www.le.ac.uk/.../ar99-00/international/ international.html Applications of Molecular Biology in Archaeology - Extraction of Ancient DNA Bradwell, S. and Nguyen, R. www.le.ac.uk/.../ar99-00/international/ international.html

BACKGROUND What is Ancient DNA? Ancient DNA refers to DNA obtained from highly degraded ancient biological remains (such as bone, teeth and hair). For a long time, scientists did not expect long-term DNA-preservation in ancient remains because degradation results shortly after individual’s death. http://www.pbs.org/wnet/pharaohs/secrets3.html

BACKGROUND Environmental Influences

BACKGROUND Early 1980’s Investigated mummies as old as 2,400 yr-old for DNA and found that DNA could be molecularly cloned in a plasmid vector. Problem: Classical molecular biological methodologies required high amounts of DNA material, but most ancient DNA are highly degraded and only minute quantities may be extracted news.nationalgeographic.com/.../ 07/0709_mummycongress.html

Polymerase-Chain Reactions Field of Molecular Archaeology was “revolutionized” by the invention of PCR Technique made it possible to detect and characterized minute traces of DNA. Ancient-DNA research has been a proven to be a powerful tool used in archaeological study. www.pbs.org/wgbh/nova/neanderthals/ mtdna.html

Extraction Teeth and bones are excellent sources for the extraction of ancient DNA. http://www.alumni.berkeley.edu/imgs/Alumni/California_Monthly/2001_Sep/talk_bones.jpg

Extraction Begin by breaking up tissue 1) Grinding in liquid nitrogen 2) Classic phenol-chloroform extraction 3) Salt-based extraction 4) A variety of commercial kits www.catgen.com/home/unduguk/ EN/100035.html www.the-scientist.com/yr1997/ mar/tools_970331.html forensic.to/webhome/dna/paternity.htm

PCR Success Successful DNA typing of both short tandem repeats loci and the hypervariable region of the human mitochondrial DNA was achieved after extracting total DNA from bones Two common methodologies employed: 1) Centrificon approach 2) QIAquick approach http://www.pbs.org/wnet/pharaohs/secrets3.html

www.pharmcentral.com/ nucleotides.htm Problems with PCR More than 99% of ancient DNA are chemically modified SOURCES OF DAMAGE: Hydrolytic damage resulting in deamination of bases 2) Oxidative damage 3) Presence of unidentified inhibitors of PCR www.pharmcentral.com/ nucleotides.htm

www.cnn.com/2000/NATURE/11/ 10/ancient.europeans.ap/ Solutions Knowledge! - The size of the amplification product inversely correlates with the amplification efficiency Gas chromatography and Mass Spectrometry allow the determination of a damage threshold Verify DNA authenticity through the estimation of D/L ratio of amino acids Simulate environmental conditions www.cnn.com/2000/NATURE/11/ 10/ancient.europeans.ap/

Risk of contamination with contemporary DNA More Problems with PCR Risk of contamination with contemporary DNA Allelic drop out www.oxoid.com/uk/ index.asp?mpage=iqualicon&c=UK

www.aero.org/facilities/ environmentalApps.html Solutions Common sense Sample pre-treatment Use of controls www.aero.org/facilities/ environmentalApps.html

Case Study The Discovery of the Kwäday Dän Ts’ìnchi Ancient Remains at the Edge of a Glacier in British Columbia’s Tatshenshini-Alsek Park. http://srmwww.gov.bc.ca/arch/kwaday/index.html

Kwäday Dän Ts’ìnchi = person long ago found Case Study Kwäday Dän Ts’ìnchi = person long ago found “Our oral history has told us about the importance of this area as a travel route and about the nearby villages on the Tatshenshini River. Now Kwaday Dän Sinchì brings these stories to life.” http://srmwww.gov.bc.ca/arch/kwaday/index.html

Case Study Initial radiocarbon dating indicates that the remains are approximately 550 years old http://srmwww.gov.bc.ca/arch/kwaday/index.html

Case Study First access was given to the researchers Monsalve et al. 2002 DNA extracted from the humerus, overlaying muscle, bone collagen Proteinaceous material was detected by measuring the % of C and N in whole bone and collagen-type residue using an elemental analyzer Denaturation step of PCR cycle lengthened to 5 min http://srmwww.gov.bc.ca/arch/kwaday/index.html

Case Study Ancient DNA authenticity determined by: 1) Verifying nitrogen and carbon % in hard and soft tissue met requirements for preservation of proteinaceous material 2) Analyzing mtDNA sequences from PCR fragments and cloning products of the hypervariable control region 3) Validating the humerus DNA results with an independent study http://srmwww.gov.bc.ca/arch/kwaday/index.html

Conclusion Ancient DNA provides insight into historical events It has been successfully applied for understanding ancient pathologies Finding may also be used for the reconstruction of population origins Ancient DNA has been applied to determine evolutionary relationships as well as paternity and maternity

The End http://srmwww.gov.bc.ca/arch/kwaday/index.html