Download Bartlett`s Lecture

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
Thornton (2004), Nature Reviews Genetics 5, p. 366.
What is aDNA, and why the interest?
• aDNA is isolated from archaeological,
paleontological remains, museum specimens, etc.
• aDNA potentially a rich source of information for
molecular evolution studies
– Compare DNA sequences of modern organisms
to ancestral organisms
– trace speciation at the molecular level
– great interest in human evolution/speciation
• aDNA can be used to define animal diets
DNA sequences from extinct animals-a time line
What happens to DNA following death?
• Typically decays quickly: nucleases,
microbial decomposition
• Occasionally DNA is spared this fate:
– Rapid dessication
– Low temperatures
– High salinity
• Slow decay:
– Depurination (loss of A and G bases)
– Oxidative damage
– Hydrolytic damage
Sites in DNA affected
by long-term chemical
processes
Effects of DNA damage
• Backbone breakage--fragmentation
• C and T residues converted to hydantoins,
blocking DNA polymerases (PCR)
• Deamination of C causes wrong base to be
added during PCR--false mutations
• Increasing time, increasing degradation,
decreasing utility
• 100,000 to 1,000,000 years considered the age
limit for DNA to yield useful sequences
SB Carroll (2003) Nature 422 p. 849
First retrievals of old DNA
• Quagga (extinct relative of the zebra) DNA cloned from
museum specimen (Higuchi et al. 1984)
• 2430 year-old Mummy DNA cloned (Paabo 1985)
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)
Revolution in ancient DNA isolation: PCR
• PCR--get a lot from a little, sequence
PCR products directly (no cloning
artifacts to worry about)
• Permits targeted studies of specific
genes or DNA regions
– Mitochondrial DNA is typical target in aDNA
PCR isolations
– Copy number of mitochondria is high
relative to nuclear DNA
Identifying ancient remains that are
likely to yield good PCR
• Amino acid racemization: conversion of
L-amino acids to D-amino acids
• Rate depends on water, temperature,
chelated metal ions (things that also
affect rate of DNA depurination)
• The higher the D/L ratio, the less likely
that DNA can be isolated: >0.08, no
DNA will be isolated
• Calculation of D/L ratios is easy, rapid
Cautions for PCR of ancient DNA
(particularly human remains)
Fossilized poo (coprolites): a rich source of information
To recover DNA that can
by amplified by PCR:
treat with a reagent that
breaks sugar cross-links
Two coprolite DNA studies
• From ground sloth coprolites: pine
forests present in south Nevada 28,500
years ago, but by 20,000 years ago pine
forests likely gone
• Analysis of diet of ancient humans: one
study found 8 different plants (looking at
mitochondrial DNA) and meat from
several types of large animals
New advances in ancient DNA sequencing
Mammoth sequencing
•Complete mitochondrial sequence obtained: “multiplex”
PCR (simultaneous amplification of many targets at once-good for minimal DNA situations)
•28 million base pairs of mammoth “metagenome”
sequence obtained, using very short sequences and the
pyrosequencing technique (massively parallel short
sequencing runs)
•“the high percentage of endogenous DNA recoverable
from this single mammoth would allow for completion of its
genome, unleashing the field of paleogenomics”
Previous sequencing techniques: 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
A
B
The readout is recorded by
a detector that measures
position of light flashes and
intensity of light flashes
A
B
25 million bases in
about 4 hours
APS = Adenosine phosphosulfate
From www.454.com
Height of peak indicates the number of
dNTPs added
This sequence: TTTGGGGTTGCAGTT
Human evolution and DNA sequencing
1) 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…. (?)
(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
neanderthal
Modern human
N = generations
Hypothetically up to 25% interbreeding rate
But there are no Neanderthal mitochondria in
modern Europeans (thousands tested)
This is consistent with a less than 0.1%
interbreeding rate
-orA higher rate of interbreeding but sterility of
interbreeding outcomes
Value of a complete Neanderthal genome?
1) Which changes in human relative to the
chimpanzee genome are recent?
2) 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)
3) 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.
65250 bp total (NE1)
Pyrosequencing versus Sanger: 99.89% agreement
How do we know it’s Neanderthal (not modern human
contaminants)?
1) Modern human mito contamination: ~2%
2) Damage “signatures” of ancient DNA
3) Hominid sequences: human-specific changes (that
differ from chimpanzee sequences) were often not
present in the Neanderthal sequence
Vi80: Vindja Cave, Croatia
1 million bp of Vi-80
were sequenced
Majority of DNA from old samples: organisms that
colonize after death…
Distribution of neanderthal DNA sequence by chromosome
matches expected frequencies based on lengths of human
chromosomes
Also--the fossil was from a male
Memories…
…of the way we were
Proposed time line for
divergence
FOXP2: a key gene in human evolution (language and
speech)?
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
Other mutations necessary as well? (brain structure/function?)
Neanderthals perhaps could talk, but may have had little
interesting to say…
Resurrecting extinct genes:
the phylogenetic approach
What is (was) the function of an ancestral/ intermediate
form of today’s genes?
Few molecular fossils exist, and they don’t go back
very far in time
BUT
•Methods exist for inferring ancestral gene sequence
•The inferred ancestral gene can be synthesized,
cloned, the gene can be overexpressed, the protein
can be purified and studied…
How to infer ancestral gene sequence?
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
(>1 billion years ago)
At which temperature does the ancestral EFTu 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:
1) How good is your Max. Likelihood prediction?
Perform many predictions and compare the
results--do they give similar results?
2) Is the function of the ancestral protein being
assayed under relevant conditions? What if the
protein functions as part of a complex?