1. mtDNA from Human Teeth Samples (05/19/2006) Gel 1: PCR Products of mtDNA Amplification Using Primer Set 1 Gel 2: PCR Products of mtDNA Amplification Using Primer Set 2 See Details and Legend Next Slide

2. Lane Identification Samples Lanes 1 and 2: 3 pieces of tooth/extraction Lanes 3 and 4: 1 piece of tooth/extraction Lane 5: 2 pieces of tooth/extraction Lane Identification for both gels: Lanes 1 to 5: PCR products of amplified mtDNA gene on purified DNA samples. Lanes 1 and 3: Extract with Acetic acid, pH 4.39. Lanes 2 and 4: Extracted with DNase and RNase-free water. Lane 5: no-PCT control (no PCT-treatment). Lanes 6 to 10: PCR products of amplified mtDNA gene on non-purified DNA samples. Lane 12: PCR positive control. Lane 11: PCR negative control.

3. Experiment Details PCT Conditions: 35 kpsi, 10 cycles, 20S up and 20 S down at 4 o C. Amplification: Mitochondrial DNA (mtDNA) was amplified with two sets of primers. Gel 1:Human mtDNA control region(HVI) was amplified using primers L16159 (5 –TACTTGACCACCTGTAGTAC-3) and H16401(5 –TGATTTCACGGAGGATGGTG-3) Reference: Genetic and Molecular Biology (Ricardo Leonart et al. vol. 22, No.3, Sept. 1999). Amplicon 287 bp. Gel II: Primers sequences are from Armed Forces Amplicon 149 bp.

4. Method used in this application This was a limited proof of principle experiment with the goal being to see if we could get mtDNA from teeth using water rather than acetic acid and to see if we could eliminate the purification step. Here is our interpretation of these data: 1. mtDNA can be extracted by PCT from teeth fragments using water as the buffer.However, we recommend to use a chaetrope such as Guanidinium HCl or GTC. The only reason we tried water was to see if we could make a rapid test, like those we did for hair. It is not necessary to use acid to decalcify the tooth. 2. We worked at 4°C. This is not necessary. Room temperature should work as well. 3. Data indicate that it will be necessary to purify the DNA.

5. Method used in this application The only reason we broke the teeth was so that they fit in the PULSE Tube. I would try an extraction without breaking the tooth if they fit since teeth have the larger root canal. We recommend the following conditions to start: A. PCT at 35 kpsi, 20 cycles, 20 seconds at high pressure, 10 seconds at low pressure. B.Use a standard DNA extraction buffer, such as GTC or GuHCL or from Qiagen or Roche. Any other standard buffer developed for the tooth using the same PCT protocol as described above should work as well. C.Purify the extract using a Qiagen column or your preferred purification method.