DNA Technology:
Revolution in Evolution
Why study (organic) evolution?
Evolution: descent with modification
• To understand natural history of life on
earth
• To understand disease processes and
public health risks
– Immunity
– Host-specificity
– Risk assessment
Observation
Propose Hypothesis
Propose Alternate
Hypothesis
Design Experiment
Redesign
Experiment
Not
repeatable
Data are Bias
Determine if Data are
Bias
Refine Hypothesis
Repeat Experiment
Repeatable
Collect and Analyze
Data
Accept as Theory
Effects of Natural Selection on Populations
Spectrum of characteristics in population
Original
population
Population after
selection
Different selection processes
Divergence in a Population
Darwin’s Disadvantages
• Didn’t have knowledge of principles of
inheritance (but made very good
predictions)
• Made analogy between natural selection
and artificial selection (a good hypothesis)
but didn’t have the skills to test it
thoroughly
Advances in Evolutionary Science:
Contributions to the Neo-Darwinian Synthesis
• Principles of inheritance
• Linkage and mutation
• ‘Genes’ are the basis for inheritance and are found
within chromosomes
• Discovery that DNA is the molecular material of genes,
cracking genetic code
• Molecular mechanisms worked out for DNA replication
and protein synthesis
• Multiple methods invented to study genetic variation
and evolution
Cross-sectional data
• Cross-sectional data (snap-shot in time) can be used
scientifically to make deductions about a process
Evolutionary science is like criminal
forensics
• The crime may not have any witnesses
• The evidence is often patchy and comes in
many forms
• In the absence of eyewitness accounts,
DNA is often the most convincing evidence
• The best evidence is based on DNA
sequence variation
DNA
Genetic Variation Results from Mutation
Most mutations are either
harmful, or neutral, but
sometimes they are beneficial.
If the mutations are not too
harmful, they will be passed on
to their progeny (offspring).
This is the hereditary basis of
evolution.
These heritable changes in a
lineage or populations of
organisms over generations
contribute to micro-evolution
Mutations Analogy
the red fox ran out
Point
mutation
the red fax ran out
Frame shift
thr edf oxr ano ut
Variation by Mutation is Compounded by
Genetic Recombination
•
•
•
•
•
Sexual reproduction
Bacterial transformation
Bacterial conjugation
Virus-mediated gene transfer
Other transfer between symbionts
DNA technology
• Facilitates the study of heritable
characteristics between individuals, within
populations and higher taxa
• Population genetics studies- field
studies of evolutionary processes
• Phylogenetics- infer evolutionary
relationships (family trees) from genetic
similarity
Longitudinal vs cross-sectional
Phylogenetic
analysis
examines
relationship from
cross-section
Population
genetic studies
can follow gene
flow over time
Molecular (DNA) Methods
• Restriction Enzyme Digestion
– RFLP
– PFGE
– Ribotyping
• PCR based methods
• DNA sequencing
• DNA microarrays
Combination of
methods
RFLP
RFLP-restriction fragment length polymorphisms
Organism B
Organism A
DNA
RE
Restriction Endonuclease- enzyme that cleaves DNA at palandromic
sequences. Example: EcoRI cuts at GAATTC
Gel Electrophoresis
DNA samples added to wells in matrix
+
Gel Made of
Translucent,
Porous Matrix
DNA migrates at
a rate inversely
related to log10
of amplicon size
EtBr binds to DNA as it travels through the gel
Visualizing DNA with UV light
_
Large pieces of
DNA
Small pieces of
DNA
+
L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 L
Ribotyping
cells
DNA
EcoRI
Transfer to nylon
membrane
(Southern Blot)
Bind labeled
16S rDNA
Probe
Anti-probe Ab and
enzyme-linked color
reaction
1%
Agarose
Gel
+
Gel
electrophoresis
PFGE
(Pulse Field Gel Electrophoresis)
+
+
Agarose
gel
-
-
PCR
polymerase chain reaction
• Invented by Kary Mullis in 1983
• As soon as 1984 it was used for
identification of unknown DNA
• Now widely used for many types of
scientific research
Concept
• Amplify small quantities of DNA by in vitro
DNA replication
Target DNA
PCR
Copies of
Target DNA
Generalized PCR cycle
repeated ca. 40 times
94 degrees
Celsiusdenaturation
72 degrees Celsiusextension
Ca.45-60 degreesprimer annealing
Taq
Target
sequence
Amplicons increase exponentially
with each cycle
Single copy of
dsDNA target
1st Cycle
2nd
Cycle
3rd Cycle
Variations of PCR technique
• Repetitive element PCR
• RAPD PCR
Primers bind where ever
there is a complemetary
DNA sequence
Can be used to generate qualitative or
quantitative data
L
1
2
3
-
Positive
L
Charge
Negative
Charge
1
2
3
-
DGGE
denaturing gradient gel electrophoresis
Gel made of substance
that denatures DNA
molecules
Denaturing agent exists
in a gradient from top to
bottom
PCR amplicons with
different sequences will
denature at different
distance from the top
DNA sequencing
• DNA usually in the form
of PCR amplicon
• One strand at a time
• Most thorough method of
studying variation
• Relatively expensive and
time consuming
Extension (polymerization)
3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’ATCGAACGGAGACTTACTCTTA T
Taq
dNTPs are randomlyincorporated into new
strand until a ‘stop’ is
added
T
A
A
G
A
C A
G
T
C
T
A
3’TAGCTTGCCTCTGAATGAGAATATGGCACCATCGAAA…
5’ATCGAACGGAGACTTA
Taq
Possible fragments
A
G
C
T
T
A
G
T
If there is contradictory info, it
will be read as ‘N’
Sequence Trace