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What, When, Why?
Who and Where?
What good is it?
What questions about this organism can DNA data help us to address?
Inference of evolutionary relationships based on molecular data
Step 1. Select a DNA region that is homologous, or similar across species due to common ancestry.
of 16S rRNA in E. coli
2. Amplify andSequence this region across isolates….
Species “I” has probably experienced a deletion event at position #6 or #7.
Implies that species “I” is divergent from the others, but this is not the case.
3. Sequence alignment is crucial for inferring how DNA sites have changed.
At variable DNA positions, related groups will tend to share the same nucleotide.
The sheer number of characters is helpful to distinguish the ‘phylogenetic signal’ from noise.
Molecular phylogeny of taxa A-I.
Colored letters = different from top sequence (taxon G)
For instance, an endosymbiotic lifestyle has evolved several times independently.
Moran and Wernegreen (2000)
Lists sequences that are most similar to yours
GENBANK = NIH genetic database with all publicly available DNA sequences. As of 2004: > 44 billion bp, and > 40 million sequences
How does this organism fit into the world of available sequence data?
Wolbachia sp. 1
Wolbachia sp. 2
Wolbachia sp. 3
Is the bacterium inside this insect really Wolbachia?
PCR and sequence a gene of interest
(e.g., 16S rDNA)
The elegant idea behind DNA sequencing
Technology changes quickly, but for many years we’ve on Sanger’s cool trick.
In the 1970’s, Sanger’s group discovered a fundamentally new method of 'reading' the linear DNA sequence using special bases called chain terminators. This method is still in use today.
What is the basis of Sanger’s method?
Shared with Walter Gilbert and Paul Berg
Figures in this and subsequent slides from Hartl and Jones,
Essential Genetics, 1999
= chain termination
clean PCR product
Step 1: Purify template (the DNA to be sequenced)
+ used Taq
+ extra primer and dNTPs
+ salts from PCR buffer, etc….
TEMPLATE DNA (e.g., YOUR PCR PRODUCT)
The dyes are “spectrally distinct,” and each has a different emission wavelength.
Step 3: Perform cycle sequencing in PCR machine
Reaction steps seems a lot like PCR, but not quite…
>> Cycle sequencing
e.g. ABI 3730
2. Set up sequencing reaction
1. Purify PCR product
3. Perform cycle sequencing
5. Read order of terminators (DNA sequence)
4. Resolve sequence fragments
Ideally, look at chromatograms and convince yourself that base calls are robust.
Sequence read #1
Sequence read #2
Sequence read #3
DNA sequence assembly: Combining sequence reads to build the entire sequence of the template DNA
+ Emulsion Oil
Link DNA fragments to beads (one fragment per bead)
Isolate DNA-containing beads
Perform emulsion PCR
Steps in “next-generation” sequencing
Attach adaptors (A and B) to ends of DNA fragments
Load Enzyme Beads
Load DNA beads
One bead per well
PicoTiterPlate(a fancy microtiter plate with >400,000 wells)
~400 wells per
Peaks in pyrogram reflect nucleotide sequence
dNTP’s are added one at a time to the reaction. Incorporation of one (or more) nucleotide(s) that are complementary to the template results in a chemiluminescent signal, which is recorded by a camera and reflected as a peak in a ‘pyrogram.’
~500 MB on one plate, in just a few hours.
Many public universities to have DNA sequencing facilities in house.
Such facilities are usually able to run small numbers of reactions for a cost of ~$10-15 per reaction, but often much less for “internal users” with ties to the university.
Ideal situation: Team up with faculty at your local university to act as facilitators.
Truro: A tranquil Cape community shaken by the murder of a young woman in January 2002
Thylacine pup preserved in alcohol in 1866. Its cells could be used for cloning. By chance this Thylacine was stored in a jar of alcohol rather than formalin, which would have destroyed the DNA.
DNA data provides new insights into evolutionary relationships, including closest matches in vast public databases.
Though technology changes rapidly, chain termination, developed in the 1970’s, remains central to much DNA sequencing.
However, much faster and cheaper approaches are gradually replacing these methods.
Increasingly accessible, DNA sequencing is already woven into the fabric of health and legal systems.
Evaluating DNA evidence is key in debates of ethics and policies surrounding medical care, individual privacy, and conservation of biodiversity.