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