Lab 8: Forensics

Purpose: The purpose of this lab is for you to understand the use of blood typing and DNA
fingerprinting for forensics and identification of individuals.
This lab will help you practice the following skills:
1. Determining blood type based on agglutination
2. Using DNA fingerprinting (DNA extraction, PCR, and gel electrophoresis) to identify a
suspect for a crime

This assignment will help you gain the following knowledge:

1. Differentiating between blood types and the science of blood typing
2. Explaining how DNA is extracted from cells for Polymerase Chain Reactions (PCR)
3. Being able to describe how PCR reactions work
4. Performing gel electrophoresis and comparing DNA fingerprints

The Case of Antonio Beaver

Simulations:

The worksheet contains links to websites with simulations that you should do to help you
understand the material. These work best with Google Chrome.

Part I – Background and blood typing

The Crime
On August 15, 1996, a 26-year-old Caucasian woman (Dr. H) drove into a St. Louis parking lot
planning to park her car and return to work. She approached
a man in the lot, thinking he was the parking attendant. The
man told her that she had to move her car immediately or it
would be towed, and he followed her back to the car. As she
got in the car to leave, the man told her she could stay. As
she got back out, the man attacked her with a screwdriver
and told her to give him the keys and her purse. She
struggled with him and then decided to flee, leaving her David Letterman
keys and purse behind. As she was exiting the car, she
noticed that the man was bleeding and that there was blood inside the driver’s side door. She ran
into a nearby parking garage and called the police as her attacker fled in her car.
The victim described her attacker to police as a clean-shaven African American man wearing a
baseball cap. She said the man was 5’10’’ tall, with a “David-Letterman-like” gap between his
teeth. The next day, the victim helped police draw a composite sketch of the attacker and police

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recovered the victim’s car in East St. Louis. Swabs of the blood stain inside the driver’s side
door were collected.
The Identification
Six days later, a detective arrested Antonio Beaver
because he thought Mr. Beaver resembled the composite
sketch in this case. However, Mr. Beaver had a full
mustache, was 6’2’’ tall and had chipped teeth. The same
detective then prepared a live lineup including Mr.
Beaver and three other men – two of them police officers.
Mr. Beaver and the other non-officer were the only two to wear baseball caps, and Mr. Beaver
was the only one with noticeable defects to his teeth. The victim identified Mr. Beaver.
The Trial
Mr. Beaver was charged with first-degree robbery and tried in April 1997. The victim testified
that Mr. Beaver was the man who attacked her, the prosecution argued that the victim’s clear
memory of the crime meant that she was better able to identify the perpetrator. The defense
presented evidence showing that fingerprints collected from the victim’s car – including prints
from the driver’s side and the rearview mirror – did not match the victim or Mr. Beaver. They
argued that prints left on the rearview mirror indicated that the person who left them must have
driven the car.
After several hours of deliberations over two days, the jury
convicted Mr. Beaver of first-degree robbery. He was sentenced to
18 years in prison.
The Biological Evidence
Claiming innocence, in 2001, Mr. Beaver filed a motion on his
own behalf requesting blood and DNA testing. The state opposed
the motion, but the court granted a hearing on the issue in 2005. Antonio Beaver
The state agreed to testing on the blood swab from inside the car
door in October 2006. In this lab, you will learn how analysis of
blood and DNA can be used in criminal cases and forensics.

Introduction to Blood Typing

ABO Blood Types:
The surfaces of red blood cells (RBCs)
contain numerous markers known as
antigens. Antigens are macromolecules that
are perceived by the body as foreign and thus
illicit an immune response such as producing
antibodies or causing an allergic reaction. In
humans and many other primates there are
four major groups of red blood cells based on

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Figure 1: Picture of A and B antigens on
red blood cells
their antigen types: A, B, AB, and O. These are the well-known blood groups or blood types.
The A blood group has antigen A, the B blood group has antigen B, the AB group has both A
and B, and the O group has neither. This does not mean that your blood causes an immune
response in you, rather, if you receive blood with a different antigen, your body will consider it
foreign and react.

Figure 2: Agglutination of red blood cells

We can test blood to see to which blood group it belongs using an antiserum (a serum
containing antibodies). Antibodies are proteins made by the immune system that recognize and
bind to one type of molecule only.
Antibodies that recognize one of the
blood type antigens will bind only to the
antigen molecules on red blood cells that
have those antigens specifically. As
antibodies can bind two molecules at a
time, they can hold the cells together as
you see in Figure 2. If the antigens are
not present on the blood cells, the
antibodies will not bind to the cells.
If a person has type A antigens on the
surface of their red blood cells, the
antibodies present in the anti-A serum
will bind to the A antigens and cause the
cells to clump together. The proper term
Figure 3: Blood typing reactions
for this clumping process is
agglutination. If the cells do not have A antigens on their surface, then agglutination will not
occur.
In Figure 3 above, anti-A serum has been added to the blood in the top left well causing
agglutination, which gives the sample a "clumped" appearance. When anti-B serum was added to
the well beneath that, containing the same type of blood, agglutination did not occur. For this
sample of blood, the results indicate that A antigens are present on the blood cells and that B
antigens are not present. Therefore, this individual has type A blood. Type B blood would clump,
or agglutinate, if anti-B serum (containing antibodies that bind to the B antigens on the cells)
were added to it. Type O blood has neither A nor B antigens on the red blood cells, so it does not

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react with either anti-A or anti-B serum. How do you think blood type AB would react with
either ant-A or anti-B serum?

Figure 4: Blood type, antigens and antibodies in plasma

The Rh Factor
The Rh factor is another type of antigen present on some red blood cells but absent on others. If a
person’s red blood cells have Rh antigens on their surfaces, the blood type is Rh+ (read: R-H-
positive). If the antigens are not present, the blood is Rh– (read: R-H-negative). If the Rh
antiserum agglutinates the red blood cells, then they have Rh antigens on their surfaces, so they
are Rh+. If the antiserum does not cause agglutination, then they are Rh–.
A person with Rh- blood does not have Rh antibodies naturally in their blood plasma (as one can
have A or B antibodies, for instance). But a person with Rh- blood can develop Rh antibodies in
their blood plasma if they receive blood from a person with Rh+ blood, whose Rh antigens can
trigger the production of Rh antibodies. A person with Rh+ blood can receive blood from a
person with Rh- blood without any problems, but not vice-versa.

The combination of the ABO blood type and the Rh factor leads to the commonly designated
blood types:

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 Blood type Antigens present
A+ Type A and Rh antigens are both present on the red blood cells
A– Type A antigens are present, but Rh antigens are not
AB+ Type A and B antigens are present, as well as Rh antigens
AB– Type A and B antigens are present, but Rh antigens are not
O+ Type A and B antigens are absent, but Rh antigens are present
O– None of the antigens are present on the red blood cells

Blood type O– does not cause clotting with any of the blood types and can be given to anyone
because it doesn’t have surface antigens. For this reason, blood type O– is considered to be the
“universal donor.”

Q1. Given this information, which blood type do you think is known as the “universal acceptor”?
AB+

Exercise 1: Blood typing

Since there is variation in blood types among people, forensic scientists can use these to identify
blood at a crime scene. Today you will analyze the blood type of Mr. Beaver and compare it to
the blood found at the crime scene. But first, you can practice blood typing and understanding
what type of blood patients need.
1. Go to this website:
https://educationalgames.nobelprize.org/educational/medicine/bloodtypinggame/
index.html
2. Click on “Play the blood typing game” to practice and test your knowledge. You can also
click on “Learn the theory” to learn more about how blood typing works.
3. Proceed to the blood typing procedure below.
Blood Typing Procedure
1) Add a drop of anti A sera (colored blue) to the well labeled A. Replace the cap on the
dropper vial.
2) Add a drop of anti B sera (colored yellow) to the well labeled B. Replace the cap on the
dropper vial.

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 3) Add a drop of anti D sera (Rh factor) to the well labeled D. Replace the cap on the
dropper vial.
4) Using the dropper vial, place one drop of Mr. Beaver’s blood in each well of the blood
typing slide. Replace the cap on the dropper vial.
5) Using a different mixing stick for each well, gently stir the blood and the antisera for 30
seconds. Discard the mixing stick after each use to avoid contamination of your samples.
6) Carefully examine the films of liquid. If the film remains uniform in appearance, there is
no agglutination (clotting). If the film appears granular, then agglutination has occurred.
7) Answer “yes” or “no” in the chart below for the agglutination reactions you observe.
8) Determine the blood type of Mr. Beaver, the victim, and the blood found at the crime
scene.
Q2. Blood Typing Table

Person Anti-A Anti-B Anti-Rh/D Blood

serum serum serum Type?

Crime Scene Yes No Yes A+

Victim (Dr. H) No Yes Yes B+

Mr. Beaver Yes No Yes A+

Q3. What can you conclude about the crime, based on the blood at the crime scene?
The blood is not from Mrs H
Q4. What can you conclude about the crime, based on the blood from the victim?
The blood at the crime scene is not from Mrs. H.
Q5. What can you conclude about the crime, based on the blood from Mr. Beaver?
Mr. Beaver could potentially have committed the crime.

DNA Analysis
DNA evidence

A blood sample was found at the scene, and a blood sample has been collected from Antonio
Beaver. Matching blood types is not enough to convict someone or prove their innocence

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because millions of people share the same blood type (see Figure 5 below). We will compare the
DNA from these samples to determine if one of them was at the scene and somehow left his or
her blood behind. Since we are only interested in the DNA from the blood sample, we must first
extract that DNA from the white blood cells (i.e., separate it from the rest of the cell).

Figure 5: Bar chart of average distribution of blood types in the United States as of 2021

What is DNA?
Deoxyribonucleic acid (DNA) can be considered the hereditary “code of life” because it
possesses the information that determines an organism’s traits and is transmitted from
one generation to the next. DNA can be compared to a recipe or a list of instructions
about how to create and maintain a specific living thing. The DNA in an individual’s
cells contains unique genetic instructions about how to make and operate that individual.
All living things are dependent on DNA, and the structure of DNA is consistent among
all species. However, the particular sequence of nitrogenous bases within DNA
molecules differs between organisms to create explicit “blueprints” that specify
individual living things. This sequence of base pairs is what makes an organism an oak
tree instead of a blue jay, a male instead of a female, and so forth.

Plants, animals, protists, and fungi are all made up of eukaryotic cells. A eukaryotic cell contains
a nucleus encased by a nuclear membrane, which houses the cell’s DNA. Bacteria are single
prokaryotic cells, which do not have nuclei (plural of nucleus). Instead, the DNA in a prokaryotic
cell is found in a long loop coiled loosely throughout the cell’s cytoplasm.

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Where exactly is the DNA found in
the blood?
If you examined a human blood slide,
you should be able to see red blood
cells and white blood cells (stained
purple for visualization). Though they
are the most numerous in blood, red
blood cells have no nucleus and no
DNA. The only DNA in blood is
found in the white blood cells, which
are part of the immune system. Blood
plasma is a clear liquid that red and
white blood cells float around in. In
addition, blood contains small
components called platelets that are responsible for preventing bleeding (clotting).

A change in the case!

So far, the blood-typing evidence we have uncovered supports Mr. Beaver’s guilt. Mr. Beaver is
still awaiting his trial and his lawyers have discovered some possible evidence to incriminate
another suspect who matches Mr. Beaver’s blood type. A different man may have been at the
crime scene, which could make a stronger case for Mr. Beaver’s defense. There is no way to
prove that this second suspect was at the crime scene without comparing the DNA from this new
person (Suspect 2) to the DNA found at the crime scene years ago. We will compare the DNA
found at the crime scene to DNA from the victim (Dr. H), Mr. Beaver, and the second suspect.
We want to be thorough and make sure that the blood at the crime scene is not from the victim
but is from a suspect.

To compare DNA from the crime scene to Dr. H, Mr. Beaver, and Suspect 2, we need to first
extract DNA from each person and from the blood collected at the crime scene.
You will now work on extracting DNA from a strawberry. The same method will be used to
extract DNA from Dr. H, Mr. Beaver, suspect 2, and the blood sample from the crime scene (but
DNA will be extracted from the white blood cells).

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Exercise 2: Procedure For Extracting DNA From a Strawberry

1) Obtain one strawberry. If you are using a fresh strawberry, remove the green sepals (tops)
from the berry.
2) Place the strawberry in a re-sealable plastic bag.
3) Close the bag slowly, pushing all the air out of the bag as you seal it.
4) Thoroughly mash the strawberry for 2 minutes.

This begins the process of lysing (or breaking open) the cell membranes of the
strawberry’s cells and nuclei exposing the DNA.

5) Pour 10 mL of extraction buffer into the bag with the mashed strawberry. Reseal the bag.
6) Mash the strawberry for 1 additional minute.

The extraction buffer (in this case a type of detergent and salt) and more mashing
further break apart the membranes of the strawberry cells and nuclei by dissolving the
phospholipids that make up the membrane. Salt in the buffer causes the broken-up parts
of the cell to separate from the DNA.

7) Place a funnel into a 50 mL conical tube. Fold

a piece of cheesecloth in half along the longer
side and place it in the funnel to create a filter.
The cheesecloth will overlap the edge of the
funnel.
8) Pour the strawberry mixture into the funnel,
filtering the contents through the cheesecloth
and into the 50 mL tube.

The cheesecloth filters the unwanted cell

fragments, which can be seen and are stuck in
the cheesecloth, from the DNA, which passes
right through. Figure 6: Diagram showing how detergent (blue)
interacts with lipids (green) and proteins (yellow)
9) Carefully pour 2 mL of the filtered contents
from the 50 mL tube into a clean 15 mL tube. Use the lines on the side of the 15 mL tube
to help measure the amount added.
10) Hold the 15 mL tube at an angle. Carefully add 5 mL of cold 95% ethanol by
running it down the inside of the tube. You should have two distinct layers.
Caution: Do not mix the strawberry extract and the ethanol!

Alcohol separates the DNA from the rest of the cell extract that made it through
the cheesecloth.

11) Watch closely as translucent strands of DNA begin to clump together where the
ethanol layer meets the strawberry extract layer. Tiny bubbles in the ethanol layer
will appear where the DNA precipitates.

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 12) Slowly and carefully rotate the wooden stick in the ethanol directly above the extract
layer to wind (or “spool”) the DNA. Remove the wooden stick from the tube and observe
the DNA. You can set the DNA on a paper towel.
Figure 7: Picture
of strawberry
This DNA comes from multiple strawberry cells. Strawberry cells are excellent sources solution with
of DNA for extraction. They are multicellular and octoploid. This means they have visible DNA
eight copies of their seven chromosomes in each of their many cells. Therefore, just one berry
will yield enough DNA to be easily seen and spooled.

Exercise 3: DNA Extraction Simulation

1. Go to this website to learn more about DNA extraction from human cells:
https://learn.genetics.utah.edu/content/labs/extraction/
2. Answer the following questions as you complete the simulation.

(You may have to enable Flash Player for it to work. Use Google Chrome since that appears to
be the most reliable browser for this. It may take a minute to load.)
Q6. List three reasons why scientists extract and isolate DNA:

1) To view under a microscope

2) To compare it to other DNA samples

3) DNA fingerprinting

Q7. What is the purpose of the detergent in DNA extraction?

To help thin out the strawberry to further break down the cells
Q8. The extraction buffer also contains salt. What does the salt do?
Help the cells separate.
Q9. What does the ethanol (or alcohol) do to the DNA?
Separates the rest of the DNA that got through the cheese cloth.

Q10. This same process for the strawberries was repeated to extract DNA from the cheek cells of
Mr. Beaver (first suspect), the second suspect, Dr. H (to confirm that any DNA at the crime
scene was not hers), and from the blood left at the crime scene. Which kind of blood cell is best
to use for DNA extraction?

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 White blood cells.

Q11. Why do you need to extract DNA from the blood left at the crime scene?
To further specify who committed the crime, multiple people can have the same boood type,
but everyone has one unique DNA.

Exercise 4: Gel Electrophoresis and DNA fingerprinting

Now that we have extracted DNA from the crime scene, Dr. H, Mr. Beaver, and Suspect 2, we
have to compare the DNA. We do this by DNA fingerprinting.
Much like the curves and loops on your fingers, DNA has a structure
that is unique to each individual (with the exception of identical twins).
Of course, rather than peaks and valleys, the differences in DNA are in
the sequence of nucleotides, the basic components of DNA. There are
four possible nucleotides:
A – Adenine C – Cytosine
T – Thymine G – Guanine
Although the basic shape of DNA is standard across all living
organisms, the sequence of nucleotides is free to vary, much like the
letters in our alphabet rearrange to form different words. Any sequence
of DNA that has a specific meaning (codes for a protein or RNA) are
called genes and act a little like the words in a sentence. Since there are
varying lengths of DNA, this simple structure can create an almost infinite number of stories.
In order to read that sequence, we must first extract and isolate the DNA (which you just did).
Then we will “fingerprint” the DNA.

This method is routinely used by biologists in several fields as well as by law enforcement to:

 Identify potential suspects whose DNA may match evidence left at

crime scenes
 Exonerate persons wrongly accused of crimes
 Identify crime and catastrophe victims
 Establish paternity and other family relationships
 Identify endangered and protected species as an aid to
wildlife officials (could be used for prosecuting poachers)
 Detect bacteria and other organisms that may pollute air,
water, soil, and food
 Match organ donors with recipients in transplant programs

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 Determine pedigree for seed or livestock breeds
 Authenticate consumables such as caviar and wine

Read more info about how DNA is used in forensics here:

https://learn.genetics.utah.edu/content/science/forensics/

DNA FINGERPRINTING
The basic idea of DNA fingerprinting is to use a sequence within the DNA to uniquely identify a
person or to match a person with some evidence. Undoubtedly, everyone’s DNA is different. If
we could easily get complete sequences of genomes, everyone would be unique (except possibly
identical twins). How different is one person’s DNA from another?

Siblings can be expected to differ by 1-2 million base pairs (out of 3.3 billion base pairs
total)

We are all uncannily similar. Compare your genes to

your classmates and on average they will be about
99.9% the same. Individuals of our species are actually
very genetically similar compared to the variation
within many other species. What endows us with our
individuality is the tiny differences in that remaining
0.1% or that one nucleotide out of every thousand. It
would be unfeasible to sequence an entire genome to
“fingerprint” an individual, thus we need to locate
regions that are highly variable among different
people.

Portions of the DNA sequence that vary the most

among humans must be used; also, portions must be
large enough to overcome the fact that human mating
is not absolutely random. Fortunately, on each Figure 7: Diagram of exons and introns in DNA
chromosome there are sequences of bases between genes that are not themselves genes called
introns.

It is believed these sequences don’t code for anything. This non-coding DNA used to be called
“junk DNA”, but we are often finding now that even though the specific sequence is not
important, their placement might be. Scientists have found that since these regions do not code
for proteins, the overall pattern and length of these regions change (mutate) a lot without any
major repercussions. Therefore, they tend to be unique to an individual (except identical twins).

We will look at a few of these sequences, called short tandem repeats (STRs), on the two
suspects and compare it to the STRs found at the crime scene. STRs are small DNA sequences,
such as GGC or ATT that are repeated numerous times. For example, a STR might be
ATTATTATTATTATT… The number of repeats (ATT) changes from person to person.

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 PCR – Making More Copies of DNA
Since the amount of DNA at a crime scene is usually very small, we can take any piece that we
find and multiply it using a method called Polymerase Chain Reaction or PCR. Through PCR,
billions of copies of a specific DNA section can be made from a single fragment, thus vastly
increasing the quantity of DNA material available to be analyzed. This short video explains how
PCR works: https://www.youtube.com/watch?v=3XPAp6dgl14

Gel Electrophoresis
We then need to determine how many repeats are in the DNA of the person of interest. In order
to do that, we use a method called gel electrophoresis, which allows us to see how long each
person’s STRs are. Gel electrophoresis works by putting the amplified bits of DNA into a semi-
solid agarose matrix (a gel). Electricity is then run through the gel. This electricity pushes the
negatively-charged DNA fragments through the pores of the gel, toward the positive end of the
apparatus. Shorter fragments of DNA (i.e., DNA with fewer repeats) move faster than
longer repeats, because they can move through the pores in the gel more easily. If you stop
the electricity before any of the fragments move completely out of the gel, the shorter ones will
have moved further than the longer ones. A dye is added to the gel in order to visualize the bands
of DNA. We can compare the distance they’ve gone to fragments of known length (a DNA
ladder) to determine how long the STRs of interest are.

DNA samples
Sample wells

Gel

DNA ladder
Movement

Electrical Longer fragment

poles

Shorter fragment

Figure 8: Diagram of gel electrophoresis

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We can take the DNA found at the crime scene and DNA from the suspects and victim, amplify
it through PCR, then run it through a gel, and compare the number of repeats (length) of their
STRs. You must look for matches at many different locations (loci) on the person's genome; one
or two (even three) aren't enough to be confident that the suspect is the right one, but thirteen loci
are used by forensic scientists. A match at all thirteen is rare enough that you (or a prosecutor or
a jury) can be very confident ("beyond a reasonable doubt") that the right person is accused.

DNA from the crime scene, Dr. H, Mr. Beaver, and Suspect 2 has already been extracted and
amplified by PCR. Now we have to conduct gel electrophoresis to separate and visualize the
DNA fragments for each person, thus creating a DNA fingerprint.

1. Go to this website: https://learn.genetics.utah.edu/content/labs/gel/

2. Start clicking through and reading the explanations.

Q12. Which DNA fragments move the quickest through the gel--short strands or long
strands?

Short strands.

Now you will make the gel and set up the gel apparatus. Follow the instructions.

Q13. Then you will load your samples into the gel, run the electrical current, then stain the
gel with ethidium bromide. Why do you need to stain the gel?
To see the DNA extractions more clearly.
Q14. Use the size standard (with known sizes of bands) to figure out the sizes of the bands in
this DNA sample. Write the sizes below.
 Top band: 900 BP
 Middle band:500 BP
 Lower band: 200BP

Now that you understand how gel electrophoresis works, let’s look at the gel from Mr. Beaver's
case. Your instructor will show you a gel from this case. Remember that the DNA samples are
put into the wells at the top of the gel and the bands form as the DNA moves down towards the
positive end of the gel.

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 Q15. The DNA bands from which two columns match?
3&4

Q16. What are your conclusions about this case and about Mr. Beaver’s role in the crime?
Mr Beaver is innocent.

In 2001, Mr. Beaver filed a motion on his own behalf requesting DNA testing. The state
opposed the motion, but the court granted a hearing on the issue in 2005. The Innocence
Project accepted Mr. Beaver’s case and filed another brief on his behalf in 2006. The state
agreed to test the blood swab from inside the car door in October 2006 and the results proved
that Mr. Beaver could not have committed this crime. The DNA testing not only excluded
Mr. Beaver as the source of the blood but led to the identification of another man already
incarcerated for other crimes. Mr. Beaver was exonerated on March 29, 2007. He was 41
years old at the time of his exoneration and had served more than a decade in prison.

The Innocence Project

The Innocence Project, founded in 1992 by Peter Neufeld and Barry Scheck at Cardozo
School of Law, exonerates the wrongly convicted through DNA testing and reforms the
criminal justice system to prevent future injustice.

The Innocence Project's mission is to free the staggering number of innocent people who
remain incarcerated, and to bring reform to the system responsible for their unjust
imprisonment. Learn more here: https://www.innocenceproject.org/about/

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