Analysis of Ancient Human DNA and Primer Contamination

1. Analysis of Ancient Human DNA and Primer Contamination By: AshneetBiln, Raelene Knight & Shauna Maracle

2. Overview • Introduction to Mitochondrial DNA • Article Overview • Primers • PCR positive and negative controls • Results • Contamination • Conclusion

3. Mitochondrial DNA • In one cell, there can be approx. 1000 mitochondria that each contain DNA • The more DNA, more risk of contamination • Older or degraded samples, use mtDNA for analysis • Each mtDNA only has 16,569 base pairs • Higher copy number relative to nuclear DNA

4. Nuclear DNA vs. mtDNA • Table 16.5 Comparison of human nuclear DNA and mitochondrial DNA markers • (Fundamentals of Forensic DNA Typing: John M. Butler)

5. Precautions • Precautions During Excavation: • Gloves • Masks • Coats • Hermetic Sterile Bags • Preserved at -20ºC • Precautions During Analyse: • Separate sterile rooms • Protective clothing • DNA free equipment & Reagents • Specific Conditions: • High-pressured system • Filtered incoming air • UV light irradiation • Laminar flow good • Bleach

6. Process • Bones/teeth reduced to powder • Decalcification & Protein digestion at 55ºC • EDTA 0.5M, pH= 8.5, Proteinase-K 1-2 mg/ml, N-laurylSarcosyl 0.5% • Extracted using phenol-chlorform-isoamyl alcohol organic extraction • concentration of 80-100 µL • Negative control • PCR

7. Preparations • Negative Controls for each set of PCR: • dNTP • MgCl2 • BSA • Taq Polymerase • PCR buffer • Source of Error Detection: • Mixture of PCR products • PCR products are copied using Invitrogen • 5 – 8 clone sequences are analysed per PCR product

8. PCR Pairs • Pair 1 HVRIaL15989 (5′-CCCAAAGCTAAGATTCTAAT-3′) H16175 (5′-TGGATTGGGTTTTATGTA-3′) • Pair 2 HVRIbL16114 (5′-TGGATTGGGTTTTATGTA-3′) H16251 (5′-GGAGTTGCAGTTGATGT-3′) • Pair 3 HVRIcL16190 (5′-CCCCATGCTTACAAGCAAGT-3′) H16322 (5′-TGGCTTTATGTACTATGTAC-3′)

9. Amplifications • 25 μl reaction volume • 6.5 mM MgCl2 • 0.4 mMdNTP • 0.66 mg/ml BSA • 1 μM of each primer • 2.5 μl GeneAmp 10× PCR buffer (Perkin-Elmer) • 0.25 μl pure DNA extract • 1.25 U AmpliTaq Gold™ • 55 cycles at 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 45 s • Types of Negative Controls: • False extracts without bone material • PCR with water instead of DNA extract

10. Results • All products were added to the BioEditprogram • Compared to mtDNA sequences • GenBank • Characterise • Contamination • Blast • Resulting conclusion

11. Results continued • Numerous contaminations have been detected in template PCR products and negative controls • Interpreted as the result of the contamination of PCR reagents, including primer lots • Contamination of PCR reagents with human DNA can be explained by the fact that they are handled and manufactured by humans • Primers that are manufactured by different companies and purified using different technologies indicate that primer contamination is very frequent

12. Contamination

13. Conclusion • Could have been systemic contamination, since it did not come from any of the researchers. • Primers & Buffers could have been contaminated. • Even with following strict and proper guidelines contamination will still occur no matter what. • Where do you think the contamination occurred? Why?

14. References • Forensic Science International, Analysis of ancient human DNA and primer contamination: one step backward one step forward, volume 210, Issues 1-3, July 15th, 2011, pages 102-109 • Fundamentals of Forensic DNA Typing: John M. Butler, pages 375-386