DNA Fingerprinting in a Forensic Teaching Experiment

Authors: Wagoner, Stacy A., and Carlson, Kimberly A.

Source: The American Biology Teacher, 70(6)
Published By: National Association of Biology Teachers
URL: [Link]

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 O N L I N E I N Q U I R Y & I N V E S T I G AT I O N

DNA Fingerprinting in a Forensic Teaching Experiment

I
STACY A. WAGONER KIMBERLY A. CARLSON

n the 1970s, the basic techniques for DNA fingerprint- referred to as short tandem repeats (STRs) and the FBI uses a
ing, Southern Blot analysis and Restriction Fragment Length standard set of 13 of these for CODIS analysis and databanking
Polymorphism (RFLP), were developed (Rudin, 2002). In 1985, (NIST, 2007). The simplicity of this assay is that the primers for
Alec Jeffreys used RFLP as a way to identify individuals from PCR for the 13 loci can be mixed into one reaction, therefore
their DNA, a process he termed DNA fingerprinting (Jeffreys et giving rise to what is known as multiplex PCR. The specificity of
al., 1985). In 1985, Kary Mullis and colleagues from the Cetus these 13 loci lies in the fact that the odds that two individuals
Corporation first published the Polymerase Chain Reaction share the same DNA profile based on these loci is about one in
(PCR) method of replicating specific regions of DNA utilizing one billion (NIJ, 2002).
gene specific primers and specific thermocycler conditions (Saiki Today DNA fingerprinting is used in a multitude of ways,
et al., 1985). including paternity testing, identifying animal versus human
The combination of RFLP with PCR led to the possibility remains, rape cases, murder trials, historical cases, military dog
of identifying individual specific regions of DNA from limited tags, missing persons, and disease/health issues (Butler, 2001).
samples, such as a single hair shaft with an intact follicle left at Television programs, such as “CSI,” have pushed forensic science
a crime scene. This technology, termed DNA profiling, would and DNA fingerprinting into every household. While doing this,
allow detectives to link a suspect to a crime scene by using the field has been glamorized, exaggerated, and oversimplified.
his/her DNA fingerprint. The United Kingdom became the first This experiment was designed to provide students, in a
country to use DNA profiling to exonerate one suspect of rape classroom laboratory setting, a hands-on demonstration of the
and to convict another for the same crime in 1987 (Canadian steps used in DNA forensic analysis by performing DNA extrac-
National DNA Database, 2003; Burns, 2005). In 1989, the tion, DNA fingerprinting, and statistical analyses of the data. The
United States (U.S.) had its first case overturned because of PCR parameters were determined by using control human DNA
DNA evidence and the U.S. federal government began develop- and first doing the reactions individually. Next, the four forward
ing regulatory standards for DNA collection and handling pro- and reverse primers were mixed to make a multiplex primer
cedures. In 1992, the National Research Council deemed DNA mix and the parameters tested using control human DNA. Once
testing a reliable method to identify a criminal suspect, which the parameters were determined, the experimental DNA was
prompted the technology to rapidly enter the mainstream court extracted from pop cans, pop bottles, plastic spoons, cigarette
system. The Federal Bureau of Investigation (FBI) established butts, and cheek swabs and used in multiplex PCR reactions.
the National DNA Index System, enabling city, county, state, The resulting PCR products were analyzed by gel electrophoresis
and federal law enforcement agencies to compare DNA profiles and visualized utilizing a gel photodocumentation system. From
electronically in 1998 (Burns, 2005). This software program is the gel, the DNA fingerprint of the individual could be deter-
referred to as CODIS (COmbined DNA Index System) and con- mined and analyzed.
tains DNA profiles from convicted offenders, missing persons,
and unsolved crimes (FBI, 2006).
In 1994, DNA profiling was further advanced by performing
Materials
PCR to evaluate specific loci that are variable between individu- • used pop cans, pop bottles, spoons, and cigarette butts
als, including monozygotic twins (NIJ, 2002). These regions are • BuccalAmp™ DNA Extraction Kit with QuickExtract™
DNA Extraction Solution and Catch-All™ Sample
STACY A. WAGONER (swagoner3@[Link]) is a former student, and KIMBERLY
Collection Swabs (EPICENTRE®)
A. CARLSON (carlsonka1@[Link]) is Associate Professor of Biology, both • Microcon centrifugal filter devices (Amicon)
in the Department of Biology, University of Nebraska at Kearney, Kearney, • GoTaq® Green PCR Master Mix (Promega)
NE 68849.

DNA FINGERPRINTING 29
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 • 10X TBE (for 1 liter: 108 g Tris base, 55 g boric acid, 40 tion and the excess solution was squeezed out by pushing the
ml 0.5 M EDTA, pH 8.0) and 1X TBE buffer swab against the inside of a 1.5 ml microcentrifuge tube. For
• 0.9% saline solution (9 g NaCl in 1 L nanopure water) the cheek sample, an individual swished 0.9% saline solution in
his/her mouth for 10-15 seconds and spit the solution into a bea-
• 500 μg/mL ethidium bromide (Fisher) – working con- ker to be discarded. The cheek, pop bottle, pop can, and plastic
centration spoon were brushed with the swab at the locations the DNA was
• TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA) likely to be found. This was inside the cheek, at the mouth of the
• 5% Chelex solution (Sigma) bottle, the lip of the can, or the bowl of the spoon. They were
swabbed at least 20 times. The swab was dried at room tempera-
• 6X orange/blue loading dye (Promega) ture for 15 minutes and placed in a microfuge tube containing
• 90% and 70% ethanol Quick Extract™ DNA Extraction Solution, rotated five times, and
• Phenol Chloroform Isoamyl alcohol (PCI; Fisher) pushed against the inside of the microfuge tube to remove excess
liquid. The microfuge tube was vortexed, placed in a heating
• 3 M sodium acetate block at 65° C for one minute, vortexed again, returned to the
• thermocycler heating block at 98° C for two minutes, vortexed a third time,
• Mini-PROTEAN® II Gel Electrophoresis Unit and 4-15% and stored at -20° C.
Tris-HCl ready-made polyacrylamide gels (Bio-Rad) All DNA sample types (pop bottles, pop cans, plastic
• 1.5 ml microcentrifuge tubes spoons, and cheek cells) were further purified by adding
Phenol Chloroform Isoamyl alcohol (PCI) in a 1:1 ratio to each
• pipets and tips microfuge tube. PCI is highly toxic. Special care and handling
• vortex and centrifuge should be taken when using it. Working in a fume hood is
• heating blocks or water baths highly recommended. Each microfuge tube was vortexed and
centrifuged at 13,000 rpm for two minutes. The top aqueous
• sterile razor blade, forceps, 100 base pair ladder (Bio- layer containing the DNA was carefully transferred into new
Rad) microcentrifuge tubes and the bottom layer discarded. The DNA
• control human DNA was precipitated by adding 3 M sodium acetate (NaAc) in a 1:10
• gel electrophoresis equipment ratio and 1 ml of 90% ethanol (EtOH) to each microfuge tube.
The microfuge tubes were mixed by vortexing and placed at -20°
• platform shaker C for approximately 24 hours. DNA samples were centrifuged at
• gel documentation system 16,000 rpm for 20 minutes at 4° C, the supernatant discarded,
• primers (Vermaas & Rhoads, 2004; Invitrogen) and 1 ml of 70% EtOH added to the pellet to wash off any excess
salt. The DNA pellets were centrifuged for five minutes at 13,000
• D7S820 rpm, the supernatant discarded, and the DNA pellet dried. The
– Forward 5’ GTC ATA GTT TAG AAC GAA CTA DNA samples were placed with caps open in a 37° C incubator
ACG 3’ for 10-15 minutes, the DNA resuspended in 25 μl of TE buffer,
– Reverse 5’ CTG AGG TAT CAA AAA CTC GA GG and placed at -20° C for storage.
3’
• CSF1PO
DNA Extraction from Cigarette Butts
– Forward 5’ CTG AGT CTG CCA AGG ACT AGC 3’ DNA was extracted from cigarette butts following the pro-
tocol outlined in Hochmeister et al. (1991). In the laminar flow
– Reverse 5’ CAC ACA CCA CTG GCC ATC TTC 3’ hood using a sterile razor blade and forceps, three cross-sections
• Y-GATA-H4 approximately 3 mm wide were cut from the filter end of a
– Forward 5’ CCT AAG CAG AGA TGT TGG TTT TC cigarette butt. The cuttings were placed in a microfuge tube with
3’ 1 ml of 5% Chelex solution and vortexed for 30 seconds. The
microfuge tube was placed in a heating block at 56° C for 30
– Reverse 5’ CTG ATG GTG AAG TAA TGG AAT minutes, vortexed, boiled by heating to 100° C for eight minutes,
TAG 3’ vortexed again, and centrifuged at 13,000 rpm for five minutes to
• HUMTH01 pellet the Chelex. The supernatant was carefully transferred to
– Forward 5’ GTG GGC TGA AAA GCT CCC GAT a centrifugal filter tube and centrifuged at 4,000 rpm. The reten-
3’ tate was washed with 2 ml of TE buffer and was stored at -20°
C. The cigarette butt DNA was further purified by PCI extraction
– Reverse 5’ CAA AGG GTA TCT GGG CTC TGG 3’ and precipitated with NaAc and EtOH as described previously.

Methods PCR Amplification & Gel Electrophoresis

PCR reactions were prepared in the laminar flow hood
DNA Extraction from Pop Bottles, Pop Cans, with DNA extracted from U937 monocytic cells as a positive
Plastic Spoons & Cheek Cells control, without DNA as a negative control, and with DNA from
the cheek sample, cigarette butt, pop can, pop bottle, or plastic
DNA extraction was done in a laminar flow hood to avoid
spoons. The positive control and negative control reactions were
contamination from other sources, including the experimenter.
prepared with both the individual primer sets and with the
The manufacturer’s protocol (EPICENTRE®) was followed for
multiplex primer mix containing all four primer sets. The experi-
DNA extraction with the following modifications. A Catch-All
mental reactions were prepared with only the multiplex primer
Sample Collection Swab was dampened with 0.9% saline solu-
mix. These were all prepared by adding 12.5 μl GoTaq® Green

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 Master Mix, 5.0 μl of U937 DNA (0.1 μg/μl; positive
control), 5.0 μl of water (negative control) or 5.0 μl Figure 1. PCR performed with individual primer sets using U937 DNA. Lane 1
of experimental DNA, 2.0 μl of individual primers = 100 bp ladder; Lane 2 = CSF1PO + U937; Lane 3 = CSF1PO no-DNA control;
sets or multiplex primer mix (final concentration
of each primer is 2.5 μM), and 5.5 μl of water for a
Lane 4 = Y-GATA-H4 + U937; Lane 5 = Y-GATA-H4 no- DNA control; Lane 6 =
final reaction volume of 25 μl. All reactions were vor- HUMTH01 + U937; Lane 7 = HUMTH01 no-DNA control; Lane 8 = D7S820 +
texed, centrifuged, and placed in the thermocycler U937; Lane 9 = D7S820 no-DNA control. CSF1PO (Lane 2) shows a PCR product at
using the following parameters: ~320 bp (arrow), Y-GATA-H4 (Lane 4) shows a PCR product at ~ 390 bp (arrow),
• Initial denaturation: three minutes at 94° C HUMTH01 (Lane 6) shows PCR products at ~180 and 190 bp (arrows), and D7S820
• Denaturation: one minute at 94° C* shows a PCR product at ~215 bp (arrow). Bands without arrows are nonspecific
• Annealing: one minute at 58° C* PCR products.
• Extension: three minutes at 72° C*
• Repeating Steps 2–4(*): 30 cycles
• Hold: 4° C (modified from Vermaas &
Rhoads, 2004).
PCR was increased to 40 cycles for pop can
and pop bottle DNA samples, and to 50 cycles for
the cigarette butt DNA samples. The increase in
cycle numbers for these DNA samples was due to
the limited concentration of DNA recovered from
these sources. The lower the concentration of DNA,
the higher the number of cycles needed to achieve
adequate amplification to observe a PCR product
on the gel.
A four to fifteen percent Tris-HCl ready-made
polyacrylamide gel was pre-run at 130 V with 1X
TBE buffer for approximately 15 minutes. Marker
DNA was made by adding 2.0 μl of 6X blue/orange
loading dye, 5.0 μl TE buffer, and 3.0 μl of Bio-Rad Once the individual primer sets were tested, the four primer
100 base pair ladder. Ten microliters of either PCR product or sets were mixed together to perform a multiplex PCR. This was
marker was loaded into the gel and electrophoresed at 100 V first done using control human U937 DNA (Figure 2). The gel
for approximately one hour at room temperature. After the bot- shows successful amplification of three of the four primer sets
tom dye band had run through the gel, the gel was placed in at once. A ~390 bp product represents CSF1PO amplification,
ethidium bromide (final concentration = 0.5 μg/mL) diluted in two products at ~180 and ~190 bp represent HUMTH01, and a
water and placed on the shaker for 30 minutes. A picture was ~215 bp product represents D7S820. Amplification for Y-GATA-
taken using a gel photodocumentation system for comparison H4 was not detected. The reaction was attempted multiple times,
and analysis of the gel. but each time Y-GATA-H4 was not detected. This suggests that
Y-GATA-H4 primer annealing is being hindered by either one of
the other primer sets or by some other factor in the PCR reaction.
Results There were no bands present in the no-DNA negative controls as
Each individual primer set was initially tested using control expected. The products for CSF1PO, HUMTH01, and D7S820
human DNA extracted from U937 monocytic cells (Figure 1). on the multiplex PCR gel (Figure 2) coincide with the same size
Any human genomic DNA would work, but U937 DNA was read- products from the gel testing the individual primer sets (Figure
ily available in the lab. The gel shows:
• a PCR product at ~320 bp for
Table 1. STR loci utilized in multiplex PCR in this study. All loci are described in Vermaas and
CSF1PO (Lane 2)
Rhoads (2004) and NIST (2007). In addition, CSF1PO is described in Hammond et al. (1994),
• a ~390 bp product for Y-GATA-
H4 (Lane 4)
D7S820 is described in Jin et al. (1997), HUMTH01 is described in Edwards et al. (1991) and
Hammond et al. (1994), and Y-GATA-H4 is described in White et al. (1999).
• two products at ~180 and ~190
bp for HUMTH01 (Lane 6) Location on # of
STR Repeat Structure PCR Product (bp)
• a ~215 bp product for D7S820 Chromosome Repeats
(Lane 8).
CSF1PO 5q33.3-34 AGAT 6-16 291-331
These product sizes are within
the expected ranges for the loci tested D7S820 7q GATA 5-15 194-234
(Table 1). No PCR products were seen AATG = bottom strand;
in the no-DNA negative control lanes. HUMTH01 11p15-15.5 3-14 171-215
TCAT = top strand
Nonspecific PCR products were detect-
ed but were outside the expected range Y-GATA- (GATA)10GAATGGATAGATTA ~360-400
of sizes for the loci tested. Y Not defined
H4 (GATA)2 AATA(GATA)4 Not defined

DNA FINGERPRINTING 31
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 1). After positive amplification Figure 2. PCR performed with multiplexed primers using In this experiment, positive
was seen with control DNA and U937 DNA. Lane 1 = 100 bp ladder; Lane 2 = multiplexed amplification was seen as bands
three of the four primer sets in on the gel when testing the individ-
the multiplex primer set mix,
primers + U937 DNA; Lane 3 = no DNA control. PCR products ual primer sets and the multiplex
the cigarette butt, the pop can, for CSF1PO (~320bp), D7S820 (~215bp), and HUMTH01 primer mix with or without DNA
the pop bottle, the cheek cells, (~180 and 190bp) are marked with arrows. The ~390bp PCR in the reaction mix. This showed
and the plastic spoon (Figure 3) product for Y-GATA-H4 was not apparent. The bands without positive PCR amplification of tar-
were tested with the multiplex arrows are nonspecific PCR products. get STRs, the specific area of DNA
primer set mix. chosen to be amplified, in three of
The cigarette butt, pop can, the four loci tested. The absence
pop bottle, and spoon PCR reac- of PCR products in the no-DNA
tions all show one PCR product negative controls demonstrates
at ~325 bp for CSF1PO, one that there was no amplification
product at ~220 bp for D7S820, occurring in these reactions and
and two products at ~200 and that there was not DNA contami-
nation of the reagents used in
~185 bp for HUMTH01. For the the PCR reactions. This verifies
cheek PCR reaction, two prod-
ucts at ~330 and ~320 bp are that amplification of the target
detected for CSF1PO, one prod- gene sequences was specific and
uct at ~230 bp for D7S820, and did not produce spurious PCR
three products (tri-allelic expres- products. The approximate PCR
sion) at ~200, ~180, and ~170 product sizes for the individual
bp for HUMTH01. These prod- primer sets coincided with the
uct sizes are within the expected PCR products with the same size
ranges for the loci tested (Table from the multiplex PCR when
1). No PCR products were seen using the control DNA for all loci
in the no-DNA negative control except for Y-GATA-H4. This sug-
lanes. Nonspecific PCR products gests that more than one specific
were detected but were outside area of DNA can be successfully
of the expected range of sizes for amplified at a time and that all of
the loci tested.
The PCR products for the three loci are the Figure 3. Multiplex PCR for cigarette butt, spoon, pop bottle, pop can, and cheek
same size for the cigarette butt, pop can, pop bottle, DNA samples. Lane 1 = 100 bp ladder; Lane 2 = cigarette butt DNA (Subject 1);
and spoon reactions, showing that the DNA origi- Lane 3 = bottle DNA (Subject 1); Lane 4 = can DNA (Subject 1); Lane 5 = spoon
nated from one individual, whereas the cheek DNA
was extracted from a sample given by a different
DNA (Subject 1); Lane 6 = cheek DNA (Subject 2); Lane 7 = negative control. C =
individual. The use of DNA samples from two differ- CSF1PO PCR products; D = D7S820 PCR products; H = HUMTH01 PCR products. The
ent individuals was done to demonstrate that differ- bands without arrows are nonspecific PCR products.
ences in banding patterns can be detected using the
methodology described. In addition, the increase in
cycle numbers for cigarette butt DNA compared to
pop can, pop bottle, and spoon DNA from the same
individual demonstrates that increasing the cycle
numbers can increase the amplification of the target
gene products. These PCR products appear brighter
and more intense. In addition to Y-GATA-H4 ampli-
fication being inhibited in the multiplex primer
set mix, we would not expect it to be amplified in
these reactions as it is a male specific marker and
all of the DNA samples were derived from female
donors.

Discussion
Television today oversimplifies much of foren-
sic science. With this experiment, students are
exposed to the actual methods of DNA analysis
used in forensics by performing DNA extraction,
amplification by PCR, and gel electrophoresis in
a laboratory. From this information, the students
could compare “suspect” DNA to DNA found at the
crime scene to determine the identity of the DNA
fingerprint.

32 THE AMERICAN BIOLOGY TEACHER, ONLINE PUBLICATION, AUGUST 2008

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 the amplification products can be individually visualized. The at Kearney (UNK) Undergraduate Research Council, the UNK
lack of amplification of Y-GATA-H4 does not negate this finding. Biology Department, and the UNK College of Natural and Social
It does suggest that a different male specific STR should be used Sciences.
if deemed necessary.
The multiplex gels with DNA extracted from a pop bottle, References
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could also be extracted from other items such as blood, hair, Hochmeister, M.N., Budowle, B., Jung, J., Borer, U.V., Comey, C.T. &
or skin to show that an individual’s DNA is the same regardless Dirnhofer, R. (1991). PCR-based typing of DNA extracted from ciga-
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the myth portrayed in popular TV shows that obtaining DNA
evidence is a guarantee that the crime will be solved in an hour.

Acknowledgments
We thank two anonymous reviewers and Darby Carlson
for their helpful comments on improving this manuscript. This
project was granted Institutional Review Board approval (IRB
#071305-1) to develop the standard operating procedure to be
used as a laboratory exercise. This project was supported by an
undergraduate research grant from the University of Nebraska

DNA FINGERPRINTING 33
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