Neanderthal DNA and origins of modern humans
What techniques are used?
- DNA extraction
- PCR
- DNA cloning and sequencing
- DNA quantification
- Authenticating and determining the true sequence
- Analysis of mitochondrial control region sequence data
Variations on these techniques used in ancient DNA are employed in conservation
genetics, forensics, biomedicine, evolution, ecology etc.
Introduction to Neanderthals and modern humans
Homo sapiens first appeared (fossil record) 120 000 years ago in Africa
Homo neandertalensis appeared earlier 200 000 years ago in Europe
Sister species to each other – evidence that there was gene fusion
There are 2 main theories about the relationship of these 2 groups:
Out of Africa Multiregionalism
Homo sapiens first lived in Africa and States that modern Homo sapiens
eventually travelled into Europe and Asia evolved in situ – from Neanderthal in
Humans had evolutionary advantages Europe and other hominid groups in Asia
that allowed them to outlive – and
perhaps cause the extinction of – all
other hominid groups (as opposed to
apes) such as Neanderthal
Obtaining material for sequencing
Neanderthal type-specimen found in 1805 in
the Neander valley near Dusseldorf (Krings et
al., 1997)
For this project, researchers took a 3.5g
sample of the right humerus
They used a 0.4g sample for DNA extraction
, DNA extraction (need to isolate DNA)
DNA is found in:
Bone is made of: - Soil compounds (humic and fulvic acids)
- an inorganic part - hydroxyapatite - - Cell components – decay products of
70 % blood, fat
- an organic part - collagen - c. 28% - Bacterial and fungal components
- other proteins, DNA and lipids - c. 2% - Glues, varnishes, fumigant residues,
dust, grime
1. Grind bone into fine powder then dissolve away the inorganic part of the bone, using
EDTA (ethylene diamine tetra-acetic acid - a chelating agent)
2. Digest proteins using a proteinase enzyme.
3. Remove proteins and soil components using solvents –phenol and chloroform.
4. Concentrate the solution using a filter (30 kD - very fine).
5. Join the DNA to silica particles using a “salt bridge” - guanidinium isothiocyanate -
and “wash” the DNA with alcohol.
6. Finally - elute the DNA off the silica at pH 8.0
*Process takes days
DNA Amplification
Problems met when ancient DNA is amplified:
Inhibition Contamination Damage
Of the polymerase caused Of the extract due to low To the DNA in the extract
by remant soil components concentration of target due to long-term burial or
etc. From handling, back- museum storage of the
migrating PCR products specimen
from earlier experiments, Mainly breaks in the DNA
poor storage (eg. vermin chain – “strand cleavage”
urine), soil microbes Most ancient <200 bp
- Mitigated by adding - Reduced by working Chemical modification of
enzyme stabilisers and cleanly (eg. separate bases during burial/storage
binding agents pre- and post- PCR labs) Mainly deamination of
Bovine serum albumin - using negative controls cytosine in uracil
(BSA), starch, other (ie. water)
serum albumins - species specific primers
(increase the
concentration of target
DNA to negate effects
of others)
What techniques are used?
- DNA extraction
- PCR
- DNA cloning and sequencing
- DNA quantification
- Authenticating and determining the true sequence
- Analysis of mitochondrial control region sequence data
Variations on these techniques used in ancient DNA are employed in conservation
genetics, forensics, biomedicine, evolution, ecology etc.
Introduction to Neanderthals and modern humans
Homo sapiens first appeared (fossil record) 120 000 years ago in Africa
Homo neandertalensis appeared earlier 200 000 years ago in Europe
Sister species to each other – evidence that there was gene fusion
There are 2 main theories about the relationship of these 2 groups:
Out of Africa Multiregionalism
Homo sapiens first lived in Africa and States that modern Homo sapiens
eventually travelled into Europe and Asia evolved in situ – from Neanderthal in
Humans had evolutionary advantages Europe and other hominid groups in Asia
that allowed them to outlive – and
perhaps cause the extinction of – all
other hominid groups (as opposed to
apes) such as Neanderthal
Obtaining material for sequencing
Neanderthal type-specimen found in 1805 in
the Neander valley near Dusseldorf (Krings et
al., 1997)
For this project, researchers took a 3.5g
sample of the right humerus
They used a 0.4g sample for DNA extraction
, DNA extraction (need to isolate DNA)
DNA is found in:
Bone is made of: - Soil compounds (humic and fulvic acids)
- an inorganic part - hydroxyapatite - - Cell components – decay products of
70 % blood, fat
- an organic part - collagen - c. 28% - Bacterial and fungal components
- other proteins, DNA and lipids - c. 2% - Glues, varnishes, fumigant residues,
dust, grime
1. Grind bone into fine powder then dissolve away the inorganic part of the bone, using
EDTA (ethylene diamine tetra-acetic acid - a chelating agent)
2. Digest proteins using a proteinase enzyme.
3. Remove proteins and soil components using solvents –phenol and chloroform.
4. Concentrate the solution using a filter (30 kD - very fine).
5. Join the DNA to silica particles using a “salt bridge” - guanidinium isothiocyanate -
and “wash” the DNA with alcohol.
6. Finally - elute the DNA off the silica at pH 8.0
*Process takes days
DNA Amplification
Problems met when ancient DNA is amplified:
Inhibition Contamination Damage
Of the polymerase caused Of the extract due to low To the DNA in the extract
by remant soil components concentration of target due to long-term burial or
etc. From handling, back- museum storage of the
migrating PCR products specimen
from earlier experiments, Mainly breaks in the DNA
poor storage (eg. vermin chain – “strand cleavage”
urine), soil microbes Most ancient <200 bp
- Mitigated by adding - Reduced by working Chemical modification of
enzyme stabilisers and cleanly (eg. separate bases during burial/storage
binding agents pre- and post- PCR labs) Mainly deamination of
Bovine serum albumin - using negative controls cytosine in uracil
(BSA), starch, other (ie. water)
serum albumins - species specific primers
(increase the
concentration of target
DNA to negate effects
of others)
