Crime Lab
A SCienceLab activity
Dr. Rekha C. Patel and Dr. Bert Ely
Department of Biological Sciences
University of South Carolina
715 Sumter St.
Columbia, SC 29208
patelr@sc.edu
ely@sc.edu
Additional Contributors: Brice Gill, Teresa Pizzuti, Karen Walton, and
Jonathon Singer
Background
DNA restriction analysis is a technique with applications in medicine, research,
and forensics. For example, in Dr. Patel’s research laboratory, we use restriction analysis
of DNA for many different reasons. One of the main uses is to create recombinant DNA
molecules. We use restriction enzymes to cut the genes of interest from the human
genome and clone them into plasmid vectors so that we can study their function at the
molecular level. Cloning involves splicing the gene we want into plasmid DNA. In this
way, we create chimeric DNA molecules, in which DNA obtained from two different
organisms is joined to give a single molecule. This new DNA molecule can be
transformed into a bacterial cell where it is replicated to produce many copies of the gene
under study. We work on genes whose expression is induced by interferons. Interferons
are antiviral and anticancer proteins produced in our bodies, and we are trying to find out
how they do their job by studying the way interferons affect gene expression.
In this laboratory activity, students will learn how DNA restriction analysis can
be used for forensic applications in this case to analyze evidence from a fictitious
crime scene. Restriction enzymes bind to specific sequences of bases and then cut the
DNA at that site. For example, restriction enzyme Apa L1 recognizes the sequence
GTGCAC cuts between the first G and the T. The DNA fragments resulting from a
restriction digest can be separated by size using agarose gel electrophoresis. Since
DNA fragments have a negative charge they are pulled through the gel by the
electric current. The gel is full of holes that are different sizes so it sorts the pieces of
DNA by size as they move toward the positive electrode. The larger the DNA
fragment, the more slowly it will pass through the gel since there will be a greater
number of holes that are too small for it to pass through. Therefore, the smaller
fragments will travel farther through the gel than the larger pieces.
Pre-Lab Activities
In preparation for the laboratory, students will work in groups to simulate DNA
restriction analysis using a DNA sequence written on paper. The paper sequence will be
cut at the restriction enzyme recognition site, and the resulting DNA fragments will be
placed on a diagram of a gel to simulate gel electrophoresis. This activity provides
students with the opportunity to investigate the use of restriction enzymes and gel
electrophoresis to see similarities and differences among DNA molecules. The
objectives are:
• To model DNA restriction analysis.
• To demonstrate how DNA restriction analysis can be used to identify DNA fragments.
•
To analyze how to analyze the results of a DNA restriction analysis.
Materials per team
•
A pairs of scissors
•
•
•
Five sets of base sequences ( paper DNA strips)
One roll of tape
A piece of poster paper
Pre-Lab Engagement (5 minutes)
Tell the students that they are now part of a forensics team and that their job is to solve
the crime on the basis of the evidence that will be given to them.
Pre-Lab Exercise (20 minutes)
Instruct each group to give a name to each of the suspect DNAs and then circle the Apa
LI restriction enzyme recognition sites (GTGCAC) on each DNA strip. Tell the
students to cut between the first G and the T of the recognition site and to label each
piece with the name given to that DNA. (Keeping all of the pieces labeled is an
important part of the exercise.) Provide each group with a piece of poster paper to serve
as a gel for displaying the DNA fragments according to their size. The DNA fragments
should be taped on the gel at positions corresponding to their size with all the fragments
that are cut from one DNA strip displayed in a single lane of the gel.
Pre-Lab Explanation (20-30 minutes)
Provide a "Final Report" handout to each group. After each group has finished putting the
DNA fragments on the chart, have each team fill out the Final Report Form. Lead a class
discussion about their conclusions, and the process they employed to obtain evidence
based on the DNA restriction analysis. Possible discussion questions include:
•
This process is often referred to as DNA fingerprinting. Why do you think this term is
used? (because different DNAs have their own unique pattern)
• Why use DNA as evidence? (because it is available and each persons DNA is unique
to them)
• What purpose do the restriction enzymes serve? (they cut specific DNA sequences)
• Does a match of the suspect DNA fragments with the crime DNA fragments mean the
suspect is guilty? Why or why not? (You need to do multiple DNA tests to get a
fingerprint that matches only one person. Also, the suspect may have left his DNA at
the crime scene before the crime was committed.)
Emphasize that the distinguishing characteristic of DNA is the sequence of nucleotide
bases. Note that the technique modeled does not involve sequencing the DNA. The
technique, DNA restriction analysis, provides indirect evidence that sequences of DNA
samples are the same or different from one another. If the restriction enzymes cut the
DNA samples into identical size fragments, the DNA samples are probably the same. If
the restriction enzymes cut the DNA samples into different size fragments, the DNA
samples are definitely different.
Assessment
Give an index card to each student. Have them describe in their own words what they
learned today and discuss briefly other applications for which DNA restriction analysis
could be used.
Predicted Results
Suspect 1:
12, 39, 9
Suspect 2:
29, 31
Suspect 3:
8, 24, 19, 13
Suspect 4:
20, 20, 24
Crime Scene: 20, 20, 24
Suspect 1
ATGGCTGAACAGTGCACTCCTCAGGCTTTGTATTTGAGCAATATGCGGAAGTGCACGAAG
TACCGACTTGTCACGTGAGGAGTCCGAAACATAAACTCGTTATACGCCTTCACGTGCTTC
Suspect 2
AAAACCATGCACCGATACACACTGGAAAGTGCACGAACTTGCCAGTTTTGTCCTCAGTTT
TTTTGGTACGTGGCTATGTGTGACCTTTCACGTGCTTGAACGGTCAAAACAGGAGTCAAA
Suspect 3
ATAGTGCACAGAACTCCAGAAGACATTGTGCACCCTACTAATGGGAGTGCACATCATTTT
TATCACGTGTCTTGAGGTCTTCTGTAACACGTGGGATGATTACCCTCACGTGTAGTAAAA
Suspect 4
CGGGAGATCATCCACGTGCACTCATCGACAGAAACGTGCACGCCACCCTGGAAAGCCAGA
GCCCTCTAGTAGGTGCACGTGAGTAGCTGTCTTTGCACGTGCGGTGGGACCTTTCGGTCT
Crime Scene
CGGGAGATCATCCACGTGCACTCATCGACAGAAACGTGCACGCCACCCTGGAAAGCCAGT
GCCCTCTAGTAGGTGCACGTGAGTAGCTGTCTTTGCACGTGCGGTGGGACCTTTCGGTCA
Suspect 1
ATGGCTGAACAGTGCACTCCTCAGGCTTTGTATTTGAGCAATATGCGGAAGTGCACGAAG
TACCGACTTGTCACGTGAGGAGTCCGAAACATAAACTCGTTATACGCCTTCACGTGCTTC
Suspect 2
AAAACCATGCACCGATACACACTGGAAAGTGCACGAACTTGCCAGTTTTGTCCTCAGTTT
TTTTGGTACGTGGCTATGTGTGACCTTTCACGTGCTTGAACGGTCAAAACAGGAGTCAAA
Suspect 3
ATAGTGCACAGAACTCCAGAAGACATTGTGCACCCTACTAATGGGAGTGCACATCATTTT
TATCACGTGTCTTGAGGTCTTCTGTAACACGTGGGATGATTACCCTCACGTGTAGTAAAA
Suspect 4
CGGGAGATCATCCACGTGCACTCATCGACAGAAACGTGCACGCCACCCTGGAAAGCCAGA
GCCCTCTAGTAGGTGCACGTGAGTAGCTGTCTTTGCACGTGCGGTGGGACCTTTCGGTCT
Crime Scene
CGGGAGATCATCCACGTGCACTCATCGACAGAAACGTGCACGCCACCCTGGAAAGCCAGT
GCCCTCTAGTAGGTGCACGTGAGTAGCTGTCTTTGCACGTGCGGTGGGACCTTTCGGTCA
DNA Instructions
1. Make up a name for each of your DNA samples. One sample is already labeled “Evidence”.
Turn the paper strip with the DNA base sequences over so the side with the bases is facing you.
Use your scissors (restriction enzymes are molecular scissors) to cut your DNA samples only
where you see this base pattern GTGCAC on the top strand. Cut between the first G and the T.
Label each of the pieces with the name of that DNA sample so that you can keep track of the
source of each fragment.
2. A base pair consists of two complementary bases. Record the number of base pairs in each
piece on the blank side of the DNA fragment.
3. Make a chart like the one below for your group. Tape your DNA sequences on the chart
according to the number of base pairs. Follow the example below.
Sample1 Sample 2 Evidence Sample 3 Sample 4 Size
30
29
28
27
26
////
5
4
3
2
1
Crime Lab Report
Names of investigators:
Name of the DNA matching the evidence:
Explain how you came to this conclusion.
Crime Lab Visit
During the visit to the University of South Carolina SCienceLab, students will have
the opportunity to perform a restriction enzyme digest and separate the resulting
DNA fragments by agarose gel electrophoresis. The class will be given a crime
scenario and asked how they can use DNA technology to determine which suspect’s
DNA matches the evidence.
I. Restriction Enzyme Digest
Add the two suspect DNAs and the DNA from the evidence to separate
microcentrifuge tubes containing water, buffer, and enzyme. Incubate the samples at
37°C for 60 minutes.
II. Gel Preparation
1) Add 50 ml of electrophoresis buffer to a flask containing 0.6 g of agarose.
III.
2)
Dissolve the agarose by boiling the mixture in a microwave oven. One of the
staff will melt the agarose for you and give it back to you when it has cooled
enough to use.
3)
Place the gel tray in the gel box so that the gaskets are pressed tight against
the sides of the gel box.
4)
Pour all 50 ml of melted agarose into the gel tray and place a comb in the
slot at one end to form the sample wells.
Prepare the gel electrophoresis box
1) After the gel hardens, turn the gel in the electrophoresis box so that the sample
wells are closer to the negative (black) pole.
2)
IV.
Slowly pour DNA electrophoresis buffer into the electrophoresis box. Fill the
electrophoresis box until the tops of the sample wells are just covered with
buffer.
Practice Loading Samples and running a gel
1) Practice loading food coloring into the wells of a test gel. Notice that it is
easier to keep the food coloring in the tubes labeled with a G (containing
glycerol) in the wells of the gel.
2) Put the lid in place and turn on the power. Set the voltage at 100 and watch
how the dyes migrate at different rates.
V.
Prepare and load the samples
1) Add 2 µl of loading dye to each of the DNA samples.
2) Carefully pipette all of the digested crime DNA and the two suspect’s
DNA into separate wells of the gel.
3) Connect the cables and run the gels at 100 volts until the blue portion of the
loading dye migrates about 30 mm from the wells.
VI.
See the DNA
Remove the gel from the electrophoresis box and place it on a light box to see the
DNA.
Interpretation
Results will vary since two different DNAs were used as the evidence. In either
case, one pattern of DNA bands resulting from the restriction analysis of the
suspects should match the pattern derived from the crime scene evidence. To
facilitate discussion, discuss a representative gel. Some sample questions include:
"What can be inferred from the results of the tests?" and "Can you presume guilt by
showing that the bands of DNA match after restriction analysis?"
Sample Preparation
A) DNA Sample Preparation
Pipette 0.5 µl of DNA (pBR322 or pUC19) into a microfuge tube containing 4.5 µl
of distilled water. Prepare one of each type per group as the two suspect DNAs.
Prepare the DNA for the evidence in the same manner. Half of the groups should get
pBR322 DNA as the DNA from the evidence and half should get pUC19 DNA.
B ) Restriction Enzyme Cocktail
Pipette 1 µl of NEB Buffer 4 and 0.5 µl of ApaL1 restriction enzyme into a
microfuge tube containing 3.5 µl of distilled water. Each group will need 3 tubes.
Laboratory Station Set-Up
• gel box with 2 combs and tray
• DNA electrophoresis buffer
• yellow tips
• micropipette
• used tip container
• tubes containing food coloring with and without added glycerol (labeled
with a G)
Data/Observation sheet
I. Restriction Digest
1. What is the function of the restriction enzyme?
2. Why are the samples put into the incubator?
II. Preparation of the Agarose Gel
1. What is the function of the agarose gel?
2. Predict what would happen if you used 0.06 g of agarose instead of 0.6 g. What effect would
that have on your experiment?
3. What is the function of the gel comb?
IV. Preparation of the Gel Electrophoresis Box:
1. Predict what would happen if you put the wells of the gel at the positive pole.
2. What is the function of the electrophoresis buffer?
V. Loading the Samples:
1. What would happen if you did not add dye to the samples?
2. Describe what is occurring in the gel when the electric current is applied.
VI. Results:
Draw a picture of the gel to record where you loaded each sample and the DNA bands that you
observed.
Data/Observation sheet with possible answers
I. Restriction Digest
1. What is the function of the restriction enzyme?
To cut the DNA at specific places in the DNA sequence
2. Why are the samples put into the incubator?
Because the restriction enzyme works better at 37 C
II. Preparation of the Agarose Gel
1. What is the function of the agarose gel?
To provide a way of separating the DNA fragments by size
2. Predict what would happen if you used 0.06 g of agarose instead of 0.6 g. What effect would
that have on your experiment?
The gel would not harden so you could not load your DNA samples
3. What is the function of the gel comb?
To make the sample wells
IV. Preparation of the Gel Electrophoresis Box:
1. Predict what would happen if you put the wells of the gel at the positive pole.
The DNA would go the wrong direction and migrate off of the gel
2. What is the function of the electrophoresis buffer?
To conduct the electricity
V. Loading the Samples:
1. What would happen if you did not add dye to the samples?
The dye makes the DNA heavy so that it does not float away
2. Describe what is occurring in the gel when the electric current is applied.
The DNA is pulled through the gel by the electric current.
Crime Lab Post-Laboratory Activities
THE GREAT DEBATE
SHOULD SOUTH CAROLINA HAVE A FORENSIC GENETIC
DATABANK?
Issues:
 Should all convicted criminals be included in the forensic genetic
database?
 Should everyone in the state be included in the forensic genetic
database?
 Should there be a forensic genetic database at all?
 When should a sample of DNA be taken? (At arrest or conviction).
Panel Discussion:
Choose one of the following roles and take the point of view you believe the
person you represent would actually take on the issues:







Public defender
Prosecutor
ACLU Spokesperson
SLED Investigator
Coroner
Mother
Civil Rights Protector
You Decide:
1. Explain the problem of deciding who should have access to genetic
test results.
2. Examine the pros and cons of keeping genetic test results private.
List reasons to maintain privacy. List reasons why test results should
be shared.
3. Create a list of rules to control access to genetic information. Who
should have access, and under what circumstances? Explain your
reasoning.
Crime Lab Post-Laboratory Activities
Deducing Family Relationships from Genetic Data
Short tandem repeat (STR or microsatellite) data are often used in paternity and missing persons
cases. Below you will find some actual STR data from 7 different people at 9 different loci. See
if you can deduce any family relationships among the subjects. Remember that each person gets
one allele from their mother and one from their father. Also, in a paternity case, if the child and
the possible father do not match at even one locus, then the father son relationship is false.
Person
M
Y
N
L
S
B
J
Sex
F
F
M
F
M
M
M
Age
76
57
36
37
38
20
16
1
15,16
15,15
15,18
15,16
15,18
14,15
15,15
2
16,18
17,17
17,17
17,17
16,18
15,17
16,17
3
22,26
21,22
22,23
21,22
21,22
22,24
21,22
4
13,14
13,15
12,13
11,13
11,13
11,13
11,11
Locus
5
29,33
31,32
27,31
32,33
28,31
32,33
31,33
6
14,14
13,18
12,18
14,18
12,17
14,21
14,17
7
11,12
9,11
9,12
11,11
11,13
11,13
11,11
8
12,13
10,14
12,14
9,14
12,12
9,12
9,12
9
7,8
9,11
11,12
7,11
8,11
7,10
7,11
Discussion of Family Relationships from STR Data
Based on age, you might hypothesize that M is the mother of Y. However, this hypothesis is
proven false by the data at locus 2, 5, 6, 8, and 9. In each case, M could not have provided one of
the alleles observed in Y. In fact, locus 2 indicates that M could not be the parent of anyone
except S and J. However, locus 5 excludes S and locus 4 excludes J. Thus, M is not the parent of
anyone on the list.
In contrast, the age and STR data are consistent with Y being the parent of N and L. At each
locus, one of the Y alleles matches one of the N alleles and one of the L alleles. Similarly, L
could be the mother of B and J. Also, S could be the father of J but not B.
Could L and S be the parents of J? In this case, J would have to have inherited one allele at each
locus from each of the presumptive parents. Careful inspection reveals that this condition did
occur. For instance, at locus one J would have inherited allele 15 from each of his parents. At
locus 2, J would have inherited a 16 from S and a 17 from L and so on. Thus, there is a good
chance that J is the son of L and S.
Crime Lab Post-Laboratory Activities
Cloning a short tandem repeat (STR) sequence
Short tandem repeat sequences mutate at a high frequency because they have a repeated
sequence that causes mistakes in DNA replication. The most common mistakes are to add or
delete a repeat unit (a GT in the sequence in this exercise). If you were easily able to sequence
the region containing the STR, you could accurately determine the number of repeats and also
determine if any other base changes were present in the population. You have a cloning vector
that contains a BamHI site. How could you clone a fragment of the sequence below that contains
the containing the STR?
1
AATCGGATGAATTCGCATTTAGCTGTTATGTAGATCTAATTGGGGCCCCAATTTGGCAAT
61 CGTGCAATCCCGGGTTAAGCTTTGTGTGTGTGTGTGTGTGTGTGTGTGACTTGAAAAATT
121 ACTTGGCATGCCTAGTGAATTCGGATACACTAGGACTAGGATCGAACCCGGGATTCCCC
181 TTTGATTAAAAGAGATCTTTAACCCAAGCTTTTACCAGTCATGACTGACCTTACCCATTC
Here are the restriction enzymes that you have available in the laboratory:
Enzyme
EcoRI
Site
G’AATTC BamHI
CTTAA’G
HindIII A’AGCTT BglII
TTCGA’A
SphI
Enzyme
Site
Enzyme
Site
G’GATCC
CCTAG’G
SmaI
CCC’GGG
GGG’CCC
A’GATCT
TCTAG’A
DraI
TTT’AAA
AAA’TTT
GCATG’C
C’GTACG
Procedure:
Find the STR sequence and mark it to highlight its position.
Find and label all the restriction sites that correspond to the enzymes listed above.
Devise a strategy for cloning a fragment of DNA containing the STR into a vector containing
a BamHI site.
Draw a diagram showing how the two sticky ends would fit together.
SOUTH CAROLINA SCIENCE STANDARDS MET BY THIS UNIT
UNIT OF STUDY: GENETICS
CLASS DESCRIPTION: HIGH SCHOOL
Science Standards addressed: (Day one and two)
I. Inquiry
II.a.1.d. Discuss uses of technologies that enable in-depth studies of the cell such as
microscopes, ultracentrifuge techniques, and radioscopy studies.
II.a.2.a. Explain the role of enzymes in chemical reactions within the cell.
II.B.1.d. Evaluate the impact of DNA technology on society (e.g., bioengineering, forensics,
genome project, DNA fingerprinting).
II.B.2.c. Discuss advancements in the study of heredity since Mendel including the chromosome
theory.
English and Social Studies Standards.
Objectives:
 Identify a need for DNA restriction analysis
 Model the concept of DNA restriction analysis.
 Apply DNA restriction analysis to the identification of DNA fragments.
 Work cooperatively to analyze the results of the DNA restriction analysis.
DAY ONE: Restriction Enzymes
Activity 1: Case of the Crown Jewels Dry Lab
DAY TWO:
Activity 2: Visit to the University Crime Lab
DAY THREE:
Activity 3: Follow-up from University Crime Lab visit to include one or more debates or Socratic
Discussion.
DAY FOUR:
Activity 4: Cloning
SOUTH CAROLINA SCIENCE STANDARDS MET BY THIS UNIT
UNIT OF STUDY: GENETICS
CLASS DESCRIPTION: MIDDLE SCHOOL
Science Standards addressed: (Day one and two)
I. Inquiry
II.c.4.e. Discuss advantages and disadvantages of selective breeding, genetic engineering, and
biomedical research.
II.c.3.c. Analyze how traits are passed from parents to offspring through pairs of genes.
English and Social Studies Standards.
Objectives:
 Identify a need for DNA restriction analysis
 Model the concept of DNA restriction analysis.
 Apply DNA restriction analysis to the identification of DNA fragments.
 Work cooperatively to analyze the results of the DNA restriction analysis.
DAY ONE: Restriction Enzymes
Activity 1: Case of the Crown Jewels Dry Lab
DAY TWO:
Activity 2: Visit to the University Crime Lab
DAY THREE:
Activity 3: Follow-up from University Crime Lab visit to include one or more debates or Socratic
Discussion.
DAY FOUR:
Activity 4: Cloning