Recovery and analysis of old
Download
1 / 40

Recovery and analysis of old/ancient DNA: molecular archaeology/anthropology - PowerPoint PPT Presentation


  • 161 Views
  • Updated On :

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

Related searches for Recovery and analysis of old/ancient DNA: molecular archaeology/anthropology

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Recovery and analysis of old/ancient DNA: molecular archaeology/anthropology' - hayes


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
Recovery and analysis of old ancient dna molecular archaeology anthropology
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 Genetics2, p. 353

    Thornton (2004), Nature Reviews Genetics5, p. 366.


What is adna and why the interest
What is aDNA, and why the interest? archaeology/anthropology

  • 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



What happens to dna following death
What happens to DNA following death? archaeology/anthropology

  • 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



Effects of dna damage
Effects of DNA damage archaeology/anthropology

  • 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


Recovery and analysis of old

SB Carroll (2003) archaeology/anthropologyNature422 p. 849


First retrievals of old dna
First retrievals of old DNA archaeology/anthropology

  • 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
Revolution in ancient DNA isolation: PCR archaeology/anthropology

  • 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
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



Recovery and analysis of old

Fossilized poo (coprolites): a rich source of information PCR

To recover DNA that can by amplified by PCR: treat with a reagent that breaks sugar cross-links


Two coprolite dna studies
Two coprolite DNA studies PCR

  • 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


Recovery and analysis of old

New advances in ancient DNA sequencing PCR

  • 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”


Recovery and analysis of old

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


Recovery and analysis of old

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


Recovery and analysis of old

Extension with individual dNTPs gives a readout base at a time in the presence of a reporter (luciferase)

A

B

The readout is recorded by a detector that measures position of light flashes and intensity of light flashes

A

B


Recovery and analysis of old

25 million bases in about 4 hours base at a time in the presence of a reporter (luciferase)

From www.454.com

APS = Adenosine phosphosulfate


Recovery and analysis of old

Height of peak indicates the number of dNTPs added base at a time in the presence of a reporter (luciferase)

This sequence: TTTGGGGTTGCAGTT


Recovery and analysis of old

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


Recovery and analysis of old

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?


Recovery and analysis of old

Model for the expansion of modern humans base at a time in the presence of a reporter (luciferase)

neanderthal

Modern human

N = generations


Recovery and analysis of old

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

-or-

A higher rate of interbreeding but sterility of interbreeding outcomes


Recovery and analysis of old

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?


Recovery and analysis of old

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


Recovery and analysis of old

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


Recovery and analysis of old

Vi80: Vindja Cave, Croatia genetics like? How did they compare to modern humans?

1 million bp of Vi-80 were sequenced



Recovery and analysis of old

Distribution of neanderthal DNA sequence by chromosome matches expected frequencies based on lengths of human chromosomes

Also--the fossil was from a male


Recovery and analysis of old

Memories… matches expected frequencies based on lengths of human chromosomes

Proposed time line for divergence

…of the way we were


Recovery and analysis of old

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)


Recovery and analysis of old

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…


Recovery and analysis of old

Resurrecting extinct genes: matches expected frequencies based on lengths of human chromosomes

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…


Recovery and analysis of old

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


Recovery and analysis of old

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


Recovery and analysis of old

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?