Broberg, Figley, Smith, Stark 1

Bailie Figley
Mrs. Reifke
AP Biology
23 February 2016
Crime Scene Suspect DNA Identification through the use of Restriction Enzymes and
Electrophoresis
Introduction:
Purpose or Problem: The purpose of this lab is to demonstrate how the restriction
enzymes in the human body can be used in biotech applications in order to cut sample DNA into
smaller pieces that can be categorized. This lab also explores the process of electrophoresis and
how this technique can be used to isolate and compare different DNA samples in order to be used
in crime scene investigation and later in criminal trials.
Background Observations/Information: Restriction enzymes are enzymes that cut
DNA molecules at a particular place. These enzymes are critical in the process of recombinant
DNA technology. The enzyme interprets certain sequences of DNA nucleotides in order to cut
the DNA into multiple pieces with sticky ends of nucleotides. Theses sticky ends are
compatible with the sticky ends of other samples and segments due to the base pairing rule. This
rule states that the pairing of corresponding nucleotides allows for the combination of otherwise
separate DNA segments. The base pairing rule is the basis behind the majority of all
biotechnologies relating to recombinant DNA. The way in which restriction enzymes cut DNA is
also crucial for the process of electrophoresis. Electrophoresis is a technique used in laboratories
in order to separate macromolecules based on size. The technique applies a negative charge so
proteins move towards a positive charge. This is used for both DNA and RNA analysis. In DNA
biotechnologies there is a technique called Restriction Fragment Length Polymorphism. This is a
technique that exploits variations in homologous DNA. In a human being, the average
percentage of DNA that any two humans have in common is approximately 99.9% similar. Due
to this similarity, the use of restriction enzymes has very similar results from each individual. To
help further identify different segments of DNA, more than one restriction enzyme is usually
used in the criminal identification process. This identification gets more difficult as the DNA
samples get more and more similar such as that between twins or siblings. This similarity cuts
the DNA into even more similar pieces that all get pulled by the charge in electrophoresis in the
same or very similar ways.
Materials and Methods:
Materials:
Restriction Enzyme Mixture
Ice
Insulation Container
Six Microcentrifuge Tubes
Marker DNA Sample
Crime Scene DNA Sample
Suspect 1 DNA Sample
Suspect 2 DNA Sample
Suspect 3 DNA Sample
Suspect 4 DNA Sample
Suspect 5 DNA Sample

Broberg, Figley, Smith, Stark 2

Micropipet
Sterile Micropipet Tips
Foam Microcentrifuge Tube Sleeve
Microcentrifuge
Water Incubator
1% Agarose Gel
DNA Loading Dye
Electrophoresis Apparatus
1x TAE Buffer
Plastic Tray
Procedure:
Restriction Digestion:
1. Place the tube containing the restriction enzyme mix, labeled ENZ, on ice.
2. Label one of each colored microcentrifuge tubes as follows:
a. green tube CS (crime scene)
b. blue tube S1 (suspect 1)
c. orange tube S2 (suspect 2)
d. violet tube S3 (suspect 3)
e. pink tube S4 (suspect 4)
f. yellow tube S5 (suspect 5)
Label the tubes with your name, date, and lab period. Place the tubes in your
microcentrifuge tube rack.
3. Using a fresh tip for each sample, pipet 10 l of each DNA sample from the stock
tubes and transfer to the corresponding colored microcentrifuge tubes. Make sure the
sample is transferred to the bottom of the tubes.
4. Pipet 10 l of enzyme mix (ENZ) into the very bottom of each tube. Use a fresh
tip to transfer the ENZ sample to each tube. Pipet up and down carefully to mix well.
5. Tightly cap the tubes and mix the components by gently flicking the tubes with
your finger. If a microcentrifuge is available, pulse-spin in the centrifuge to collect all the
liquid in the bottom of the tube. Otherwise, gently tap the tube on the table top.
6. Incubate the tubes for 45 min at 37C or overnight at room temperature in a large
volume of water heated to 37C.
7. If required, follow the instructor's directions to pour a 1% agarose gel.
8. After the incubation period place the tubes in the refrigerator until the next
laboratory period. If there is sufficient time to continue, proceed directly to step 2 of
Lesson 2.
Agarose Gel Electrophoresis:
1. Remove the digested DNA samples from the refrigerator (if applicable).
2. If a centrifuge is available, pulse spin the tubes in the centrifuge to bring all of the
liquid into the bottom on the table top.
3. Using a separate tip for each sample, add 5 l of loading dye "LD" into each tube.
Cap the tubes and mix by gently flicking the tube with your finger. Collect the sample at
the bottom of the tube by tapping it gently on the table or by pulse-spinning in a
centrifuge.

Broberg, Figley, Smith, Stark 3

4. Remove the agarose gel from the refrigerator (if applicable) and remove the
plastic wrap.
5. Place the agarose gel in the electrophoresis apparatus. Fill the electrophoresis
chamber with 1x TAE buffer* to cover the gel, using approximately 275 ml of buffer for
a Bio-Rad Mini-Sub Cell, horizontal electrophoresis chamber.
6. Check that the wells of the agarose gels are near the black () electrode and the
bottom edge of the gel is near the red (+) electrode.
7. Using a separate tip for each sample, load the indicated volume of each sample
into 7 wells of the gel in the following order:
a. Lane 1: S, DNA size standard, 10 l
b. Lane 2: CS, green tube, 20 l
c. Lane 3: S1, blue tube, 20 l
d. Lane 4: S2, orange tube, 20 l
e. Lane 5: S3, violet tube, 20 l
f. Lane 6: S4, red tube, 20 l
g. Lane 7: S5, yellow tube, 20 l
8. Carefully place the lid on the electrophoresis chamber. The lid will attach to the
base in only one orientation. The red and black jacks on the lid of the horizontal
electrophoresis chambers will match with the red and black jacks on the base. Plug the
electrodes into the power supply, red to red and black to black.
9. Turn on the power and electrophorese your samples at 100 V for 20 minutes.
10. Turn off the power after the 20 minutes and remove the lid from the
electrophoresis apparatus.
11. Remove the gel from the apparatus and place in plastic tray. Pour Dye into tray
with gel and agitate for two minutes, or until gel is completely stained.
12. Pour excess dye back into beaker or container to be reused or stored for later
experimentation.
13. Wash gel under running water until the DNA fragments are clearly visible. This
will take time and the gel is fragile. Remain patient and if necessary leave gel to soak in
water bath to further remove stain.
14. Record results.
Results:
Qualitative Observations: The agarose gel is very fragile and soft. It tended to tear
along the edges of the rectangle as the gel was being washed. Once the gel had dried, it became
very hard and began to wrinkle.
Data Tables:
Table 1: Distance Traveled by DNA Fragments and Actual/Approximate Size of
Fragments
Lambda/H Size
indill
Standard

Crime

Scene

Suspect

Band

Distance
(mm)

Actual Size
(base pairs)

Distance
(mm)

Approximate
Size (base pairs)

Distance
(mm)

Approximate
Size (base pairs)

23

23,130

39

3,700

44

2,475

Broberg, Figley, Smith, Stark 4

2

27

9,916

43

2,600

58

1,400

30

6,557

64

1,250

62

1,300

36

4,361

45

2,322

48

2,027

Suspect

Suspect

Suspect

Suspect

Distance
(mm)

Approximate
Size (base
pairs)

Distance
(mm)

Approximate
Size (base
pairs)

Distance
(mm)

Approximate
Size (base
pairs)

Distance
(mm)

Approximate
Size (base
pairs)

43

2,600

39

3,700

43

2,600

44

2,400

51

1,700

42

2,700

59

1,300

48

2,027

57

1,450

64

1,250

73

900

59

1,200

74

815

Graphs:
Figure 1:

Discussion: Due to the nature of this lab there was no hypothesis and therefore there was no
evidence to either confirm or reject the hypothesis. In this lab the independent variable was the
size of the DNA fragments. This is shown because the smaller the fragment, the further it could
travel through the gel in the allotted time. The dependent variable was the distance that the
fragments had traveled. There were several circumstances that may have led to potential error.
The first is that sample 4 received double the normal amount of restriction enzyme than the rest
of the samples. Another is that the centrifuge may have been used incorrectly. Lastly the samples
and the restriction enzyme may not have mixed more a long enough amount of time for the two
to react. In this lab we were trying to determine which suspect had DNA at the crime scene
through the use of electrophoresis DNA fragment identification. Every sample of DNA was
fragmented by the restriction enzymes as shown by multiple fragments being represented in the
agarose gel. If the DNA had not been cut, the DNA would only be shown as a solid bar very
close to the original well that it was deposited in because it would be too large to move through
the gel. The DNA was cut into its fragments by the restriction enzymes mixed with the DNA
samples in this lab. These restriction enzymes read the DNA and locate specific locations with
certain nucleotides in order to cut them and create sticky ends. If two different DNA samples
were to be cut at the same place on their molecules, that would indicate that that was a site with

Broberg, Figley, Smith, Stark 5

the corresponding nucleotide sequence. Suspect 3 appears to have an almost identical cutting of
the DNA as that of the DNA sample located at the crime scene as shown in Table 1 with the
distance traveled for the CS being 39, 43, 64 and that of suspect 3 being 39, 42, 64. This slight
difference is most likely due to the rough estimation done during the interpretation. The
similarity between these two samples gives adequate evidence to suggest that suspect 3 was at
the scene of this specific crime.