1. By: Ashneet Biln, Raelene Knight & Shauna Maracle

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

3.   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 Mitochondrial 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 -20 º C  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.  - Bones/teeth reduced to powder - Decalcification & Protein digestion at 55 º C o EDTA 0.5M, pH= 8.5, Proteinase-K 1-2 mg/ml, N- lauryl Sarcosyl 0.5% - Extracted using phenol-chlorform-isoamyl alcohol organic extraction o concentration of 80-100 µL - Negative control - PCR Process

7.  Preparations  Negative Controls for each set of PCR: - dNTP - MgCl 2 - 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.   Pair 1 HVRIa L15989 (5′-CCCAAAGCTAAGATTCTAAT-3′) H16175 (5′-TGGATTGGGTTTTATGTA-3′)  Pair 2 HVRIb L16114 (5′-TGGATTGGGTTTTATGTA-3′) H16251 (5′-GGAGTTGCAGTTGATGT-3′)  Pair 3 HVRIc L16190 (5′-CCCCATGCTTACAAGCAAGT-3′) H16322 (5′-TGGCTTTATGTACTATGTAC-3′) PCR Pairs

9.  Amplifications  25 μ l reaction volume  6.5 mM MgCl2  0.4 mM dNTP  0.66 mg/ml BSA  1 μ M of each primer  2.5 μ l GeneAmp 10× PCR buffer (Perkin-Elmer)  0.25 μ l 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.  - All products were added to the BioEdit program - Compared to mtDNA sequences - GenBank - Characterise - Contamination - Blast - Resulting conclusion Results

11.   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 Results continued

12. Contamination

13.   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? Conclusion

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