PCR ELECTROPHORESIS
BY C. KOHN, AGRICULTURAL SCIENCES,
WATERFORD WI
GENETIC TESTING
• The Sanger Method enables scientists to
read DNA letter by letter.
• This test made the Human Genome Project,
and the sequencing of many other species,
possible.
• The problem with the Sanger Method is
that it is slow and expensive.
• It wouldn’t work to use it for day-to-day
needs.
• The Sanger Method provides scientists
with every possible letter in a genome.
• This amount of information is not always
necessary.
Source: en.wikipedia.org
DNA FINGERPRINTING
• When we simply need to identify the source of DNA, or
a few genes, we can use simpler and less-expensive
tests.
• Often we don’t need to know the entire genome of
person – we just need to know if their DNA is a match
to another sample of DNA, or if their DNA contains a
specific set of genes.
• To determine this, we would create
a DNA Fingerprint.
Source: buzzle.com
HUMAN DNA
• DNA fingerprinting works for any kind of DNA, even
human DNA.
• This is important because 99.9% of human DNA is the same.
• This means that you have 99.9% of the same DNA as the person
sitting next to you.
• The 0.1% difference between two peoples’ DNA means that 3
million bases are different – this is more than enough to allow us
to pinpoint the source of DNA down to a specific individual.
• DNA Fingerprinting is when
a geneticist examines the 3
million base pairs that could
be different for any variations
that are unique to an individual.
CREATING A DNA FINGERPRINT
• To create a DNA fingerprint, we have four steps:
•
•
•
•
1. Collect a sample of DNA
2. Amplify specific variable regions of the DNA
3. Count repeated sequences (STRs)
4. Look for matches from collected samples to determine what
samples are from the same sources
• For example, if you found
DNA at the scene of a
crime, you could collect
DNA from a suspect and
see if it matches the DNA
found at the crime scene.
COLLECTION & AMPLIFICATION
• The first step in solving this hypothetical crime would be
to collect a pure, untainted sample of DNA.
• We can’t have multiple sources of DNA in the same sample.
• Next, scientists have to amplify regions of the DNA that
have a lot of variety from person to person.
• We can’t look at regions of DNA that are similar from person to
person.
• Typically, exons are going to be very similar from person to person.
• Vice versa, introns (sections of DNA that do not code for
proteins) have a lot more variability and work better for DNA
fingerprinting.
• Introns tend to have small sections that repeat over and over.
Source: simple.wikipedia.org
STR’S
• When scientists compare the intron sampling regions of
the DNA, they are comparing Short Tandem Repeats
of DNA, or STRs.
• An STR is a short unit of DNA (4-5 bases long) that
repeats multiple times in the DNA.
• For example, you might a “AATG” section of DNA that repeats
over and over  ATCT.AATG.AATG.AATG.AATG.CAG…”
• In this case, there would be 4
Source: nfstc.org
STR repeats of AATG.
IMPORTANCE OF STR’S
• STRs are critical because they
represent the areas of greatest
variability from person to person.
• While the DNA that codes for
functional proteins will be very similar
from person to person, the STRs in
introns are the areas of greatest
variety and are the only way to
create a unique DNA fingerprint for
each person.
• While you may have the same
number of STRs at one particular
region in your DNA as someone else,
the likelihood of having the same
number of STR repeats at every region
in your DNA and someone else’s DNA
is next to impossible.
Source: bcs.whfreeman.com
EXAMPLE
• For example, if you look in the chart below, you can
see 5 regions where STRs were measured and counted.
• In the first column, you can see the number of repeats
at each STR location.
• For each suspect, you can see if their number of STR
repeats at each location matches.
• In this case, the second suspect’s DNA is a match to the crime
scene.
STR Region
1
2
3
4
5
# Repeats
(Crime Scene Sample) # Repeats (Suspect 1)
7
8
4
11
14
13
11
18
8
7
# Repeats (Suspect 2)
7
4
14
11
8
HOW WE DO IT
• So how can we tell how many STRs each person has at
each sampling site? How can we determine a person’s
or organism’s DNA fingerprint?
• The key to determining this information is a test called
Polymerase Chain Reaction, or PCR.
• PCR is sort of like a molecular
copy machine.
• It can quickly, easily, and
cheaply make many copies
of a specific section of DNA.
• It does not work to read all of
an individual’s genome, but it
does work well for small
sections of DNA.
Source: scq.ubc.ca
•PCR works first by separating the dual-strand of
DNA into two individual strands using heat.
•Each side can then be copied by polymerase
enzymes, creating two strands from one.
•This process is repeated over and over (2
becomes 4, 4 becomes 8, 8 becomes 16…...)
•It can amplify a specific sequence of DNA
billions of times!
PCR Animations 1, 2, 3, 4, 5
TAQ POLYMERASE
• To denature the DNA, or separate the double
strand into two single strands, we have to heat
the DNA.
• One problem with this method was that the heat also
destroyed a normal polymerase enzyme.
• To make this technique work, scientists
use the Taq polymerase.
• Taq polymerase is found in bacteria that live
in the hottest environments on earth (such as
the inside of the geysers at Yellowstone where
it was first discovered).
• The Taq polymerase is able to function in the
hot/cold/hot kind of conditions used for PCR.
After amplification by Taq
polymerase, each DNA sample is
cut up into fragments by restriction
enzymes (chemical scissors)
Remember that restriction
enzymes recognize and cut
within specific DNA sequences.
DNA
Seq. 1
DNA
Seq. 2
These specific DNA sequences
are located at different places
along the strand of DNA,
depending on the source.
Because of this, each DNA
sample will have a unique
banding pattern.
The same
restriction
enzyme will cut
the same gene
from different
people in
different ways.
Chunks of DNA
will be larger if
they have longer
STR repeats.
RESTRICTION FRAGMENTS
• For example, EcoRI is a restriction fragment that always
looks for the GAATTC sequence.
• It will always cut DNA between the G and the first A.
• This sequence is likely to show up multiple times in a
normal DNA segment.
• Because this segment will occur different places in
different DNA, it will create uniquely sized ‘chunks’ of
DNA for each person depending on the number of STRs.
GEL ELECTROPHORESIS
• Once we’ve made billions of copies of the same
section of DNA and have cut them with a restriction
enzyme, we have to run the DNA through a gel.
• This is necessary in order for scientists to determine how much
DNA was in that particular section that was copied.
• If we know how much DNA
was in that section, we know
how many STR repeats were
in that section.
• Shorter fragments of DNA will
travel further than longer chunks.
• Known quantities of DNA provide
a basis for comparison in the gel.
GEL ELECTROPHORESIS
• A few drops of each amplified and
cut DNA sample are placed in a
small pocket, or well, at one end of
the gel.
• The other end of the gel has a positive
charge.
• All DNA molecules are negatively
charged, so they move through
pores in the gel toward the positive
pole.
• The phosphate in the DNA gives it a
negative charge.
GEL ELECTROPHORESIS
• The shorter DNA fragments slip more easily through the
pores of the gel than the longer fragments.
• I.e. it would be easier for you to swim to the other side of the
pool if you were wearing a Speedo than if you were wearing
baggy shorts.
• After a set period of time, the
shorter DNA fragments will travel
farther through the gel.
• The shorter fragments will be closer to
the positive end of the gel than the
longer fragments.
TREATING/READING THE SAMPLES
• In the last step, the gel is treated with a stain that
makes the DNA visible under ultraviolet light.
• The DNA fragments show up as a series of bands in
each "lane" of the gel. Each band consists of many
DNA fragments of one particular size.
• That size is determined by the
number of STR repeats.
• The pattern of bands will be
different for each sample
because each sample has a
unique set of DNA fragment
lengths.
GENETIC MARKERS
• A genetic marker is a gene that is found at a known location and
produces a predictable pattern.
• When DNA fingerprints are used in court as evidence, genetic
markers from these highly variable noncoding regions are used.
• Genetic markers can occur in alleles for
diseases or other traits.
• We can compare the banding patterns of a patient
to known patterns from people with and without the
disease to see if they match one or the other.
• If the patterns of the patient match the markers from
the known samples with the disease, we know that
that patient would also have that genetic disease
• Whenever we analyze the banding patterns
from PCR/Electrophoresis, this is known as
Restriction Fragment Analysis.
SUMMARY
1. DNA sections are amplified tens of billions of times by Taq
polymerase in the process of Polymerase Chain Reaction,
or PCR.
2. The amplified DNA is cut into chunks using a restriction
enzyme.
3. The cut pieces of DNA are put into wells in a gel and
pulled through the gel using electricity in a process called
Gel Electrophoresis
4. As the fragments move through the gel, they create a
unique banding pattern. This is due to the fact that the
larger pieces of DNA move more slowly than the small
pieces.
• Larger chunks have more repeating sections, or STRs, than smaller
chunks.
5. The unique banding pattern (Restriction Fragment
Analysis) enables scientists to determine the source of a
sample of DNA by comparing it to collected samples.
EXTRA RESOURCES
•
•
•
•
•
Gel Electrophoresis Virtual Lab
DNA Detective; Who Dun It ?
Polymerase Chain Reaction Quiz 1, 2, 3
DNA Fingerprinting Tutorial & Quiz (advanced)
DNA Fingerprint Quiz 1, 2 ,3 , 4, 5