Recovery and analysis of old/ancient DNA: molecular archaeology/anthropology. Why study ancient DNA (aDNA)? Obtaining aDNA Early studies of aDNA Guidelines for studying aDNA Reconstructing extinct gene sequence Hofreiter et al .(2001) Nature Reviews Genetics 2 , p. 353
Hofreiter et al.(2001) Nature Reviews Genetics2, p. 353
Thornton (2004), Nature Reviews Genetics5, p. 366.
DNA sequences from extinct animals--a time line archaeology/anthropology
Sites in DNA affected by long-term chemical processes archaeology/anthropology
SB Carroll (2003) archaeology/anthropologyNature422 p. 849
1) Isolate DNA (20 micrograms/gram mummy tissue
2) Treat with Klenow enzyme (DNA polymerase) to make DNA fragments blunt ended
3) Cloned into alkaline phosphatase treated pUC8 (pMUM plasmids)
***But cloning presents problems, eg. repair of DNA following transformation (leading to false mutations)
To recover DNA that can by amplified by PCR: treat with a reagent that breaks sugar cross-links
Previous sequencing techniques PCR: one DNA molecule at a time
New: many DNA molecules at a time -- arrays
One example: “pyrosequencing”
Cut a genome to DNA fragments 300 - 500 bases long
Immobilize single strands on a very small plastic bead (one piece of DNA per bead)
Amplify the DNA on each bead to cover each bead to boost the signal
Separate each bead on a plate with up to 1.6 million wells
Sequence by DNA polymerase -dependent chain extension, one base at a time in the presence of a reporter (luciferase)
Luciferase is an enzyme that will emit a photon of light in response to the pyrophosphate (PPi) released upon nucleotide addition by DNA polymerase
Flashes of light and their intensity are recorded
Extension with individual dNTPs gives a readout base at a time in the presence of a reporter (luciferase)
The readout is recorded by a detector that measures position of light flashes and intensity of light flashes
25 million bases in about 4 hours base at a time in the presence of a reporter (luciferase)
APS = Adenosine phosphosulfate
Height of peak indicates the number of dNTPs added base at a time in the presence of a reporter (luciferase)
This sequence: TTTGGGGTTGCAGTT
Human evolution and DNA sequencing base at a time in the presence of a reporter (luciferase)
Compare sequences among all living humans (gene flow, origins, the sum total of human variation)
2) Compare sequences between us and closest living ancestor -- Pan troglodytes, chimpanzee -- where are the differences, which genes were selected during this divergence?
3) Compare sequences between us and other extinct hominids -- Homo neanderthalensis
The way we were…. (?) base at a time in the presence of a reporter (luciferase)
(The genome(s) of Homo neanderthalensis)
Mitochondrial genome: Paabo et al, 1997
Neanderthals split from modern human lineage ~500,000 years ago
(Homo sapiens: about 200,000 years ago, Homo sapiens sapiens, about 45,000 years ago)
Neanderthals and humans: coexisted until about 30 - 40,000 years ago
Did interbreeding occur?
Model for the expansion of modern humans base at a time in the presence of a reporter (luciferase)
N = generations
Hypothetically up to 25% interbreeding rate base at a time in the presence of a reporter (luciferase)
But there are no Neanderthal mitochondria in modern Europeans (thousands tested)
This is consistent with a less than 0.1% interbreeding rate
A higher rate of interbreeding but sterility of interbreeding outcomes
Value of a complete Neanderthal genome? base at a time in the presence of a reporter (luciferase)
Which changes in human relative to the chimpanzee genome are recent?
Where have “selective sweeps” occurred in the human genome since divergence of Neanderthals? (selective sweep: reduction of variation in genomic DNA adjacent to a mutation that is under powerful selective pressure)
What was Neanderthal biology like?
Neanderthal nuclear sequences -- what were Neanderthal genetics like? How did they compare to modern humans?
“Amplification-independent direct cloning” (no PCR artifacts)
“Metagenome” from 38,000 year old bone
Average fragment size ~ 50 bp. genetics like? How did they compare to modern humans?
65250 bp total (NE1)
Pyrosequencing versus Sanger: 99.89% agreement
How do we know it’s Neanderthal (not modern human contaminants)?
Modern human mito contamination: ~2%
Damage “signatures” of ancient DNA
Hominid sequences: human-specific changes (that differ from chimpanzee sequences) were often not present in the Neanderthal sequence
Vi80: Vindja Cave, Croatia genetics like? How did they compare to modern humans?
1 million bp of Vi-80 were sequenced
Distribution of neanderthal DNA sequence by chromosome matches expected frequencies based on lengths of human chromosomes
Also--the fossil was from a male
Memories… matches expected frequencies based on lengths of human chromosomes
Proposed time line for divergence
…of the way we were
FOXP2: a key gene in human evolution (language and speech)? matches expected frequencies based on lengths of human chromosomes
Mutation of this gene leads to deficits in “linguistic processing” and “orofacial movements”
Two specific mutations in FOX2P are “fixed” in humans compared to chimpanzees
There was a relatively recent (200,000 years ago) “selective sweep” in the FOX2P region of the human genome
(selective sweep: a region of the genome that stays relatively unchanged because of selective pressure on beneficial mutations within that region)
Implication: FOXP2 variants not a guarantee of survival matches expected frequencies based on lengths of human chromosomes
Other mutations necessary as well? (brain structure/function?)
Neanderthals perhaps could talk, but may have had little interesting to say…
Resurrecting extinct genes: matches expected frequencies based on lengths of human chromosomes
the phylogenetic approach
How to infer ancestral gene sequence? matches expected frequencies based on lengths of human chromosomes
Maximum likelihood analysis:
A phylogenetic tree is constructed
At any internal node, each possible ancestral state is evaluated for its likelihood of yielding the present day sequences
Highest likelihood gives the best guess for ancestral sequence
A success story: ancestral bacterial EF-Tu matches expected frequencies based on lengths of human chromosomes
(>1 billion years ago)
At which temperature does the ancestral EF-Tu function best (were early bacteria thermophiles?)
The reconstructed ancestral EF-Tu binds to GTP best at 65°C, suggesting a thermophilic ancestor to bacteria
Caveats: matches expected frequencies based on lengths of human chromosomes
How good is your Max. Likelihood prediction? Perform many predictions and compare the results--do they give similar results?
Is the function of the ancestral protein being assayed under relevant conditions? What if the protein functions as part of a complex?