-129. 3. Urbani C, Lastrucci RD, Kramer B (1999). The effect of temperature on sexdetermination using DNA-PCR analysis of dental pulp. The Journal of Forensic Odonto-stomatology 17:35-39

1.   Macroscopic changesThe changes in color observed turned from brown, black, grey/blue and finally chalky white; that corresponds to a calcinated stage of burning. Calcination in the root was observed from 400ºC and 5 minutes of fire exposure, while calcination in the crown was observed from 500ºC and 10 minutes of fire exposure.When calcination of the surface of crown and root, inner layers (dentine, pulp chamber and root canal showed a lower degree of alteration by the fire, due to the protection of the more affected outer layers. Fractures due to fire were observed from 300ºC and 10 minutes of fire exposure in the crown and 400ºC and 10 minutes of fire exposure in the root. Crown fragmentation and separation from the root was observed from 300ºC and 15 minutes of fire exposure.

F igure 2 . C rown co lo r changes due to Temperature exposure at different times.

Figure 1. Color changes due to Temperature exposure at different times. Temperature in Celsius degrees and time in minutes.

1 minute 5 minutes 10 minutes 15 minutes

100ºC

200ºC

300ºC

400ºC

500ºC

600ºC

700ºC

TEMPERATURE((ºC)( TIME((minutes)( CROWN(FX( ROOT(FX(

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Figure 3 . Roots co lor changes due to Temperature exposure at different times.

F i g u r e 4 . F r a c t u r e s d u e t o Temperature exposure at different times.

Sample DNA concentration Temperature (ºC) Time (minutes) mg/ml

Control 1 RT 32.8 Control 2 RT 60

100 1 31.8 100 5 19 100 10 0.257 100 15 0.231 200 1 0.828 200 5 0.387 200 10 0.589 200 15 0.346 300 1 0.665 300 5 0.447 300 10 0.336 300 15 0.130 400 1 0.230 400 5 0.104 400 10 <0.05 400 15 <0.05 500 1 0.169 500 5 0.068 500 10 0.1 500 15 <0.05 600 1 0.055 600 5 <0.05 600 10 0.102 600 15 <0.05 700 1 0.142 700 5 <0.05 700 10 <0.05 700 15 <0.05

Qubit® Fluorometric DNA Quantitation system and ViiATM 7 Real-time PCR system are For Research Use Only. Not for use in diagnostic procedures.