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Analysis of Ancient Human DNA and Primer Contamination . By: Ashneet Biln , Raelene Knight & Shauna Maracle. Overview. Introduction to Mitochondrial DNA Article Overview Primers PCR positive and negative controls Results Contamination Conclusion . Mitochondrial DNA. - PowerPoint PPT Presentation
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Analysis of Ancient Human DNA and Primer Contamination
By: Ashneet Biln, Raelene Knight & Shauna Maracle
Introduction to Mitochondrial DNA Article Overview Primers PCR positive and negative controls Results Contamination Conclusion
Overview
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
Nuclear DNA vs. mtDNA
Table 16.5 Comparison of human nuclear DNA and mitochondrial DNA markers
(Fundamentals of Forensic DNA Typing: John M. Butler)
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
- Bones/teeth reduced to powder- Decalcification & Protein digestion at 55ºC
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 extractiono concentration of 80-100 µL
- Negative control- PCR
Process
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
Pair 1
HVRIa L15989 (5′-CCCAAAGCTAAGATTCTAAT-3′)H16175 (5′-TGGATTGGGTTTTATGTA-3′)
Pair 2HVRIb L16114 (5′-TGGATTGGGTTTTATGTA-3′)H16251 (5′-GGAGTTGCAGTTGATGT-3′)
Pair 3HVRIc L16190 (5′-CCCCATGCTTACAAGCAAGT-3′)H16322 (5′-TGGCTTTATGTACTATGTAC-3′)
PCR Pairs
Amplifications
25 μl reaction volume 6.5 mM MgCl2 0.4 mM dNTP 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
- All products were added to the BioEdit
program- Compared to mtDNA sequences - GenBank - Characterise - Contamination- Blast- Resulting conclusion
Results
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
Contamination
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
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
References
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