ASSESSING DAMAGES: TESTING THE ASSUMPTIONS OF A NON-DESTRUCTIVE PROTOCOL FOR DNA EXTRACTION FROM MODERN HUMAN TEETH. Christopher Bowers, Graciela S. Cabana, and Bridget F.B. Algee-Hewitt Department of Anthropology, University of Tennessee, Knoxville Introduction DNA extraction is a routine procedure in the biological and forensic anthropological fields, yet techniques necessitating destruction of the analyzed sample often deter its implementation. When skeletal material is scarce, or when the question of whether later testing will be required becomes relevant, destructive DNA extraction methods may not be an attractive option. For this reason, non-destructive techniques are routinely explored. Recently Bolnick et al. (2011) offered a compelling non- destructive extraction technique, but without extensive testing of the destructive potential. In particular, possible chemical effects that could compromise the integrity of the sample were not investigated. Project Goals The goal of this project is to provide a meaningful, supplemental analysis of the destructive effects of DNA extraction techniques by investigating the chemistry involved in the bone-solution interface. The Bolnick et al. (2011) procedure involves soaking the material to be analyzed in an extraction solution for twenty-four hours. This soaking process has the most potential for harm, due to the sample being in contact with highly concentrated EDTA, a strong chelating agent; for this reason, this is the process which received the most attention throughout this project. A few of the questions that one should ask are: -How can possible unobserved chemical damage be determined if there is no gross damage to the material? -What elements of the soaking procedure are the most destructive? -Can these potentially destructive steps be improved? Samples and Methods Teeth were used as the sample material, and were weighed during each step of the procedure to measure gross changes in the samples. Following the procedure laid out in Bolnick et al. (2011), any teeth selected for the experiment were submerged in full strength bleach; washed with HPLC-grade water; each side was irradiated with 254 nm ultraviolet (UV) light for ten minutes; and, finally, each tooth was soaked for twenty-four hours in a 10 mL solution of 0.5 M EDTA and 150 uL 0.25 mg/mL proteinase K at a pH of 8.0 with gentle rocking. After soaking overnight, the solution was carefully poured into a new 15 mL conical tube, and the teeth were washed three times with HPLC-grade water and air dried at room temperature. Also any bleach used, and one solution which was not used to soak a tooth (a blank) was kept for each set of teeth soaked overnight. Solutions were then sent to Vanderbilt University's Department of Civil and Environmental Engineering where they were run through ICAP (Inductively Coupled Argon Plasma) to determine the solution concentrations of magnesium (Mg), phosphorous (P), and calcium (Ca) that was in solution. P and Ca were selected for testing because they are the main constituents in calcium hydroxyapatite (tooth enamel), Mg was selected for being the target of EDTA used in the extraction protocol. Preliminary testing was done using pig's teeth due to their availability. Pig's teeth were selected because they are entirely covered with enamel, similar to human teeth, making them suitable for chemical observations. Five pig's teeth were put through the protocol. Later testing on six human teeth was conducted, with two teeth subjected to EDTA concentrations of 0.1 M and 0.25 M. Dentometrics were taken on the human teeth before and after soaking. Results After soaking several pig's teeth, one of which was soaked twice, it was found that all of the teeth lost mass and were significantly affected physically. Any teeth which had cracks previously were now entirely broken apart, and much of the surface had changed color. The teeth were left in conical test tubes, and after some time it was noted that they had become severely discolored, more so than immediately following the soaking procedure (see Fig. 1). Regardless of their original size, all the teeth lost about 0.2 grams of mass, and had similar amounts of Ca, P, and Mg (~6700 to 9400 mg/L as Ca) left over in solution. The human teeth fared much better than the pig's teeth, with only a few showing significant damage; there was also significantly less Ca, P, and Mg (850 to 1350 mg/L as Ca) left in solution after soaking. The two teeth which were soaked using lower concentrations of EDTAhad much less Ca, P, and Mg (200 and 850 mg/L as Ca) in solution than the others. As with the pig's teeth, any human teeth that had surface damage before soaking were more affected by the procedure (see Figures 3 and 4). Much of the enamel had seemed to wear off so that the crowns had become a similar color to the roots, making dentometric measurement very difficult. The measurements taken were more affected by this change than by actual changes to the dimensions of the teeth. One tooth had a filling which was very strongly bound to the tooth before soaking, after soaking the filling dropped out of the tooth, and severe degradation could be observed where some surface damage was present before. None of the human teeth lost mass in the same way that the pig's teeth did. Discussion It has become very evident that surface damage and wear has a major impact on the gross damage to the material; however, chemical effects stayed roughly the same regardless of the amount of observable damage. Moreover, the solution concentrations were directly related to the concentration of EDTA used, with lower concentrations having the least effect on the teeth. This would suggest that much less EDTA is required to chelate with the DNase than is actually used, and that leftover EDTA is reacting with the hydroxyapatite in the tooth (or the leftover EDTA could be reacting with the DNase). While this would be ideal for a destructive method where the integrity of the sample is irrelevant; in a non-destructive method refinements would be needed to determine an optimal amount of EDTA to use for sequestering the DNase. There is a large discrepancy between the results from the pigs' teeth and the human teeth which can be explained by the differences in condition prior to soaking, as well as differences in surface chemistry. The pigs' teeth were somewhat worn before the samples arrived at the lab, while the human teeth were in fairly good shape. A likely cause for the difference in chemical effects is due to the common addition of fluoride to drinking water and tooth paste used by humans. The fluoride reacts with hydroxyapatite to form fluoroapatite, a significantly less reactive compound. This makes the procedure better suited for use with modern samples than it would be for older, more worn samples. The differences in Mg in solution for pigs' teeth and the human teeth indicates that it is in fact from the tooth. The amount of DNase present in the soaking solution is minute, its contribution to Mg in the samples insignificant. Conclusions and Future Direction The method proposed by Bonick et al. (2011) is a good attempt at a practical, non-destructive DNA extraction technique, but it is not perfect. Surface damage can be exacerbated during the procedure, and particularly worn samples can experience a loss of material due to chemical reactions with the soaking solution. Future studies should try to refine the technique, determining optimal concentrations and pH's for the procedure so that the reaction with skeletal material is minimized, while maintaining good results from the extraction procedure. Soak in Bleach UV Each Side Incubat e Overnig ht Add EDTA And proK Rinse Rinse Or, Rinse and Repeat Figure 5. Testing Procedure References 1. S. Chandler abd D.W. Fuerstenau, Solubility and Interfacial Properties of Hydroxyapatite , Adsorption On and Surface Chemistry of Hyrdoxyapatite 2. Martell, A.E. And R.M. Smith, 1974, Critical Stability Constants , Volume 1, Amino Acids, Plenum Press, NY 3. Stumm, W. and J.J. Morgan, 1996, Aquatic Chemistry , 3 rd Edition, John Wiley & Sons Inc., NY 0.5 M EDTA Pig's Teeth Human Teeth Mg (mg/L) 214.3 to 303 12.8 to 21.5 Ca (mg/L) 6737 to 9372 947 to 1349 P (mg/L) 2507 to 2713 468.6 to 634 Comments Pig's teeth lost 0.2 g, human teeth lost none but had some gross damage 0.25 M EDTA Human Premolar with crack 8.79 858 414 No observable gross damage to this one, despite having been cracked. 0.1 M EDTA Human Premolar light wear 5.13 224 112 Zero damage, almost as if it had not undergone soaking EDTA Metal Chelate Figure 1. Pig's Molar Post Soaking Figure 2. Pig's Premolar Post Soaking Figure 3. Human Premolar With Filling Before (Left) and After (Right) Soaking Figure 4. Human Premolar With Wear Before (Left) and After (Right) Soaking ICAP Solubility of calcium hydroxy apatite, Ca5OH(PO4)3, 1.45 mg/l @ pH 8.0 Aged Pig Teeth (6,787 to 9,373 mg/L) Maximum solubility in equilibrium with 0.5M EDTA (Ca = 20,050 mg/L) Human Teeth (947.3 to 1,348.8 mg/L) Expected Range of Values Figure 6. Ca Conc. vs. pH