AP BIOLOGY
DNA Fingerprinting & Forensics
Introduction
Technicians working in forensic labs are often asked to do DNA profiling or "fingerprinting" to
analyze evidence in law enforcement cases and other applications.1 The various exercises performed
in this lab are only a small fraction of techniques for recovering information for law enforcement
purposes. The evidence needed for DNA fingerprinting can be obtained from any biological material
that contains DNA: body tissues, body fluids (blood and semen), hair follicles, etc.
Hair
Some individuals are known as hair follicle experts. By looking at the follicle, one can
determine if the hair has been pigmented or if the natural color is at all present. Hair grows
approximately ½ inch in one month so if the pigmented portion is 1 inch from the root area, the
individual had their hair colored approximately two months prior to the event in question. This
information could be useful with questioning other people about the possible suspect or victim.
Depending on the individual, hair strands will have either fragmented fibers or continuous fibers.
Simply wipe the hair strand with ethanol and observe it under 40x on a compound microscope.
Light micrographs of three human hairs. The left example illustrates dark hair with a typical fragmentary
medulla. The middle is a blonde hair with no medulla. The right coarser hair is white with a continuous medulla.
Blood
The DNA analysis can even be done from dried material, such as blood stains, burned or
mummified tissue. If a body has been burned, flexor muscles will cause the body to curl, called
pugilistic posture. Blood has more surface tension than water. Therefore, when looking at blood
spatters, the investigating person should look at the diameter of the drop, location in regards to other
drops, and if the spatter is elongated in any way. Blood is not affected by age, sex, disease, body
temperature, alcoholic content, atmospheric pressure, temperature or humidity. The blood droplet is
circular in shape when falling and will not break up into smaller droplets by simply falling through air.
Blood will spatter to a greater extent on porous surfaces than nonporous surfaces. When hitting a
surface, the droplet is cast off in a backlash action and it strikes across the surface. Cast off always
comes from the larger drop. The pointed end of cast-off points back to its source.6 Most people fail
to look up a wall or even on the ceiling. This information becomes invaluable, especially when
perpetrators forget to clean blood or tissue from high places.
Crime Scene
Equipment to think about having on hand when investigating a crime scene may include:
Gloves
Watch
Medical equipment
Writing implements
Paper bags
Tape
Flashlight
Specimen containers
Thermometer
Camera
Blood Collection tubes
Plastic trash bags
Batteries for camera
Paper envelopes
Photo placards
Measurement instruments
Evidence tape
Magnifying glass
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Documentation of a scene is extremely important. If the investigator did not write it down or
take a picture of something, it didn’t happen. To investigate a crime scene, one must write
descriptions of the scene, the victim, other information. The photographic documentation of the scene
creates a permanent historical record of the scene. Photographs provide detailed corroborating
evidence that constructs a system of redundancy should questions arise concerning the report,
witness statements, or position of evidence at the scene.7 Things to remember when photographing
a crime scene:
1. Remove all nonessential personnel from the scene.
2. Obtain an overall (wide-angle) view of the scene to spatially locate the
specific scene to the surrounding area.
3. Photograph specific areas of the scene to provide more detailed views of
specific areas within the larger scene.
4. Photograph the scene from different angles to provide various perspectives
that may uncover additional evidence.
5. Obtain some photographs with scales to document specific evidence.
6. Obtain photographs even if the body or other evidence has been moved.
Chain of custody will determine if a case can properly go to trial and be successful. Always
make sure to sign for evidence and to have the next person sign for materials, documents, etc.
DNA
If a sample of DNA is too small it may be amplified using polymerase chain reactions (PCR)2
techniques. The DNA is then treated with restriction enzymes that cut the DNA into fragments of
various length.3
Restriction Enzymes
In 1968, Dr. Werner Arber at the University of Basel, Switzerland and Dr. Hamilton Smith at
the Johns Hopkins University, Baltimore, discovered a group of enzymes in bacteria, which when
added to any DNA will result in the breakage (hydrolysis) of the sugar-phosphate bond between
certain specific nucleotide bases (recognition sites). This causes the double strand of DNA to break
along the recognition site and the DNA molecule becomes fractured into two pieces.
Restriction enzymes sit on a DNA molecule and slide along the helix until they recognize
specific sequences of base pairs that signals the enzyme to stop sliding. The enzymes then digest
(chemically separate) the DNA molecule at that site - called a "restriction site" - acting like molecular
scissors, cutting DNA at a specific sequence of base pairs. These molecular scissors or “cutting”
enzymes are restriction endonucleases.
If a specific restriction site occurs in more than one location on a DNA molecule, a restriction
enzyme will make a cut at each of those sites, resulting in multiple fragments. Therefore, if a given
linear piece of DNA is cut with a restriction enzyme whose specific recognition code is found at two
different locations on the DNA molecule, the result will be three fragments of different lengths. The
length of each fragment will depend upon the location of restriction sites on the DNA molecule.
These "chemical scissors" recognize a particular four - or six - base pair (bp) recognition
sequence on a segment of DNA. It cuts the DNA molecule at that point. The enzymes used in this
experiment are EcoRI and PstI. The place on the DNA backbone where the DNA is actually cut is
shown below for the enzyme:
Like all enzymes, restriction enzymes function best under specific buffer and temperature conditions.
The proper restriction enzyme buffer has been included with the DNA sample, so that when the
rehydrated DNA and enzymes are mixed, the ideal conditions are created for the enzymes to function
optimally.
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Agarose Gel Electrophoresis
DNA that has been cut with restriction enzymes can be separated and observed using a
process known as agarose gel electrophoresis. Electrophoresis separates DNA fragments
according to their relative size. DNA fragments are loaded into an agarose gel slab, which is placed
into a chamber filled with a conductive liquid buffer solution. A direct current is passed between wire
electrodes at each end of the chamber. DNA fragments are negatively charged, and when placed in
an electric field will be drawn toward the positive pole. The matrix of the agarose gel acts as a
molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over
a period of time smaller fragments will travel farther than larger ones. Fragments of the same size
stay together and migrate in single "bands" of DNA.
Visualizing DNA Restriction Fragments
DNA is colorless so DNA fragments in the gel can't be seen during electrophoresis. A blue
loading buffer, containing two blue loading dyes, is added to the DNA solution. The loading dyes do
not stain the DNA but make it easier to load the gels and monitor the progress of the DNA
electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA
fragments. The "faster" dye co-migrates with smaller DNA fragments while the "slower" dye comigrates with larger DNA fragments.
Staining the DNA pinpoints its location on the gel. When the gel is immersed in a dilute
solution of Bio-Safe DNA stain, the dye molecules attach to the DNA molecules trapped in the
agarose gel. To enhance contrast and to easily visualize the DNA bands, excess background stain
can be removed from the gel by destaining the gel with distilled water. When the bands are visible,
one can compare the DNA restriction patterns of the different samples of DNA.
The gel below shows a possible DNA pattern. The technician’s job is to determine which
banding patterns match those at the crime scene as well as how “weak or strong” the evidence is.
Remember, the DNA evidence may place the suspect at the scene, but other evidence may be
needed to prove him or her guilty.5,6 In actual DNA fingerprinting, technicians analyze much larger
segments of DNA and many more bands and lanes are produced. These technicians are looking for
a specific DNA segment, common to a given population that will produce a unique banding pattern for
each individual.
Reliability of DNA Evidence
Two major factors affecting the reliability of DNA fingerprinting technology in forensics are
population genetics and genetic statistics. Depending on demographic factors such as ethnicity or
geographic isolation, some segments will show more variation than others.
Because of this, some populations show much less variation in particular DNA segments than
others. The degree of variation will affect the statistical odds of more than one individual having the
same sequence. If 90% of a given population has the same frequency in its DNA fingerprinting
pattern for a certain DNA segment, then very little information will be attained. But if the frequency of
a DNA pattern turning up in a population for a particular segment is extremely low, then this segment
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can serve as a powerful tool to discriminate between individuals in that population. Different
populations show different patterns in their genotypes due to the contributions made to their individual
gene pools over time. Therefore, in analyzing how incriminating the DNA evidence is, one needs to
ask the question: “Statistically how many people in a population may have the same pattern as that
taken from a crime scene: 1 in 1,000,000? 1 in 10,000? Or, 1 in 10?”
References
1. DNA Profiling Fast Becoming Accepted Tool For Identification, Pamela Zurer, Chemical and Engineering News, Oct. 10, 1994.
2. PCR menas polymerase chain reaction; it is a technique used to amplify small amounts of DNA (in this case so that further
analysis of the DNA can occur).
3. An excellent resource for the classroom teacher is Genetic Fingerprinting, Pauline Lowrie and Susan Wells, New Scientist, 16
November 1991.
4. Is DNA Fingerprinting ready for the courts?, William C. Thompson and Simon Ford, New Scientist, March 31, 1990.
5. When Science Takes the Witness Stand, Peter Neufeld and Nevelle Coleman, Scientific American, Vol. 262: 5, May 1990.
6. Flight Characteristics and Stain Patterns of Human Blood, Herbert MacDonnell and Lorraine Bialousz, LEEAA, U.S. Government
Printing Office, Superintendent of Documents, Washington, D.C. , 1971.
7. Death Investigation: A Guide for the Scene Investigator, U.S. Department of Justice, Office of Justice Programs, 1998.
Lesson 1: Restriction Endonucleases
To better understand how EcoRI/PstI may help you in performing your DNA fingerprinting test,
first you must understand and visualize the nature of the “cutting” effect of a restriction endonuclease
on DNA:
ATG AATTCTCAATTACCT
TACTTAA GAGTTAATGGA
The line through the base pairs represents the sites where bonds will break if a restriction
endonuclease recognizes the site GAATTC. The following analysis questions refer to how a piece of
DNA would be affected if a restriction endonuclease were to “cut” the DNA molecule in the manner
shown above.
1. How many pieces of DNA would result from this cut? __________
2. Write the base sequence of both the left and right side DNA fragments.
Left:
Right:
3. What differences are there in the two pieces?
4. DNA fragment size can be expressed as the number of base pairs in the fragment. Indicate
the size of the fragments (mention any discrepancy you may detect).
a. The smaller fragment is _____________ base pairs (bp).
b. What is the length of the longer fragment? ______________
Lesson 2: Restriction Digestion of DNA Samples and Preparing Agarose Gels
(DNA GROUP)
Upon careful observation, it is apparent that the only difference between the DNA of different
individuals is the linear sequence of their base pairs. In the lab, your team will be given 6 DNA
samples. Recall that your task is to determine if any of them came from the same individual or if they
came from different individuals.
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Thus far your preliminary analysis has included the following:
 The similarities and differences between the DNA from different individuals.
 How restriction endonucleases cut (hydrolyze) DNA molecules.
 How adding the same restriction endonuclease to two samples of DNA might provide some
clues about differences in their linear base pair sequence.
Now that you have a fairly clear understanding of these three items you are ready to proceed to the
first phase of the DNA fingerprinting procedure – performing a restriction digest of your DNA samples.
MATERIALS FOR RESTRICTION DIGESTION OF DNA SAMPLES
pipet tips (at least 15)
crime scene DNA
EcoRI/PstI enzyme mix (labeled ENZ)
suspect 1 DNA
P-10 and P-20 micropipets
suspect 2 DNA
color coded microtubes
suspect 3 DNA
incubator (set at 37o C)
suspect 4 DNA
Styrofoam microtube rack
suspect 5 DNA
sharpie
ice bowl
goggles
latex gloves
**Note: ALWAYS place cap of any vial upside down on lab table. NEVER allow the inside of
any cap to touch the lab table!! ALWAYS recap all vials.
**Note: ALWAYS change the pipette tip before obtaining a different sample. If you are unsure,
change the tip.
PROCEDURE FOR RESTRICTION ENZYMES OF DNA SAMPLES
1. Label the 6 colored microtubes as follows:
Green:
Blue:
______________________________
______________________________
Orange: ______________________________
Violet:
______________________________
Pink/Red: ______________________________
Yellow: ______________________________
b. The restriction digests will take place in these tubes. These tubes may now be kept in your
rack.
2. Obtain your DNA samples.
Using a fresh tip for each sample, transfer 10 ul of each DNA sample from the colored stock tubes
into each of the corresponding labeled colored tubes. Place liquid in the bottom of the microtube.
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Observations
1) Describe the samples of DNA (physical properties)
2) Is there any observable differences between the samples of DNA?
3) Describe the appearance of the restriction endonuclease mix.
3. Combine and react.
a. Locate the clear microtube (in fridge) that contains the restriction enzyme mix, labeled
“ENZ”. ENZ = Enzyme mix
b. Using the micropipet, and a new pipet tip for each sample, transfer 10 ul of the enzyme mix
“ENZ” to each reaction tube.
** NOTE: Change tips whenever you switch reagents, or, if the tip touches any of the liquid in
one of the tubes accidentally. When in doubt, change the tip! DNA goes in the tube before the
enzyme. Always add the enzyme last.
4. Now your DNA samples should contain a total of 20 ul (10 ul of crime scene or suspect DNA and
10 ul of EcoRI/PstI Enzyme mix).
5. Mix the contents.
a. Close the caps on all the tubes.
b. Mix the components by gently flicking the tubes with your finger.
c. Place the tubes in the centrifuge with the hinges up. Pulse for two seconds to force the
liquid into the bottom of the tube to mix and combine reactants. (Be sure the tubes are in a
balanced arrangement in the rotor).
d. Don’t open until it comes to a complete stop.
Review Questions for Lesson 2: Restriction Enzymes of DNA Samples
1) Before you incubated your samples, describe any visible signs of change in the contents of
the tubes containing the DNA after it was combined with the restriction enzymes.
6. Incubate the samples.
a. Place the tubes in the styrofoam rack and incubate them at 37oC for 45 minutes.
b. After the incubation, store the DNA digests in the refrigerator.
2) Can you see any evidence to indicate that your samples of DNA were fragmented or
altered in any way by the addition of EcoRI/PstI? Explain.
3) In the absence of any visible evidence of change, is it still possible that the DNA samples
were fragmented? Explain your reasoning.
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MATERIALS FOR AGAROSE GEL (GEL GROUP)
gel tray
distilled water
electrical tape
scoopula
agarose powder
paper towels
50x TAE buffer
thermometer
electronic scale
hot gloves
microwave
leveling bubble
weighing dish
2,500 mL Erlenmeyer flask
100 mL graduated cylinder
electrophoresis comb
refrigerator
latex gloves
goggles
PROCEDURE FOR AGAROSE GEL
1. Prepare electrophoresis buffer.
a. TAE (Tris, acetate, EDTA) electrophoresis buffer is available as a 50x concentrated
solution. In addition to the 1x TAE buffer that will be used for the gel, approximately
275mL is also required for each electrophoresis chamber.
b. Measure 7mL of 50x TAE and pour into a 500mL Erlenmeyer flask marked TAE buffer. Do
Not pour extra liquid back into the stock container. Ask if anyone else needs any. If not,
pour the extra 50x TAE buffer liquid in the waste container. Don’t pour the liquid down
the drain!
c. Fill TAE Buffer Erlenmeyer flask with distilled water up to 375 mL mark. You now have
375mL of 1x TAE buffer.
d. Swirl until dissolved.
e. Using a clean scoopula, measure out 0.5 gram of agarose powder. Do Not place the extra
agarose powder back into the stock container. Ask if anyone else needs any. If not, put
the extra powder in waste container. Don’t pour the liquid down the drain!
f. Place agarose powder into a 500mL Erlenmeyer flask marked Agarose gel.
g. Measure 50mL of the 1x TAE buffer solution and add this to the flask with the agarose
powder.
h. Swirl to suspend the powder in the buffer solution.
i. Take the agarose/TAE buffer solution to your instructor. They will microwave the flask for
1 minute, swirl, then microwave for 20 seconds more.
j. Wearing hot gloves, take the dissolved agarose/TAE buffer solution back to your station.
k. Insert the thermometer and wait until it reads 55-60o C. When the temperature is in this
range, the gel is ready to be poured.
l. While waiting for the gel to cool, tape up the sides of the gel tray with electrical tape. Press
the tape firmly to the edges of the gel tray to form a fluid-tight seal.
m. Level the gel tray on a leveling table or workbench using the leveling bubble.
n. Place the gel comb into the appropriate slot of the gel tray. Gel combs should be placed
within ¾ of an inch of the end of the gel casting tray (not in the middle of the gel).
o. Wearing the hot gloves, pour the agarose/TAE buffer solution into the gel tray until ½ of the
comb teeth are covered.
p. Allow the gel to solidify at room temperature for 25 minutes – it will appear cloudy, or
opaque, when ready to use.
q. Once the gel has solidified, very carefully, remove the gel comb by lifting it straight up.
Gently rinse the comb with water.
r. Do not remove tape yet.
s. Carefully transport your gel to the electrophoresis box.
Lesson 3: Electrophoresis of DNA Samples (DNA GROUP)
Observation
1) After incubation, are there any visible clues that the restriction enzymes may have in some
way changed the DNA in any of the tubes? Explain your reasoning.
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MATERIALS
electrophoresis box
digested DNA samples
500x DNA Bio-safe stain “S”
500mL Erlenmeyer flask
distilled water
power supply
DNA sample loading dye “LD”
P-10, P-20 and P-50 micropipettes
1x TAE electrophoresis buffer
HindIII DNA size markers “M”
Styrofoam microtube rack
gel staining tray
sharpie
pipette tips
latex gloves
plastic wrap
agarose gel
goggles
PROCEDURE
1. Obtain your gel and remove the tape from the sides.
2. Place the casting tray with the gel in it, into the platform in the electrophoresis box. The wells
should be at the (-) cathode end of the box, where the black lead is connected.
3. Add and mix loading dye.
a. Using a new tip for each sample, add 5 ul of sample loading dye “LD” to each DNA
sample.
b. Close the caps on all the tubes. Mix the components by gently flicking the tubes with your
finger.
c. Make sure hinges are facing up and that the centrifuge is balanced. Centrifuge for 1
second, twice.
4. Loading samples.
a. Pour approximately 275mL of 1x TAE electrophoresis buffer into the electrophoresis
chamber. Pour buffer in the gel box until it just covers the wells.
b. Locate your lambda HindIII DNA size marker in the tube labeled “M” (in fridge).
c. Using a separate pipette tip for each sample, load your gel as follows:
**Note: Gels are read from left to right. The first sample is loaded in the well at the left
hand corner of the gel. Pipetting strategies: elbow on table, steady hand with opposite
hand, don’t puncture gel, and don’t release top until pipette is complete out of box.
Lane 1:
Lane 2:
Lane 3:
Lane 4:
Lane 5:
Lane 6:
Lane 7:
M, HindIII DNA size marker, clear microtube, 10 ul
CS, green microtube, 20 ul
S1, blue microtube, 20 ul
S2, orange, 20 ul
S3, violet, 20 ul
S4, red microtube, 20 ul
S5, yellow microtube, 20 ul
d. Make a sketch of the gel to ensure the proper reading of the samples.
5. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red to red and
black to black. Connect electrical leads to the power supply.
6. Set the power supply for 100 V and turn on power supply. Make sure green light is on. If not,
check outlet reset button. The green light should be on. If not, push reset button in.
7. Run the electrophoresis for 40 minutes.
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8. Preparing 1x Bio-Safe stain. (STAIN GROUP)
a. Obtain a clear microtube marked “S” which has 500x Bio-Safe stain (in fridge).
b. Pour the stain into a 500mL Erlenmeyer flask marked gel stain.
c. Add distilled water until you reach the 500mL mark.
d. Swirl to ensure maximum diffusion. The stain in nontoxic but will stain hands and clothes.
e. Cover the flask with plastic wrap until samples are complete.
9. Staining the gels.
a. When the electrophoresis is complete, turn off the power and remove the lid from the gel
box.
b. Have the staining tray ready before you remove the gel tray and gel.
c. Carefully remove the gel tray and the gel from the gel box. Gels will slide out and
possibly crack unless you have your fingers at the edge of the tray to keep from
dropping the gel.
d. Slide the gel into the staining tray non-well end first.
e. Pour 65mL of 1x Bio-Safe DNA stain into the plastic staining tray (enough to cover the gel).
f. Cover with plastic wrap and let the gel stain overnight. Make sure the gel will not be in
direct sunlight as this will fade the bands.
10. Pour electrophoresis buffer into the designated waste container that your instructor has provided.
Don’t pour the liquid down the drain!
11. Gently rinse gel box, being careful not to get the electrodes wet.
12. Gently rinse and dry the gel trays.
14. Wash and dry all used equipment.
Review Questions
1) After DNA samples are loaded into the sample wells, they are “forced” to move through
the gel matrix. What size fragments (large vs. small) would you expect to move toward
the opposite end of the gel most quickly? Explain.
2) Which fragments (large vs. small) are expected to travel the shortest distance from the
well? Explain.
Lesson 4: Photographing and Analyzing the DNA Patterns (STAIN GROUP DAY 2)
Unaided visual examination of gels indicates only the positions of the loading dyes and not the
positions of the DNA fragments. DNA fragments are visualized by staining the gel with a blue dye.
The blue dye molecules have a high affinity for the DNA and strongly bind to the DNA fragments,
which makes them visible. These visible bands of DNA may then be photographed to be retained as
evidence. For fingerprinting analysis, the following information is important to remember:
 Each lane has a different sample of DNA.
 Each DNA sample was treated with the same restriction endonucleases.
MATERIALS
digital camera
distilled water
2 millimeter rulers
linear graph paper
semi-log graph paper
white paper
PROCEDURE FOR PHOTOGRAPHING GEL (Stain and Gel Group)
1. Pour off the Bio-Safe DNA stain into the designated waste container. Be careful to hold the gel
in place so as to not allow the gel to become cracked or ripped.
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2.
3.
4.
5.
Fill the staining tray with distilled water and allow destaining to occur for 5-10 minutes.
While holding the gel, pour off the water into the sink.
Place gel on a piece of white paper.
You may want to photograph your gel with the millimeter ruler placed next to the HindIII marker
side. The millimeter ruler must start at the bottom of the loading well.
Quantitative Analysis of DNA Fragment Sizes
If you were on trial, would you want to rely on a technician’s eyeball estimate of a match, or
would you want some more accurate measurement? In order to make the most accurate comparison
between the crime scene DNA and the suspect DNA, other than just a visual match a quantitative
measurement of the fragment sizes needs to be created.
PROCEDURE FOR ANALYZING GEL
1. Using the ruler, measure the migration distance of each band. Measure the distance in millimeters
from the bottom of the loading well to each center of each DNA band and record your numbers in the
data table. You may use a second ruler to lay horizontally across the gel to ensure accurate readings.
The data in the table will be used to construct a standard curve and to estimate the sizes of the crime
scene and suspect restriction fragments.
2. To make an accurate estimate of the fragment sizes for either the crime scene or the suspects, a
standard curve is created using the distance (x-axis) and fragment size (y-axis) data from the
Lambda/HindIII size marker. Graph the Lambda/HindIII marker and then measure the distance for all
bands and annotate on your table. To do this, locate the distance of the band; go vertically to the line
on the graph. Then using both linear and semi-log graph paper, plot distance versus size for bands 26. Use a ruler and draw a line to the base pair number. Extend the line all the way to the right hand
edge of the graph.
3. To estimate the size of an unknown crime scene or suspect fragment, find the distance that
fragment traveled. Locate that distance on the x-axis of your standard graph. From that position on
the x-axis, read up to the standard line, and then follow the graph line to over to the y-axis. You might
want to draw a light pencil mark from the x-axis up to the standard curve and over to the y-axis
showing what you’ve done. Where the graph line meets the y-axis, this is the approximate size of
your unknown DNA fragment. Do this for all crime scene and suspect fragments.
Which graph provides the straightest line that you could use to estimate the crime scene or the
suspect’s fragment sizes? Why do you think one graph is straighter than the other?
4. Decide which graph, linear or semi-log, should be used to estimate the DNA fragment sizes of the
crime scene and suspects. Justify your selection.
5. Compare the fragment sizes of the suspects and the crime scene. Is there a suspect that matches
the crime scene? How sure are you that this is a match? Describe the scientific evidence that
supports your conclusion.
6. What would your gel look like if the DNA were not fragmented?
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7. What caused the DNA to become fragmented?
8. A restriction endonuclease “cuts” two DNA molecules at the same location. What can you assume
is identical about the molecules at that location?
9. So who committed the murder?
10. Will your evidence stand up in court?
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