Analyzing DNA
Polymerase Chain Reaction (PCR)
The most prolific tool used to
analyze an organism’s DNA is called
How do we polymerase chain reaction (PCR)
analyze a PCR is a method whereby
genome? investigators can search for specific
known sequences of genetic
information
This is done by:
1. Isolating an organism’s DNA sample
How does it 2.Adding in specially engineered RNA
work? primers to a solution containing the DNA
(Overview) 3. Replicating the sequence to which the
primers binds, millions of times
High volume replication of a DNA sequence
allows it to be detected
Why perform PCR?
PCR might be used to:
Search for a specific DNA
sequence like:
A gene known to predispose
someone to disease
In an ancestry/heritage
investigation (“23 And Me”
etc.)
Compare two sequences of
Uses of PCR DNA to one another for:
Forensic analysis (“DNA
evidence”)
Paternity tests (or a long lost
sibling?)
Analyze identity of a virus or
bacterium
Evolutionary studies
How closely related are two
or more organisms? How
many DNA sequences to
they share?
In order to perform PCR, a sample of
extracted DNA must be exposed to a
master mix. This is a solution that
contains:
Materials of 1. DNA Polymerase enzyme
This enzyme builds new DNA
PCR 2. Free DNA nucleotides
To be built into new DNA
3. Specially engineered RNA primers
These can be purchased from scientists who
build them to your specifications
PCR Steps:
1.Denaturation
Steps of PCR
2.Annealing
3.Extension
Sample is heated, separating the two strands of DNA.
Strands gain enough energy to overcome the H-bonding
between them.
Works without helicase enzyme!
Denaturation
Sample is cooled, allowing primers to attach to DNA.
Primers are smaller than the two DNA strands. They attach first.
Attached primers prevent original DNA from joining together again.
Annealing RNA Primer
Polymerase binds to primers, creates new DNA!
DNA Polymerase enzyme we add replicates DNA at the
primers (amplification)
Builds new strands out of free nucleotides we add
Sample is heated again during this step to promote function
of the polymerase enzyme
DNA
Extension Polymerase
RNA Primer
PCR Steps:
1.Denaturation
2.Annealing
Steps of PCR
3.Extension
Process is repeated to make over a
million copies of the target sequence!
Polymerase
Chain Reaction
Developed by
Kary Mullis
Nobel Prize in
Chemistry in
1993
ß PCR Temperatures/times
Gel Electrophoresis
Visualizing PCR
A technique called gel electrophoresis is
used to visualize our DNA sample after
PCR
This can be used to:
Gel Confirm if we successfully performed
Electrophoresis PCR
Look to see if our target sequence was
present
See all replicated sequences to compare
to another sample
How do we tell what we replicated?
We can’t zoom in and count the nucleotides,
or read the DNA piece by piece
We can separate a jumble of DNA based
on size
Gel Size in this case means length of the
Electrophoresis sequences (measured in number of
nucleotides or base pairs).
Gel electrophoresis allows us to force different
lengths of DNA apart from one another so that
we can see them inside of a gooey gel substance
called a gel.
Gel 1. DNA is added to chambers in an agarose gel
Electrophoresis 2. Electric current is passed through the gel
(Overview) 3. DNA moves along the gel, pushed by current
4. Smaller fragments move farther in same amount of time
– DNA is separated based on length!
5. Can be seen inside the gel as “bands”
6. Compare bands from test sample to known bands in
control samples called a molecular weight ruler or
ladder
Gel with nothing loaded in the wells
DNA will move
this way when
electric current
is applied.
DNA is
Gel negatively
charged. Will be
Electrophoresis
pushed by
electric current.
Loading dyed samples into wells
Gel
Direction
of Electric
Movement of Current
DNA
Amplified
DNA
Shorter pieces of DNA go farther in the same amount of time.
Results?
A couple is considering having children and want to see if they
carry certain genetic markers for two heritable genetic disorders.
Both disorders (A and B) are recessive, so if both parents are
carriers, there’s a chance their child might inherit the disorder.
We perform PCR using primers for the two recessive alleles and
amplify them.
We perform electrophoresis to visualize the samples.
Example
Control (Both Mother’s Father’s
Alleles) Sample Sample
According to these data:
The mother carries the recessive allele for disorder A.
The father carries the recessive allele for disorder B.
Primers for both alleles were added to both samples, but since the
mother doesn’t carry the allele for disorder B, no DNA was present to be
amplified. Because the father doesn’t carry the allele for disorder A, not
DNA was present to be amplified.
Allele ‘a’
Example
Allele ‘b’
Control (Both Mother’s Father’s
Alleles) Sample Sample
Control
Samples
What is in lane 1?
This is called the molecular weight ruler/ladder.
This is a sample always added to gels
Contains premade sequences of DNA that are of specifically engineered
lengths.
Why add the ladder?
This allows us to roughly analyze the lengths of the replicated sequences in
our tested samples!
This sample gel compares human DNA
samples to those of other species.
What might the PCR products in the
human samples represent?
Specific genetic sequences present in
all humans. These are being compared
to the DNA other other animals.
Which animal appears to be the most
closely related to humans?
Sample 4!
Which lane contains the highest number
of successfully replicated PCR products?
The human sample.
Sample Gel Which DNA sequence might be the
oldest?
The sequences at ~800 bp and ~1,300
bp long appears in the most organisms.
Sample 5 does not contain the 800 bp
sequence. What might this tell you
about this organism’s evolutionary
history?
This organism likely evolved either
before that sequence developed, or
after the rest of the organisms and it
lost that sequence.
What is the approximate length of the
shortest human DNA sequence?
Approximately 90 bp.
