PCR Electrophoresis
by Mike Belmonte, Muskegon Michigan
Edited by C. Kohn, Waterford, WI
Objectives
Describe the technique that enables scientists to mass-produce specific segments of
DNA in a test tube.
Describe a technique used to compare DNA samples.
Key Terms
polymerase chain reaction (PCR)
gel electrophoresis
genetic marker
DNA fingerprint
DNA Profiling
How to solve a crime or determine a parent based on DNA

99.9% of human DNA is exactly the same
from person to person
◦ As a species, we are more like each other than
most other living species.
 Sidebar: racism is not at all scientific – one race cannot
be biologically better than another if we are 99.9% the
same

However, the 0.1% difference between
individuals equates to 3 million bases that
are different between you and the person
next to you.
◦ This is not enough to make them very different
from you.
◦ This is enough to distinguish your DNA from their’s
We’re very similar!

DNA Profiling is when a geneticist
examines the 3 million base pairs that
could be different for any variations that
are unique to an individual.
◦ This is also know as DNA Fingerprinting

When we determine how your DNA is
different from everyone else’s, we create
a genetic profile that is specific only to
you.
DNA Profiling

To create a DNA fingerprint, we have four
steps:
◦ 1. collect a sample of DNA
◦ 2. amplify specific regions of the DNA that
typically have a lot of variety from person to
person
◦ 3. count repeated sequences (STRs)
◦ 4. look for matches from collected samples
(e.g. blood drops)
Steps of DNA Profiling
Let’s imagine we have a crime scene and
we want to match blood found on the
scene to a suspect.
 If the suspect drank from a cup, the saliva
on the cup would have a tiny amount of
cheek cells.

◦ This is enough to create a profile.

We would collect DNA from
the crime scene as well as DNA
from the cells in the saliva on
the cup.
Step 1: Collect a sample

Next, we use Polymerase Chain Reaction
(PCR) to amplify specific regions (we’ll
cover this process later in this PPT).
◦ PCR can make billions of identical copies from a
single snippet of DNA

Geneticists look for Short Tandem
Repeats
◦ STRs are short units of DNA (4-5 bases) that
repeat over and over in a stretch of DNA.
Step 2: Amplify DNA Regions
STRs are critical to gene research because
they are the source of most of the
variation in DNA
 Individual A might have 20 more STRs for
one region than Individual B.

This creates a basis for
comparing which DNA samples
came from which people (or
suspects)
In this case, STR1 has 7
repeats
STR2 has 4 repeats at the
same spot.
STRs

3. The PCR process (gene copying) allows us to
see to compare how long each STR is.
◦ In criminal forensics, we look at 13 STR regions

4. We then look for a match.
◦ If all 13 STR regions match, we know that the DNA found
at the scene of a crime is from that person.
◦ The odds of it being from someone else are 1 in 1 billion.
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
…
Polymerase Chain Reaction
A copy machine for DNA
Mass-Producing DNA in a
Test Tube

The polymerase chain reaction (PCR) is a
technique that makes many copies of a certain
segment of DNA without using living cells.

Starting with a single DNA molecule, PCR can
generate 100 billion identical copies of a gene in
just a few hours.

A great advantage of PCR is that it can copy one
specific segment of DNA from within a tremendous
amount of DNA.

The technique is so precise and powerful that its
starting material does not even have to be purified
DNA.
PCR Animations 1, 2, 3, 4, 5
•PCR works by separating the dual-strand of
DNA into two individual strands so that each
side can then be primed (by primers) and
copied by polymerase enzymes.
•This process is repeated over and over.
•It can amplify a specific sequence of DNA by
as many as one billion times!

For PCR, we use a special
polymerase enzyme called
the Taq Polymerase

The Taq polymerase was
discovered in bacteria in a
hot spring such as those
you’d find in Yellowstone
Park)

The Taq polymerase can
work in very hot conditions
that would denature
normal proteins (including
enzymes like Polymerase)
Taq Polymerase

A heat-resistant polymerase enzyme is
necessary because of the constant reheating of the DNA to denature it.

The process involves constant heating,
cooling, and re-heating.

This would destabilize any normal
polymerase enzyme.

Taq does everything a normal
polymerase would do (i.e. add
complementary bases), but remains
stable at very high temperatures.
Taq Polymerase

Only a tiny amount (less than
one millionth of a gram) of DNA
needs to be present in the
starting material.

Using PCR to produce multiple
copies of a DNA sample can
make analysis of that sample
much easier.
◦ It’s easier to investigate something if
there is a lot of it!

It also enables scientists to
make copies of very rare DNA.
◦ For example, scientists have used
the technique to clone DNA pieces
recovered from…
 5,000-year-old human remains
 A 40,000-year-old woolly mammoth
frozen in a glacier
 A 30-million-year-old plant fossil.

PCR also has applications in
medicine.
◦ For example, PCR makes it possible
to detect viral genes in cells infected
with the virus that causes AIDS.
PCR Steps
Comparing DNA
•Often scientists want to compare PCR results of different
DNA samples.
•One useful technique for sorting molecules or fragments of
molecules by length is called gel electrophoresis.
First, each DNA sample is cut up
into fragments by a group of
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.
Gel Electrophoresis

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.
Restriction Fragments

Next, a few drops of each DNA
sample are placed in a small
pocket, or well, at one end of a
thin slab of gelatin-like
material called a gel. The other
end of the gel has a positive
charge.

All DNA molecules are
negatively charged (DNA has a
negative charge because of
the phosphate ions in its
chemical "backbone“) , so they
move through pores in the gel
toward the positive pole.
Gel
Electrophoresis
Gel Electrophoresis

The shorter DNA fragments
slip more easily through
the pores of the gel.

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.

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.

The pattern of bands will be different for each of the four
samples because each sample has a unique set of DNA
fragment lengths.
Gel Electrophoresis
Animations 1, 2 ,3, 4, 5, 6
DNA Fingerprinting 1, 2, 3

Just as the skin patterns on
your fingertips make up your
particular set of fingerprints,
you have a particular banding
pattern produced by your
restriction fragments called a
DNA fingerprint.
◦ The fingerprint is created by a
combination of your STRs at
different regions and where the
restriction enzyme cut the DNA.
◦ STR creates the size of the band
◦ Restriction Enzyme creates the
number of the bands

Unless you have an identical
twin, no one else is likely to
have the exact same DNA
fingerprint as you.
Genetic markers can help to pick out the
differences between 2 DNA fingerprints.

A genetic marker is a gene that is found at a known location and
produces a predictable pattern.

Genetic markers can occur in alleles for diseases or other traits.
◦ Markers can also be located in one of the many noncoding
stretches of the human genome (introns).
◦ Introns account for about 97% of the human
genome.

When DNA fingerprints are used in court as
evidence, genetic markers from these highly
variable noncoding regions are used.

Why is this important?
◦ To use DNA to identify a specific person
accurately, you want to compare genetic
markers that are unlikely to be shared
with any other person.
 Introns are least likely to be repeated among
different people

Using PCR and gel electrophoresis, a DNA fingerprint can be made
from cells in a single drop of blood or from a hair follicle. DNA is
extracted from the small sample, and multiple copies are made using
PCR.

Genetic markers (usually from introns) are then compared.

In most cases, the probability of two people having identical genetic
markers is small—somewhere between one chance in 100,000 and one
chance in 1,000,000,000 (one billion), depending on the number of
genetic markers compared.
1. Name one application of the massproduction of DNA using PCR.
2. Explain how gel electrophoresis
compares DNA samples.
3. Why are genetic markers from
noncoding regions useful in distinguishing
DNA fingerprints?
Concept Check 13.4
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

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
Extra Resources