PCR is the simplest method to use to produce multiple copies of DNA.^{2,5–7,10,11} The strands of DNA are unwound and duplicated by a polymerase using each strand as a template. PCR has great sensitivity and applicability in analyzing DNA from limited biological material.^10,11 Gall and colleagues¹⁰ and Dimo-Simonin and colleagues¹¹ reported that DNA could be amplified from cytological stained smears. PCR also is an important technique for amplifying DNA that may be old and partially degraded.⁵

The real-time polymerase chain reaction (RT-PCR) assay has the ability to monitor the progression of DNA quantification. Reactions are characterized at the point during cycling when amplification of a PCR product is first detected rather than by the amount of PCR product accumulated after a fixed number of cycles. RT-PCR assays are sensitive and require minimal attention. They also are cost-effective, fast and accurate.^12,13

The Quant-iT PicoGreen (Invitrogen, Carlsbad, Calif.) assay is another method used to quantify DNA. It is a nonspecific method that relies strictly on the total amount of DNA present rather than the presence of a specific gene.

Toothprints (Kerr, Orange, Calif.) dental impression wafers are a commercial product that has been reported to be able to register patients’ unique bite characteristics, as well as capture their DNA.¹⁴ The developer, however, has stated that "no specific DNA tests have been done" to verify the amount or quality of the DNA present.¹⁵ It also is unclear how long the DNA will be able to be extracted from the bite registrations stored in the plastic bags that are provided (this issue was beyond the scope of this study). The manufacturer recommends that Toothprints be used to make bite registrations when the children are 3, 8 and 13 years of age to correspond to the three main stages of dentition development: primary, mixed and adult.

There are no data that verify the product’s ability to capture DNA or to provide accurate impressions for use in identifying people. Therefore, we conducted a study to test the ability of the dental impression wafer to capture DNA, to analyze the quantity and quality of that DNA and to analyze any inaccuracies in the impression technique. We used the Quant-iT PicoGreen assay to determine the total amount of DNA and the RT-PCR assay to determine the overall quality of the DNA. Establishing the validity of Toothprints as an effective tool may help with the genetic and dental matching processes that are used to identify recovered living and deceased children.⁴

	SUBJECTS, MATERIALS AND METHODS

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Subjects. We recruited 20 healthy patients (nine boys and 11 girls) with mixed dentition who ranged in age from 7 to 12 years from Riley Hospital for Children, Indianapolis. None of the subjects had systemic disease or oral pathological lesions. The Indiana University Institutional Review Board approved the study, and we obtained informed consent from the children’s parents or guardians.

Sample collection. Each of the 20 subjects completed all four steps of the study during one appointment. We assigned each subject a number to conceal his or her identity.

In the first step, we made one bite registration by using a dental impression wafer per the manufacturer’s instructions, placed it in the provided plastic zipper bag and stored it in the dark at room temperature for seven days. In the second step, we obtained a saliva sample by having the subject rinse once with 10 milliliters of mouthwash for 15 seconds. Third, we collected buccal mucosa cells by twisting a cytology brush while moving it up and down on the inside of the subject’s cheek. Finally, we made a maxillary alginate impression for each patient. All of the steps were carefully demonstrated and then observed or performed by a pediatric dentist (M.E.) trained in these techniques.

We poured the alginate impressions by using cast stone, following manufacturer’s instructions, to create dental models and allowed them to dry. We then trimmed the casts and scanned them at 300 dots per inch (DPI) by using an American Board of Forensic Odontology (ABFO) no. 2 ruler to maintain scale. We also scanned in the bite registrations by using an ABFO no. 2 ruler at 300 DPI. We superimposed the scans of the dental models over the scans of the bite registrations and recorded the number of matched and unmatched teeth (Figure). We calculated a ratio of matched to unmatched teeth. The method we used to compare the bite registration and dental model was a modification of the technique developed by Johansen and Bowers.¹⁶ This was a two-dimensional comparison. A positive match occurred when the incisal or occlusal outlines on the transparency of the model matched the incisal or occlusal outlines on the bite registration.

View larger version (71K): [in this window] [in a new window]   Figure. A. Image of a bite registration with tooth imprints made with a Toothprints dental impression wafer (Kerr, Orange, Calif.). B. Scan of patient dental model. C. Flipped image of dental model. D. Superimposition of flipped image of dental model over imprinted bite registration.

The DNA purification procedure we used for the oral rinse and cytology brush collection was from that described by the manufacturer of the Gentra Puregene Buccal Cell Kit (Qiagen, Valencia, Calif.). We lysed the cells with the cell lysis solution and precipitated the proteins with the protein precipitate solution as described by the manufacturer. We transferred the DNA supernatant to fresh tubes containing 1 mL of 100 percent isopropanol and 5 microliters of the glycogen solution supplied by the manufacturer. After we centrifuged the pellets, we washed them with 1 mL of 70 percent ethanol. After we air-dried the DNA pellets, we rehydrated them with 100 µL of the hydration solution supplied by the manufacturer for the mouthwash and 20 µL for the buccal swab samples. We collected cells by vigorously washing the dental impression wafer after it was stored for seven days with 10 mL of sterile phosphate buffered saline solution.

We then extracted the DNA by using the protocol described above and used 100 µL of the DNA hydration solution to rehydrate the DNA pellets from the dental wafer impression samples. We stored the DNA extracted from each source at – 70 C until we performed the DNA analyses.

DNA quantification. We measured the DNA concentrations by using a nonspecific method (Quant-iT PicoGreen dsDNA Quantitation Reagent, Invitrogen) per the manufacturer’s instructions. We mixed the collected DNA samples with buffer containing 10 millimolar of Tris-hydrochloride with 1 mM ethylenediaminete-traacetic acid, pH 7.5 and Quant-iT PicoGreen reagent to a total volume of 200 µL per well in 96 well plates. We then analyzed the plates by using a fluorimeter at a wavelength excitation of 480 nanometers and emission of 520 nm. We generated a DNA standard curve by using a Lambda DNA standard and used it to calculate the DNA concentration of each of the samples. We analyzed each sample three times, calculated the average and used it for statistical analysis.

We measured the concentration of intact double-stranded DNA by using RT-PCR using TaqMan RNase P detection reagent (FAM Dye; Applied Biosystems, Foster City, Calif.) with an ABI Prism 7000 Sequence Detection System (Applied Biosystems). We mixed the DNA samples with TaqMan Universal Master Mix (Applied Biosystems), RNase P primers (Applied Biosystems) and RNase probe (Applied Biosystems) in a total volume of 25 µL per the manufacturer’s instructions. The PCR parameters were set for 15 seconds at 95 C and 60 seconds at 60 C for 50 cycles. We used human genomic DNA to generate a standard curve to determine the DNA concentration in each of the samples. We ran the samples three times, calculated the average and used it for statistical analysis.

Statistical analysis. We compared the DNA yields of the DNA isolated from the dental impression wafer, mouthwash and buccal swab by using analysis of variance and accounted for the within-subject correlations. We performed pairwise comparisons between the methods by using the Fisher protected least significant differences method to control the overall significance level. Because of several outliers, we analyzed the DNA yield data by using the ranks of the data. We computed Spearman rank correlation coefficients to determine if there were any associations between the DNA yields for the three isolation methods.

We computed an exact 95 percent confidence interval for the percentage of acceptable bite registrations to determine if the dental impression wafer provided accurate bite registrations compared with scanned stone models made from the alginate impressions.

	RESULTS

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Quant-iT PicoGreen DNA yield. Use of the dental impression wafer resulted in significantly lower DNA yields (1.03 µg) than did the mouthwash (509.57 µg, P = .0001) and buccal swab (113.61 µg, P = .0001) methods (Table 1). The buccal swab samples had significantly lower DNA yields than did the mouthwash samples (P = .0012). Correlations between the DNA yield measurements of the buccal swab, mouthwash and dental impression wafer were too low to be considered clinically or statistically significant: the buccal swab and mouthwash correlation was –0.03, the buccal swab and dental impression wafer correlation was –0.15 and the mouthwash and dental impression wafer correlation was 0.23. Therefore, there was little correlation between a patient’s having a high yield of DNA from the mouthwash method and either of the other two methods.

	DISCUSSION

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In our study, we tested the ability of a dental impression wafer to capture DNA, analyzed the quantity and quality of that DNA and analyzed any inaccuracies in the impression technique. Mouthwashes and buccal swabs have been the primary methods for acquiring DNA for forensics. However, these methods are almost never used with children.

The amount of DNA we recovered from the dental impression wafer samples in most of the cases was sufficient for amplification and identification using the PCR method. The RT-PCR method yielded similar results to those of King and colleagues⁶ for buccal swab samples (11.5240 versus 12 µg) and mouthwash samples (22.2540 versus 15.8 µg). The amount of DNA recovered in our study from the buccal swab and mouthwash samples was higher than that from the dental impression wafer samples (0.0279 µg). According to the Federal Bureau of Investigation, the typical amount of DNA sought for analysis is 0.001 µg, but samples of 0.0002 µg can be typed (Dr. Bruce Budowle, Senior Scientist, Laboratory Division, Federal Bureau of Investigation, written communication, November 2005). Therefore, the average yield of 0.0279 µg isolated from the dental impression wafer samples is adequate for analyses.

The dental impression wafer can yield enough DNA for forensic analysis, although the quantities were not as high as those obtained from mouthwash and buccal swab samples.

The variability between samples from each method is evident by the large ranges in DNA concentrations, especially with the mouthwash samples. We gathered samples whenever a patient had an appointment and met the inclusion criteria. Therefore, there could be numerous variables to explain these differences in DNA concentrations, such as that some of the patients may have eaten before we collected their samples but others did not. A larger study would be needed to address this issue properly.

The number of subjects whose samples we quantified by using RT-PCR was 19 (Table 2), and the number of subjects whose samples we quantified by using Quant-iT PicoGreen was 20 (Table 1). The difference was due to the fact that there was an insufficient amount of one subject’s sample. The order of acquiring samples was the same (dental impression wafer, mouthwash, buccal swab) for the last 16 of the 20 subjects. We acquired the first four subjects’ samples in a different order (mouthwash, dental impression wafer, buccal swab), which may have led to variations in the DNA yield for dental impression wafer samples.

In our study, the accuracy of the bite registrations made using the dental impression wafer was 86 percent. This level of accuracy was not sufficient to be used for identification purposes. The time between bite registration and analysis was minimal in this study. Therefore, the accuracy will be less across time. There is no question that the bite impression’s accuracy would diminish across time owing to the changes in children’s dentitions during the age span of 3 to 13 years identified by the dental impression wafer’s manufacturer.

Future investigations are needed to determine how long the DNA that remains on the bite registration when it is stored in a dark location at room temperature is of acceptable quality. The bite registration also should be examined for any material distortions across time when it is stored per the manufacturer’s instructions. Future studies should determine if the wafers contain human scents that may be used by dogs in search-and-rescue operations. These issues were beyond the scope of this our study.

	CONCLUSIONS

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The results of our study demonstrate that the dental impression wafer can yield enough DNA for forensic analysis, although the quantities were not as high as those obtained from mouthwash and buccal swab samples. Also, the dental impression wafer in our study did not appear to make a bite registration with enough accuracy to be used for identification purposes.