Download Applications of Molecular Biology in Archaeology

Applications of Molecular
Biology in Archaeology
- Extraction of Ancient DNA
Bradwell, S. and Nguyen, R.
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
Problems with PCR
More than 99% of ancient DNA are chemically modified
SOURCES OF DAMAGE:
1) Hydrolytic damage resulting in deamination
of bases
2) Oxidative damage
3) Presence of unidentified inhibitors of PCR
www.pharmcentral.com/ nucleotides.htm
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/
More Problems with PCR
Risk of contamination with
contemporary DNA
Allelic drop out
www.oxoid.com/uk/ index.asp?mpage=iqualicon&c=UK
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
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