Ancient DNA

Question 1

Define Ancient DNA

Answer 1

Extraction, analysis and interpretation of DNA from ancient (or historical) sources (>= 50 years)

Question 2

Sources of ancient DNA

Answer 2

Bones (e.g. petrous bone)
Teeth
Skin
Hair
Blood
Tissue (both mummified and recent)
Fabric
Urine
Plants (e.g. pollen) (non-human DNA source)

Question 3

Neanderthals

Answer 3

Lived 30,000 to 40,000 years ago in Europe to West Asia

Not human ancestors, but interbred with humans - this was discovered by PCR

Question 4

Mitochondrial DNA

Answer 4

16,568 base pairs
Circular
Only inherited from the mother

Question 5

Why is mitochondrial DNA used for ancient DNA studies?

Answer 5

There are 2 copies of genomic DNA in the nucleus of each cell
There are 100-1000 mitochondria in each cell, with each mitochondrion containing 1-10 copies of mitochondrial DNA
Therefore mitochondrial DNA is more abundant than nuclear DNA
Therefore there is a better chance of amplifying the DNA by PCR

Furthermore, the small size and circular form of mtDNA make it more resistant to degradation than nuclear DNA

Question 6

Using mitochondrial DNA to compare humans to Neanderthals

Answer 6

Mitochondrial DNA contains 3 main polymorphic/hypervariable regions: HV1, HV2 and HV3
HV1 = 379 base pairs long
This region of the mitochondrial DNA sequence was compared between a modern human and ancient Neanderthal DNA, after sequencing both regions
=< 8 differences = Neanderthals were human ancestors
> 8 differences = Neanderthals were not human ancestors

Question 7

Ideal preservation conditions for DNA

Answer 7

Cold, dry, alkaline

Question 8

Condition of ancient DNA

Answer 8

Typically <150 nt long and single stranded

also can’t isolate DNA that is more than 1 million years old

Question 9

Sources of DNA contamination

Answer 9

Bacterial DNA
Fungal DNA
Insect DNA
Human DNA e.g. from skin, sweat, saliva, blood, hair (strands contain mtDNA)

Question 10

Counter-measures for avoiding contamination

Answer 10

Protective clothing e.g. lab coat, double gloves, hat, visor, mask
A dedicated room for the procedure that has been thoroughly cleaned e.g. filtered air, bleached benches, benches bathed in UV overnight
Independent PCRs
Independent laboratories

Question 11

Conclusions on ancestral relationship between Neanderthals and humans

Answer 11

Researchers ground up a chunk of bone from the upper right arm of an original Neanderthal specimen
Checked for amino acid racemisation (indicates whether the DNA has survived) - lots of AA racemisation = lots of water = DNA likely to be damaged
DNA washed from bone
Independent PCRs in 2 different labs were carried out, with approx. 50 mtDNA molecules per PCR
Gave 123 overlapping 50 bp fragments covering 379 bp
27 differences, therefore Neanderthals were not our ancestors

Question 12

Problems with obtaining nuclear Neanderthal DNA

Answer 12

1. Low percentage of Neanderthal DNA is in their bones (4 % maximum - the rest is bacterial)
   Solution = obtain lots of bones - from the Vindija cave in Croatia
2. Contaminating DNA - the other 96 % of DNA was from bacterial sources, as well as some fungal and some human DNA
   Solution = digest bacterial DNA with 8 different restriction enzymes
3. The yield of DNA after purification was very poor (up to 95 % of the DNA was lost in the final steps)
   Solution = replace alkaline treatment with thermal treatment (the alkaline treatment used during the ‘melting’ step was found to be denaturing the DNA)
4. Standard DNA sequencing (Sanger) is very slow
   Solution = pyrosequencing

Question 13

Restriction enzymes

Answer 13

Recognise and cut specific base sequences

e.g. HindIII

Question 14

Pyrosequencing

Answer 14

= a sequencing method based on real-time pyrophosphate