ARCHAEOGENETICS

Ancient and Modern DNA and Archaeology

possibilities and limitations

Seminari Ragusa 11/13 Aprile 2012Josef Caruana

What is ancient DNA

• Ancient DNA is DNA which has suffered degradation

• Normally more than 100 years old

• Usually found in short fragments

History of aDNA• In 1984 traces of DNA from a Quagga specimen were cloned

and sequenced. (Higuchi et al. 1984)

• Pääbo(1985) cloned DNA from human mummified specimen

• Many of the earlier work done on ancient DNA was later

shown to be contamination, or was not reproducible.

Fig 1: The only picture of a living quagga. This was taken in 1870

(After http://www.petermaas.nl/extinct/speciesinfo/quagga.htm)

The Polymerase Chain Reaction: the technique that makes ancient DNA analysis

possible

• Denaturation (Breaks Hydrogen bonds)

• Annealing (primers attach to annealing positions)

• Elongation (Taqpolymerase synthesis a new DNA strand complementary to the template)

A technique which amplifies

trace amounts of DNA

Fig 2: Diagram showing the different steps of PCR

amplification. (After: http://genome.wellcome.ac.uk/)

How ancient DNA is analysed

• Sampling

• Preparation for extraction

• DNA Extraction

• PCR

• Cloning

• Sequencing

• Analysis

• Authentication

Figure 3: The write-up area of the MIB

Protocols for working with aDNA

• Samples come from a well known provenance

• As few people as possible handle the samples

• Work is conducted in specialised clean

extraction and PCR rooms

• Constant irradiation with UV light and cleaning

with DNA destroying reagents.

• Personal protective clothing is worn

• No person who has been in the main lab can go into the clean rooms for the remainder of the day

• Cloning of PCR products

• Ideally results are replicated in an independent laboratory

Figure 4: A person wearing

personal protective clothing when

conducting ancient DNA

research.

Contamination – the major issue

when working with Human aDNAArchaeological samples are very easy to contaminate

This can happen-1. In ancient times

2. In situ - microorganisms

3. During the excavation

4. During post excavation

5. During sampling

6. During work in the lab

Fig 5: Excavation at a Greek site called Kouphovouno(After http://www.nottingham.ac.uk/archaeology/research/scape_kouphovouno.php)

Survival of aDNA

Environmental conditions determine the speed of DNA degradation.

These include:• The level of microorganism activity in the soil.

• The pH value of the soil.

• The thermal age of the site.

• Post excavation handling.

• Time since excavation

Survivability of ancient DNA

• Pre-excavation

– Dna degrades at a fast rate initially

– The rate of Degradation slows down over time

• Post excavation

– As soon as material is exposed the rate of DNA

degradation increases

– The longer it takes for DNA analysis the less

chance of successful amplification

Applications of aDNA analysis

• Sex identification

• Kinship analysis

• Species

Identification

• Palaeodisease

• Migratory patterns

Fig 6:Skeleton of a Child(After http://www.jiaa-kaman.org/anthro.html)

Fig 7:A Multiple Burial

(After http://www.jiaa-kaman.org/anthro.html)

Fig 8: A midden showing bones from different animal

species in it (After http://www.labradorvirtualmuseum.ca/wem/AdlavikArch.html)

These studies are done by the

analysis of:

• Mitochondrial DNA

• X/Y chromosomal and autosomal DNA

What are the most important tools of these studies?

•Single nucleotide polymorphisms

•Short tandem repeats

Single Nucleotide Polymorphisms

(SNPs)

• A single base change in a strand of DNA

• This leads to the formation of two alleles, the

original sequence and the one with the new

mutation.

• In aDNA analysis only SNPs from non coding

regions are used.

Short Tandem Repeat analysis

•A short repeated

sequence which is found in

the non coding regions of

DNA.

•If enough STR positions

are analysed a unique

genetic fingerprint of an

individual can be mapped. Fig 9: showing both SNPs and STRs. This STR is made up of a three base pair sequence which is repeated 5 times in

Male 1, 6 times in Male 2, and 7 times in male 3.(After: http://www.le.ac.uk/ge/maj4/NewWebSurnames041008.html)

The importance of mitochondrial

DNA

• Passed on in a matrilineal manner

• High copy number when compared to nuclear DNA

• Higher mutation rate then nuclear DNA

• Hypervariable regions 1 and 2 are non-coding regions.

Fig 10: The mtDNA Genome. HVR 1 and 2

are the ones amplified in ancient DNA

studies as they have a higher rate of

mutation. (After http://clanhaley.com/dna/index.htm)

Haplogroup/Haplotype

• mtDNA and Y Chromosomal DNA results are classified into haplogroups.

• A Haplotype is a group of alleles which are usually inherited together. (Brown: 2002)

• Haplotypes that share a common ancestor form a haplogroup

Figure 11: Simplified phylogenetic tree showing mitochondrial eve

(After: http://www.genebase.com/image/mtdnaSnpBackboneChart01.jpg)

The Neolithic Revolution

• Was it a cultural change or did people migrate

• MtDNA haplotype analysis was conducted on modern Europeans

• The results have shown that a lot of haplotypes found in modern Europe today entered Europe in several waves during the Upper Palaeolithic (Richards et al.2000)

Neolithic migrations into Europe

• A bottleneck occurred around 20,000 years ago

• Then new haplogroups entered Europe

• 20-25% of modern day European mtDNAlineages are descended from the Neolithic migrations.

• Most present Europeans are matrilinealydescended from Palaeolithic and Mesolithic lineages.

Sex identification

• Regulates a protein (Amelogenin) which is found in developing tooth enamel

• The gene is found both in the X and Y chromosome

• The Y chromosome gene produces a 112bp amplification whilst the X chromosome gene produces a 106bp amplification because of a 6bp deletion.

The Amelogenin gene

Fig 13/14: Shows the six base pair deletion on the X chromosome and how this is visualised on a gel. (After http://www.cstl.nist.gov/)

Mycenae Grave Circle B(Bouwman et al. 2008)

•Study to see if the 35 inhumations found in Grave circle B came from the same

important family.

•No nuclear DNA was retrieved from the 22 skeletons sampled.

• mtDNA sequences were retrieved for 4 of the inhumations• The mtDNA haplotype of 2 of the skeletons in addition to facial

reconstruction led the scientists to conclude that the two are brother and sister

• The long time elapsed between excavation and DNA analysis had hampered the amount of aDNA successfully retrieved from the site.

Fig 16/17: Facial reconstructions of individuals buried together in Grave

circle B at Mycenae. aDNA analysis indicates that samples 55 and 58 might

be siblings. (After Bouwman et al. 2008)

Domestication of Bos

• Modern mtDNA samples of cattle coming from Europe, Africa and the Near East were compared with four mtDNA sequences of extinct British wild Oxen

• mtDNA haplotype grouping supports the idea that European cattle originated from the Near East

• The results showed that the centre of origin has a higher haplotypicdiversity then the regions into which the cattle were then taken.

• European cattle are descended from Near Eastern animals and not domesticated locally in Europe. Fig 18: mtDNA reduced median

networks (After Troy et al. 2001)

Both Ancient DNA and DNA from

present day pathogens can give

insights into palaeodisease

Diseases studied via ancient DNA include

• Tuberculosis

• Leprosy

• Syphilis

• Plague

• 1918 Influenza

• HIV from 1959

How can Ancient DNA contribute to

Palaeodisease ?

• Provides time depth to studies carried out with modern DNA

• Can test hypotheses derived from modern DNA studies

• May find variants now lost

• May be able to show how loss or increase of virulence has happened

• May be able to pinpoint human genetic response to disease

• Can identify disease in past populations

• Can reveal prevalence of disease in a population

Tuberculosis: DNA can also be

detected in bones without lesions

• Only 5 – 7% of present-day TB victims develop bone lesions

• Was this the case in the past ?

• If so then TB prevalence is seriously underestimated

• A DNA-based approach can indicate the presence of the pathogen on otherwise asymptomatic individuals

• Two studies suggest TB in bones lacking lesions.

• Ancient DNA analysis may reveal the presence of pathology were these are not visible as lesions in osteological studies.

• aDNA of Mycobacterium tuberculosis which cause tuberculosis has successfully been amplified from archaeological specimen. (Bouwman and Brown 2005)

• Some paleodisease DNA survives in the archaologicalrecord whilst others does not, thus whilst the mycobacteria which cause tuberculosis and leprosy has been succesfully amplified, A study on Syphillis did not obtain any results, probably due to the cell structure of the organism.

• In their study Bouwman and Brown(2005) tried to

obtain Mycobacterium tuberculosis. and Treponema

pallidum sequences from 46 bones (9th – 19th century)

• 25 of the bones yielded mtDNA

• Of these, 7 bones gave M. tuberculosis PCR products

• No Treponema sequences were obtained.

Plant domestication

• aDNA was successfully amplified from charred and desiccated wheat.(Allaby et al. 1994)

• Studies are being conducted in order to determine the original point of domestication/s of several plant species

• These studies are showing that cereal domestication occurred in more than one region (Brown et al. 2009, in press)

• The studies also indicate that tetraploid wheat had a minimum of two domestications. ( Brown et al. 2009, in press)

Fig 19: The origins of Agriculture in the

Fertile Crescent and its development

throughout time (After Brown et al.

2009)

Contamination

• The most problematic issue when dealing with

human ancient DNA

• Safeguards are needed to reduce the possibility

of contamination

• Renders results invalid

How should archaeologists avoid

contamination

• Face Mask

• Gloves

• Full Body suite

• Minimal handling of the material

• Appropriate storage of material

• Ideally the biological material is excavated by the person doing the analysis

Acknowledgements

• My Supervisor Terry Brown and the people in his group

• University of Manchester

• Heritage Malta

• University of Malta - Genetics Laboratory

• Superintendence of Cultural Heritage Malta