DescriptionDNA Forensics lab
Week 1
Week 2
Week 3
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: https://doi.org/10.1662/0002-7685(2008)70[29:DFIAFT]2.0.CO;2
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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 fingerprinting, Southern Blot analysis and Restriction Fragment Length
Polymorphism (RFLP), were developed (Rudin, 2002). In 1985,
Alec Jeffreys used RFLP as a way to identify individuals from
their DNA, a process he termed DNA fingerprinting (Jeffreys et
al., 1985). In 1985, Kary Mullis and colleagues from the Cetus
Corporation first published the Polymerase Chain Reaction
(PCR) method of replicating specific regions of DNA utilizing
gene specific primers and specific thermocycler conditions (Saiki
et al., 1985).
The combination of RFLP with PCR led to the possibility
of identifying individual specific regions of DNA from limited
samples, such as a single hair shaft with an intact follicle left at
a crime scene. This technology, termed DNA profiling, would
allow detectives to link a suspect to a crime scene by using
his/her DNA fingerprint. The United Kingdom became the first
country to use DNA profiling to exonerate one suspect of rape
and to convict another for the same crime in 1987 (Canadian
National DNA Database, 2003; Burns, 2005). In 1989, the
United States (U.S.) had its first case overturned because of
DNA evidence and the U.S. federal government began developing regulatory standards for DNA collection and handling procedures. In 1992, the National Research Council deemed DNA
testing a reliable method to identify a criminal suspect, which
prompted the technology to rapidly enter the mainstream court
system. The Federal Bureau of Investigation (FBI) established
the National DNA Index System, enabling city, county, state,
and federal law enforcement agencies to compare DNA profiles
electronically in 1998 (Burns, 2005). This software program is
referred to as CODIS (COmbined DNA Index System) and contains DNA profiles from convicted offenders, missing persons,
and unsolved crimes (FBI, 2006).
In 1994, DNA profiling was further advanced by performing
PCR to evaluate specific loci that are variable between individuals, including monozygotic twins (NIJ, 2002). These regions are
STACY A. WAGONER (swagoner3@cox.net) is a former student, and KIMBERLY
A. CARLSON (carlsonka1@unk.edu) is Associate Professor of Biology, both
in the Department of Biology, University of Nebraska at Kearney, Kearney,
NE 68849.
referred to as short tandem repeats (STRs) and the FBI uses a
standard set of 13 of these for CODIS analysis and databanking
(NIST, 2007). The simplicity of this assay is that the primers for
PCR for the 13 loci can be mixed into one reaction, therefore
giving rise to what is known as multiplex PCR. The specificity of
these 13 loci lies in the fact that the odds that two individuals
share the same DNA profile based on these loci is about one in
one billion (NIJ, 2002).
Today DNA fingerprinting is used in a multitude of ways,
including paternity testing, identifying animal versus human
remains, rape cases, murder trials, historical cases, military dog
tags, missing persons, and disease/health issues (Butler, 2001).
Television programs, such as “CSI,” have pushed forensic science
and DNA fingerprinting into every household. While doing this,
the field has been glamorized, exaggerated, and oversimplified.
This experiment was designed to provide students, in a
classroom laboratory setting, a hands-on demonstration of the
steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analyses of the data. The
PCR parameters were determined by using control human DNA
and first doing the reactions individually. Next, the four forward
and reverse primers were mixed to make a multiplex primer
mix and the parameters tested using control human DNA. Once
the parameters were determined, the experimental DNA was
extracted from pop cans, pop bottles, plastic spoons, cigarette
butts, and cheek swabs and used in multiplex PCR reactions.
The resulting PCR products were analyzed by gel electrophoresis
and visualized utilizing a gel photodocumentation system. From
the gel, the DNA fingerprint of the individual could be determined and analyzed.
Materials
• used pop cans, pop bottles, spoons, and cigarette butts
• BuccalAmp™ DNA Extraction Kit with QuickExtract™
DNA Extraction Solution and Catch-All™ Sample
Collection Swabs (EPICENTRE®)
• Microcon centrifugal filter devices (Amicon)
• GoTaq® Green PCR Master Mix (Promega)
DNA FINGERPRINTING
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29
• 10X TBE (for 1 liter: 108 g Tris base, 55 g boric acid, 40
ml 0.5 M EDTA, pH 8.0) and 1X TBE buffer
• 0.9% saline solution (9 g NaCl in 1 L nanopure water)
• 500 μg/mL ethidium bromide (Fisher) – working concentration
• TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA)
• 5% Chelex solution (Sigma)
• 6X orange/blue loading dye (Promega)
• 90% and 70% ethanol
• Phenol Chloroform Isoamyl alcohol (PCI; Fisher)
• 3 M sodium acetate
• thermocycler
• Mini-PROTEAN® II Gel Electrophoresis Unit and 4-15%
Tris-HCl ready-made polyacrylamide gels (Bio-Rad)
• 1.5 ml microcentrifuge tubes
• pipets and tips
• vortex and centrifuge
• heating blocks or water baths
• sterile razor blade, forceps, 100 base pair ladder (BioRad)
• control human DNA
• gel electrophoresis equipment
• platform shaker
• gel documentation system
• primers (Vermaas & Rhoads, 2004; Invitrogen)
• D7S820
– Forward 5’ GTC ATA GTT TAG AAC GAA CTA
ACG 3’
– Reverse 5’ CTG AGG TAT CAA AAA CTC GA GG
3’
• CSF1PO
– Forward 5’ CTG AGT CTG CCA AGG ACT AGC 3’
– Reverse 5’ CAC ACA CCA CTG GCC ATC TTC 3’
• Y-GATA-H4
– Forward 5’ CCT AAG CAG AGA TGT TGG TTT TC
3’
– Reverse 5’ CTG ATG GTG AAG TAA TGG AAT
TAG 3’
• HUMTH01
– Forward 5’ GTG GGC TGA AAA GCT CCC GAT
3’
– Reverse 5’ CAA AGG GTA TCT GGG CTC TGG 3’
Methods
DNA Extraction from Pop Bottles, Pop Cans,
Plastic Spoons & Cheek Cells
DNA extraction was done in a laminar flow hood to avoid
contamination from other sources, including the experimenter.
The manufacturer’s protocol (EPICENTRE®) was followed for
DNA extraction with the following modifications. A Catch-All
Sample Collection Swab was dampened with 0.9% saline solu-
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tion and the excess solution was squeezed out by pushing the
swab against the inside of a 1.5 ml microcentrifuge tube. For
the cheek sample, an individual swished 0.9% saline solution in
his/her mouth for 10-15 seconds and spit the solution into a beaker to be discarded. The cheek, pop bottle, pop can, and plastic
spoon were brushed with the swab at the locations the DNA was
likely to be found. This was inside the cheek, at the mouth of the
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 temperature for 15 minutes and placed in a microfuge tube containing
Quick Extract™ DNA Extraction Solution, rotated five times, and
pushed against the inside of the microfuge tube to remove excess
liquid. The microfuge tube was vortexed, placed in a heating
block at 65° C for one minute, vortexed again, returned to the
heating block at 98° C for two minutes, vortexed a third time,
and stored at -20° C.
All DNA sample types (pop bottles, pop cans, plastic
spoons, and cheek cells) were further purified by adding
Phenol Chloroform Isoamyl alcohol (PCI) in a 1:1 ratio to each
microfuge tube. PCI is highly toxic. Special care and handling
should be taken when using it. Working in a fume hood is
highly recommended. Each microfuge tube was vortexed and
centrifuged at 13,000 rpm for two minutes. The top aqueous
layer containing the DNA was carefully transferred into new
microcentrifuge tubes and the bottom layer discarded. The DNA
was precipitated by adding 3 M sodium acetate (NaAc) in a 1:10
ratio and 1 ml of 90% ethanol (EtOH) to each microfuge tube.
The microfuge tubes were mixed by vortexing and placed at -20°
C for approximately 24 hours. DNA samples were centrifuged at
16,000 rpm for 20 minutes at 4° C, the supernatant discarded,
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
rpm, the supernatant discarded, and the DNA pellet dried. The
DNA samples were placed with caps open in a 37° C incubator
for 10-15 minutes, the DNA resuspended in 25 μl of TE buffer,
and placed at -20° C for storage.
DNA Extraction from Cigarette Butts
DNA was extracted from cigarette butts following the protocol outlined in Hochmeister et al. (1991). In the laminar flow
hood using a sterile razor blade and forceps, three cross-sections
approximately 3 mm wide were cut from the filter end of a
cigarette butt. The cuttings were placed in a microfuge tube with
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
minutes, vortexed, boiled by heating to 100° C for eight minutes,
vortexed again, and centrifuged at 13,000 rpm for five minutes to
pellet the Chelex. The supernatant was carefully transferred to
a centrifugal filter tube and centrifuged at 4,000 rpm. The retentate was washed with 2 ml of TE buffer and was stored at -20°
C. The cigarette butt DNA was further purified by PCI extraction
and precipitated with NaAc and EtOH as described previously.
PCR Amplification & Gel Electrophoresis
PCR reactions were prepared in the laminar flow hood
with DNA extracted from U937 monocytic cells as a positive
control, without DNA as a negative control, and with DNA from
the cheek sample, cigarette butt, pop can, pop bottle, or plastic
spoons. The positive control and negative control reactions were
prepared with both the individual primer sets and with the
multiplex primer mix containing all four primer sets. The experimental reactions were prepared with only the multiplex primer
mix. These were all prepared by adding 12.5 μl GoTaq® Green
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
of experimental DNA, 2.0 μl of individual primers
sets or multiplex primer mix (final concentration
of each primer is 2.5 μM), and 5.5 μl of water for a
final reaction volume of 25 μl. All reactions were vortexed, centrifuged, and placed in the thermocycler
using the following parameters:
• Initial denaturation: three minutes at 94° C
• Denaturation: one minute at 94° C*
• Annealing: one minute at 58° C*
Figure 1. PCR performed with individual primer sets using U937 DNA. Lane 1
= 100 bp ladder; Lane 2 = CSF1PO + U937; Lane 3 = CSF1PO no-DNA control;
Lane 4 = Y-GATA-H4 + U937; Lane 5 = Y-GATA-H4 no- DNA control; Lane 6 =
HUMTH01 + U937; Lane 7 = HUMTH01 no-DNA control; Lane 8 = D7S820 +
U937; Lane 9 = D7S820 no-DNA control. CSF1PO (Lane 2) shows a PCR product at
~320 bp (arrow), Y-GATA-H4 (Lane 4) shows a PCR product at ~ 390 bp (arrow),
HUMTH01 (Lane 6) shows PCR products at ~180 and 190 bp (arrows), and D7S820
shows a PCR product at ~215 bp (arrow). Bands without arrows are nonspecific
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
100 base pair ladder. Ten microliters of either PCR product or
marker was loaded into the gel and electrophoresed at 100 V
for approximately one hour at room temperature. After the bottom dye band had run through the gel, the gel was placed in
ethidium bromide (final concentration = 0.5 μg/mL) diluted in
water and placed on the shaker for 30 minutes. A picture was
taken using a gel photodocumentation system for comparison
and analysis of the gel.
Results
Each individual primer set was initially tested using control
human DNA extracted from U937 monocytic cells (Figure 1).
Any human genomic DNA would work, but U937 DNA was readily available in the lab. The gel shows:
• a PCR product at ~320 bp for
CSF1PO (Lane 2)
• a ~390 bp product for Y-GATAH4 (Lane 4)
• two products at ~180 and ~190
bp for HUMTH01 (Lane 6)
• a ~215 bp product for D7S820
(Lane 8).
These product sizes are within
the expected ranges for the loci tested
(Table 1). No PCR products were seen
in the no-DNA negative control lanes.
Nonspecific PCR products were detected but were outside the expected range
of sizes for the loci tested.
Once the individual primer sets were tested, the four primer
sets were mixed together to perform a multiplex PCR. This was
first done using control human U937 DNA (Figure 2). The gel
shows successful amplification of three of the four primer sets
at once. A ~390 bp product represents CSF1PO amplification,
two products at ~180 and ~190 bp represent HUMTH01, and a
~215 bp product represents D7S820. Amplification for Y-GATAH4 was not detected. The reaction was attempted multiple times,
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.
There were no bands present in the no-DNA negative controls as
expected. The products for CSF1PO, HUMTH01, and D7S820
on the multiplex PCR gel (Figure 2) coincide with the same size
products from the gel testing the individual primer sets (Figure
Table 1. STR loci utilized in multiplex PCR in this study. All loci are described in Vermaas and
Rhoads (2004) and NIST (2007). In addition, CSF1PO is described in Hammond et al. (1994),
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).
STR
Location on
Chromosome
Repeat Structure
# of
Repeats
PCR Product (bp)
CSF1PO
5q33.3-34
AGAT
6-16
291-331
D7S820
7q
GATA
5-15
194-234
HUMTH01
11p15-15.5
AATG = bottom strand;
TCAT = top strand
3-14
171-215
Y-GATAH4
Y
(GATA)10GAATGGATAGATTA
(GATA)2 AATA(GATA)4
Not defined
~360-400
Not defined
DNA FINGERPRINTING
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31
1). After positive amplification
was seen with control DNA and
three of the four primer sets in
the multiplex primer set mix,
the cigarette butt, the pop can,
the pop bottle, the cheek cells,
and the plastic spoon (Figure 3)
were tested with the multiplex
primer set mix.
Figure 2. PCR performed with multiplexed primers using
U937 DNA. Lane 1 = 100 bp ladder; Lane 2 = multiplexed
primers + U937 DNA; Lane 3 = no DNA control. PCR products
for CSF1PO (~320bp), D7S820 (~215bp), and HUMTH01
(~180 and 190bp) are marked with arrows. The ~390bp PCR
product for Y-GATA-H4 was not apparent. The bands without
arrows are nonspecific PCR products.
The cigarette butt, pop can,
pop bottle, and spoon PCR reactions all show one PCR product
at ~325 bp for CSF1PO, one
product at ~220 bp for D7S820,
and two products at ~200 and
~185 bp for HUMTH01. For the
cheek PCR reaction, two products at ~330 and ~320 bp are
detected for CSF1PO, one product at ~230 bp for D7S820, and
three products (tri-allelic expression) at ~200, ~180, and ~170
bp for HUMTH01. These product sizes are within the expected
ranges for the loci tested (Table
1). No PCR products were seen
in the no-DNA negative control
lanes. Nonspecific PCR products
were detected but were outside
of the expected range of sizes for
the loci tested.
The PCR products for the three loci are the
same size for the cigarette butt, pop can, pop bottle,
and spoon reactions, showing that the DNA originated from one individual, whereas the cheek DNA
was extracted from a sample given by a different
individual. The use of DNA samples from two different individuals was done to demonstrate that differences 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 amplification 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.
In this experiment, positive
amplification was seen as bands
on the gel when testing the individual primer sets and the multiplex
primer mix with or without DNA
in the reaction mix. This showed
positive PCR amplification of target STRs, the specific area of DNA
chosen to be amplified, in three of
the four loci tested. The absence
of PCR products in the no-DNA
negative controls demonstrates
that there was no amplification
occurring in these reactions and
that there was not DNA contamination of the reagents used in
the PCR reactions. This verifies
that amplification of the target
gene sequences was specific and
did not produce spurious PCR
products. The approximate PCR
product sizes for the individual
primer sets coincided with the
PCR products with the same size
from the multiplex PCR when
using the control DNA for all loci
except for Y-GATA-H4. This suggests that more than one specific
area of DNA can be successfully
amplified at a time and that all of
Figure 3. Multiplex PCR for cigarette butt, spoon, pop bottle, pop can, and cheek
DNA samples. Lane 1 = 100 bp ladder; Lane 2 = cigarette butt DNA (Subject 1);
Lane 3 = bottle DNA (Subject 1); Lane 4 = can DNA (Subject 1); Lane 5 = spoon
DNA (Subject 1); Lane 6 = cheek DNA (Subject 2); Lane 7 = negative control. C =
CSF1PO PCR products; D = D7S820 PCR products; H = HUMTH01 PCR products. The
bands without arrows are nonspecific PCR products.
Discussion
Television today oversimplifies much of forensic 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.
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the amplification products can be individually visualized. The
lack of amplification of Y-GATA-H4 does not negate this finding.
It does suggest that a different male specific STR should be used
if deemed necessary.
at Kearney (UNK) Undergraduate Research Council, the UNK
Biology Department, and the UNK College of Natural and Social
Sciences.
The multiplex gels with DNA extracted from a pop bottle,
pop can, cigarette butt, plastic spoon, and cheek swab also show
successful amplification of target STRs as seen by the presence
of PCR products that fall within the expected size range for
the loci as shown in Table 1. Since the PCR product sizes for
CSF1PO, HUMTH01, and D7S820 for the cigarette butt, pop
can, pop bottle, and spoon samples were estimated to be the
same size, we can deduce that they are from one individual. This
is in contrast to the PCR product sizes for these three loci from
the cheek sample, which was from a different individual. The
comparison shows that the multiplex PCR protocol can be used
to distinguish between individuals.
References
DNA could be extracted from more individuals to further
exemplify the differences in the DNA fingerprints. DNA as a
molecular fingerprint for each individual could be discussed, as
well as how DNA fingerprinting is utilized in different areas such
as the criminal justice system, military dog tags, organ donor
compatibility, and species genome projects. Since the DNA
extracted in this experiment was primarily from saliva, DNA
could also be extracted from other items such as blood, hair,
or skin to show that an individual’s DNA is the same regardless
of the source. To differentiate between females and males, a different male specific STR marker could be used and put into the
multiplex PCR primer set.
As a teaching experiment in a laboratory setting, a mock
crime scene could be set up so that students could collect samples for DNA extraction from items such as blood on a shirt, the
rim of a ball cap, or a used cigarette. The DNA could be extracted
from these samples and each student could also extract his/her
own DNA utilizing the cheek swab method. Amplification and
gel electrophoresis could be done comparing the PCR products
to determine if one of the students committed the crime. The
activity teaches students how long the process of DNA fingerprinting takes. Connecting a DNA fingerprint to the perpetrator
of a crime is not something that can be done in minutes or even
hours, as portrayed on television. Many times in real-life crime
scenes, there is no known match to the DNA tested and the
crime becomes a “cold case” until a match in CODIS is made.
This is not popular opinion based on television shows but, in
reality, it is a common occurrence of which students should be
aware. These are a few of the topics that could be discussed. If
the instructor wants to incorporate probability testing into the
laboratory exercise, the DNA profiles can be used to determine
allele frequencies and to perform Hardy-Weinberg analysis. This
laboratory exercise demonstrates how DNA fingerprinting is
performed and how long it takes. It also shows that DNA fingerprinting is a useful tool in forensic science. Lastly, it debunks
the myth portrayed in popular TV shows that obtaining DNA
evidence is a guarantee that the crime will be solved in an hour.
Burns, K.S. (2005). Criminal Investigations, DNA & Forensic Science.
Available online at: http://www.karisable.com/crdna1.htm.
Butler, J.M. (2001). Overview and history of DNA typing. Forensic DNA
Typing: Biology and Technology Behind STR Markers, 1st Edition.
Burlington, MA: Elsevier Academic Press.
Canadian National DNA Database. (2003). Forensic history of DNA:
highlights. Available online at: http://www.nddb-bndg.org/foren_
history_e.htm.
Edwards, A., Civitello, A., Hammond, H.A. & Caskey, C.T. (1991). DNA
typing and genetic mapping with trimeric and tetrameric tandem
repeats. American Journal of Human Genetics, 49, 746-756.
Federal Bureau of Investigation (FBI). (2006). CODIS: Combined DNA
Index System. Available online at: http://www.fbi.gov/hq/lab/
codis/index1.htm.
Hammond, H.A., Jin, L., Zhong, Y., Caskey, C.T. & Chakraborty, R.
(1994). Evaluation of 13 short tandem repeat loci for use in personal identification applications. American Journal of Human Genetics,
55, 175-189.
Hochmeister, M.N., Budowle, B., Jung, J., Borer, U.V., Comey, C.T. &
Dirnhofer, R. (1991). PCR-based typing of DNA extracted from cigarette butts. International Journal of Legal Medicine, 104, 229-233.
Jeffreys, A.J., Wilson, V. & Thein, S.L. (1985). Individual-specific “fingerprints” of human DNA. Nature, 316, 76-9.
Jin, L., Underhill, P.A., Buoncristiani, M.R., & Robertson, J.M. (1997).
Defining microsatellite alleles by genotyping global indigenous
human populations and non-human primates. Journal of Forensic
Science, 42, 496-499.
National Institutes of Justice (NIJ). (2002). Using DNA to solve
cold cases. Available online at: http://www.ncjrs.gov/pdffiles1/
nij/194197.pdf.
National Institute of Standards and Technology (NIST). (2007). Short
tandem repeat DNA internet database. Available online at: http://
www.cstl.nist.gov/div831/strbase/index.htm.
Rudin, N. (2002). A short history of DNA typing. An Introduction to
Forensic DNA Analysis, 2nd Edition. Boca Raton, FL: CRC Press.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A.
& Arnheim, N. (1985). Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science, 230, 1350-1354.
Vermaas, W. & Rhoads, D. (2004). Experiment IV: short tandem repeats
DNA profiling. Available online at: http://photoscience.la.asu.edu/
Photosyn/courses/BIO_343/lab/Experiment-IV.html.
White, P.S., Tatum, O.L., Deaven, L.L. & Longmire, J.L. (1999). New,
male-specific microsatellite markers from the human Y chromosome. Genomics, 57, 433-437.
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
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33
DNA Forensics Lab
Instructions on How to Use OligoAnalyzer
OligoAnalyzer is a free bioinformatics tool used to analyze DNA primers. You will be using it to
analyze the primers you have created in this lab. This tool calculates the physical characteristics
of any oligo sequence, including length, GC content, melting temperature (Tm) and molecular
weight.
1. Access OligoAnalyzer at https://www.idtdna.com/calc/analyzer. You would have to create a
free account to access the tool. The home screen will look as follows:
2. Enter your sequence into the “Sequence” box in the 5’ to 3’ orientation. The following DNA
primer sequence will be used in this tutorial: CGGGAAGGGAACAGGAGTAAG.
Note: to get the correct Tm, it is important to enter the Mg2+ and dNTP concentrations you will use
in your experiment. We will only focus on the understanding the fundamentals of primer design of
this lab and will use SpecSheet settings for all calculations.
1
3. Click on the “Analyze” button (colored in orange) to calculate physical characteristics of DNA
your primer. Sample results of this calculations are shown below.
X
2
4. Click on the “Hairpin” button to calculate thermodynamic parameters of your DNA primer
sequence.
The Gibbs free energy (ΔG) value gives an indication of the strength of the secondary structure.
IDT recommends the ΔG value be more than –9 for self-dimers and hetero-dimers. Sample
thermodynamic values and hte resulting hairpin structure are shown below:
3
5. Click on the “Homo-Dimer Analysis” button to predict all homo-dimers that could possibly
form and calculate their free energies. A sample output of this calculation is shown below:
Note: homo-dimers form between two identical DNA primer sequences.
6. Find another student in the class whose primer has a similar Tm. Create a multiplex of two
primers by checking them against each other via the “Hetero-Dimer Analysis” tool. Using the
remaining time of this lab, make adjustments to make the best multiplex of two primers. Share
the results with the lab section.
4
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Neg Control multiplex AG/CG
Experimental DNA Multiplex
AC/CG
U937 DNA Multiplex
AC/CG
Extracted DNA multiplex Kate/Ellie
Negative control multiplex Sierra/Hunter/Nicole
U937 multiplex Sierra/Hunter/Nicole
Experimental multiplex Sierra/Hunter/Nicole
Experimetnal DNA multiplex GCW/CC
U937 multiplex ECW
Experimental DNA multiplex ECW
Negative control Multiplex ECW
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Experimental DNA Multiplex Dylan/Josi
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Neg Control Multiplex Collin Perry
U937 CSF1Po
Exp DNA multiplex
Exp DNA CSF1PO
U937 multiplex
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U937 CSF1PO
CET
Ladder
Neg Control CSF1PO CET
Exp CSF1PO CET
U937 D75820
ME
Neg Control D75820 ME
Exp DNA D75820 ME
U937 CSF1PO KAL
Neg control CSF1PO KAL
Exp DNA CSF1PO
KAL
Exp DNA HUMTH01 ——– A10
Neg Control HUMTH01 ——— A11
U937 HUMTH01 ———– A12
Neg control Y-Gata-H4
C1
Exp DNA Y-Gata-H4
C2
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Neg Control Y-GATA-H$ —B1
U937 Y-GATAH4 — —- B2
Exp DNA Y-GATA H4 ——- B3
Exp HumTH01 ———- B4
U937 DNA Overflow of Ladder Sorry — B5
Ladder
Negative Control HUMTH01 ———- B6
U937 D7s820—— B7
Neg control D7S820—— B8
Exp DNA D7S820 —– B9
Exp DNA Y-Gata ———- B10
U937 Y-GATA——— B11
Neg Control Y-GATA H4 ——B12
U937 Y-GATAH$———- C3
LABORATORY EXPERIMENT
pubs.acs.org/jchemeduc
Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the
Forensic Laboratory
Kelly M. Elkins*
Chemistry Department and Criminalistics Program, Metropolitan State College of Denver, Denver, Colorado 80217-3362,
United States
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bS Supporting Information
ABSTRACT: The polymerase chain reaction (PCR) is a common experiment in upper-level
undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and
thermocyclers have become more affordable for institutions. Typically, instructors design PCR
primers to amplify the region of interest and the students prepare their samples for PCR and analyze
the results. However, primers can also be designed in undergraduate laboratories with students at
this level. In a course that focuses on forensic DNA molecular biology for forensic chemistry
students, students have used the Applied Biosystems AmpFlSTR SGM Plus kit that amplifies DNA
at eleven regions in a single test tube. It is important for forensic chemistry students to be able to
design and analyze a single set of primers and, more importantly, create multiplexes of primers. This
enables students to more fully understand how the primers and the kits that are routinely employed
by the crime laboratories function. Creating a single set of primers does not demonstrate the extent
of design and engineering inherent in creating multiplexes or adequately prepare students for
research and careers in the field. The in silico method described herein uses free bioinformatics tools
and results in student-designed multiplexes for Combined DNA Index System (CODIS) loci. Sample student data are shown.
KEYWORDS: Upper-Division Undergraduate, Biochemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Internet/WebBased Learning, Problem Solving/Decision Making, Biotechnology, Forensic Chemistry, Molecular Biology, Nucleic Acids/DNA/RNA
T
he polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry and molecular biology courses as reagents and thermocyclers have
become more affordable for institutions. There are numerous
reports of the use of PCR in undergraduate laboratories with a
forensic application. For example, PCR can be used to amplify a
369 801 base pair D1S80 locus of the human genome,1 to
amplify hypervariable regions of DNA from dog hair and saliva,2
to amplify a 440 base pair hypervariable region of human mtDNA
from a simulated crime scene,3 and to amplify a 192 base pair
DNA segment present in genetically modified foods.4 In the
undergraduate lab, PCR has also been demonstrated to differentiate bacterial species,5 to genotype the normal variation in
human color vision,6 to evaluate a metabolic polymorphism,7 in
diagnostics,8 and to test for genetically modified organisms in
foodstuffs.9,10 In all of these examples, the instructors designed
the experiments and created and secured the primers, and the
students prepared their PCR samples using the procedure as
written.
In the second semester of a two-semester forensic science
course that focuses on DNA molecular biology for forensic
chemistry students, students employ the Applied Biosystems
AmpFlSTR SGM Plus multiplex kit that amplifies DNA at 11
regions (D21S11, FGA, TH01, vWA, D8S1179, D18S51,
D3S1358, D19S433, D16S539, D2S1338, and the gender marker, amelogenin) including nine of the thirteen short tandem
repeat (STR) loci of the U.S. Combined DNA Index System
(CODIS) (all of the above except D19S433 and D2S1338) for
Copyright r 2011 American Chemical Society and
Division of Chemical Education, Inc.
PCR and DNA typing (Table 1). Most U.S. crime laboratories
utilize 16-plex kits for human casework for sample completion so
that the data can be entered into CODIS, as necessary. It is
important for forensic chemistry students to be able to employ a
kit for PCR and mimic crime lab work and, more importantly, to
design and analyze a single set of primers and create primer
multiplexes. This enables students to more fully understand how
the primers and the kits that are routinely employed by the crime
laboratories function and extends their research skills.
Primers can be designed in undergraduate laboratories with
students at this level.11 14 In a single three-hour laboratory
session, students design PCR primers in silico using free bioinformatics tools available on the World Wide Web and collaborate
with a peer to create a multiplex PCR primer set for two loci.
Creating only a single set of primers does not demonstrate the
extent of design and engineering inherent in creating multiplexes
or adequately prepare students for jobs in forensics or even
modern clinical testing in medicine.
’ MATERIALS AND METHODS
This study was approved by Metro State’s Institutional Review
Board (IRB)—Human Subjects Review Committee (HSRC),
and the students consented to participate in the study and to the
release of the anonymous data shown here.
Published: August 10, 2011
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LABORATORY EXPERIMENT
Table 1. CODIS STR Loci by NCBI Accession Number and Repeata
a
Data from ref 20.
Table 2. Sample PCR Primer Results
Figure 1. Genome sequence for TPOX CODIS locus region on human
chromosome 2 thyroid peroxidase (hTPO) reference sequence and
published validated forward and reverse primers (arrows and bold)
producing a 64 base pair product.
This in silico lab does not require specialized materials,
expensive chemicals, or extensive preparation. A reliable Internet
connection and student laptops or computers (via a computer or
department lab) are required. The utilized Web sites include
NCBI,15 IDT’s OligoAnalyzer,16 NCBI Primer-Blast,17 and
NIST’s STRbase.18
An example of the type of data collected and evaluated in this
lab is presented to the students during the prelab lecture. The
primers shown as an example (TPOX locus) are used in the same
course in a subsequent laboratory (real-time PCR amplification)
(Figure 1 and Table 2),19 and amplify a nonvariable portion of
the locus and student-designed primers from this lab. Students
are shown how to navigate to the Web sites and locate a STR
repeat. The differences between the forward and reverse primers
(both 50 to 30 but located on the plus and minus strands,
respectively) and the STR repeat region for a sample locus from
NCBI (Table 1) are highlighted prior to inputting the primers in
the analysis Web sites, collecting the output to be saved in a text
file, and creating multiplexes.
Working in alone or in pairs, the students obtain the DNA
sequence for a CODIS STR locus (Table 1)20 from the National
Center for Biotechnology Initiative (NCBI)15 using mini-Mac
computers in one of the department computer laboratories. After
locating the STR repeat (Figure 2), they initially try to create the
primers from the 50 region directly upstream from the repeats
and the region directly downstream for the 30 complement using
the given guidelines for optimal primers including the following:
40 60% guanine cytosine (GC) content, melting temperatures (Tm) within 5 °C, less than four identical base repeats,
only weak primer dimer and hairpin formation, a length between
18 30 bases, and overall Tm values in the range of 50 72 °C.
The amplicon should not exceed 500 base pairs. The primers are
evaluated in Integrated DNA Technologies’ (IDT) OligoAnalyzer 3.116 by pasting the primer sequence into the sequence box
and clicking on the Analyze, Hairpin: Submit and Self-Dimer
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Figure 2. Genome sequence for TH01 CODIS locus region on
human chromosome 11 tyrosine hydroxylase sequence, AATG
(named for 30 strand) STR repeat (underlined), and student designed
forward and reverse primers (arrows and bold) producing an 87
base pair product given the 9 repeats observed in the NCBI
reference sequence (or 90 base pair product using K562 with the 9.3
repeat allele).
LABORATORY EXPERIMENT
boxes on the right. The defaults are used for all calculations. The
Hetero-Dimer: Calculate calculation is used to evaluate the two
primers together. Then the primer sequences are submitted to
NCBI Primer-Blast (Homo sapiens)17 to evaluate specificity by
entering the NCBI accession number in the PCR template box
and the designed primers in the forward and reverse primer boxes
under primer parameters. The students also indicated the primer
region for the NCBI accession number by base number in the
boxes to the right of the PCR template box and the expected
product size below the primer sequence boxes.
After the students completed a reasonable single primer set
(Table 3), the students paired themselves or their groups to
create a multiplex of the two sites by first locating other students
with similar melting temperatures for their primers. The multiplex primers were checked against each other in OligoAnalyzer
using the Hetero-Dimer function. The students made adjustments to the primers to create their best multiplex using the
remaining lab time or out of class while recording all attempts in
the text file (Table 3).
In a follow-up laboratory experiment, the students tested the
primers they designed experimentally using real-time PCR19 in a
Table 3. Sample Student PCR Primer Multiplex Results
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Table 4. Student Designed STR PCR Primers
a
Size based upon NCBI sequence. b Size based upon K562 DNA
sequence (both alleles indicated).
25 μL reaction. The students amplified human K562 DNA
(using 1 ng total in the 25 μL reaction) (Promega, Madison,
WI) using the published and validated TPOX primers21 and their
designed primers (Table 4) (purchased from IDT, Coralville, IA)
(1 μL each of 5 μM primers) in a real-time PCR experiment
conducted on a Bio-Rad (Hercules, CA) iQ5 instrument using
the iQ5 software (v. 2.0) and 12.5 μL of 2 iQ SYBR Green
Supermix (Bio-Rad, Hercules, CA) and nuclease-free water to
total 25 μL on a 96-well plate according to the following protocol
for a gradient plate: an initial 3 min denaturation at 95 °C and 40
cycles of denaturation at 95 °C for 15 s using an annealing extension gradient at 50 65 °C for 60 s. A melting curve
was produced by conducting 91 cycles of 30 s of melting every
0.5 °C from 50 to 95 °C. The SYBR Green I excitation
wavelength is 497 nm, and the emission wavelength is 522 nm.
Three primer sets from classes of previous years were also tested.
A total of ten primer sets were evaluated. The production of the
single reaction and multiplex PCR products was evaluated by the
shape of the melt peaks from real-time PCR and post-PCR
agarose gel electrophoresis using a 2% gel run in 1 TAE
(40 mM Tris-acetate, 1 mM EDTA, pH 8.0) (National Diagnostics, Atlanta, GA) using the DNA Logic (Lambda Biotech, St.
Louis, MO) and Lonza (Basel, Switzerland) 20 bp (base pair)
ladders. Upon completion, digital photographs were recorded of
each gel upon illumination under UV light using a digital
photography system equipped with a SYBR filter.
Hazards
No chemicals are needed, and there are no intrinsic chemical
hazards associated with this laboratory experiment as it is
performed entirely on a computer. For the follow-up lab, SYBR
Green I is a weak mutagen and may be an intercalating agent and
should be handled with care. Gloves, goggles, long pants, and
closed-toed shoes should be worn at all times in the laboratory.
The digital camera should be set to UV vis safety at all times,
and gel pictures should be recorded with the illuminator door in
the closed position.
’ RESULTS
Sample results for the example TPOX primers are shown in
Table 2. The TPOX PCR primers amplify a nonvariable, 64 base
LABORATORY EXPERIMENT
pair amplicon of the Homo sapiens thyroid peroxidase on
chromosome 2 (NCBI Accession number: NG_011581) from
bases numbered 81262 81325 (inclusive) (Figure 1). An analysis of these primers yields that the primer length varies by one
base pair, the GC content varies by 7.1%, the Tm vary by 0.2 °C,
and the best self-dimers and hairpins were 2 3 base pairs. The 30
primer Tm had the lower GC content, but the overall Tm was
almost identical to that of the 50 primer due to the one base
increase in length. The hairpins and self-dimers exhibited sufficiently low melting temperatures as compared to the annealing
temperature of the primer to the template. The best heterodimer
formed is four base pairs, which is more than optimal (three or
less). The primers were determined to be specific for only this
region in the human genome.
Students work individually or in pairs in the lab. Each student or
group of students chooses a different locus from the thirteen
CODIS loci (Table 1). After inputting the accession number and
locating the STR repeats from the NCBI nucleotide file for a
CODIS locus (Table 1), the students evaluate the 50 region
directly upstream from the repeats as a potential 50 primer and
the complementary region directly downstream for the 30 complement. This is advantageous for forensic use as the recovered DNA
is often degraded by environmental exposure, arson, or mass
disaster events and shorter DNA segments are more easily
amplified (e.g., mini-STRs). Then the students use currently
available Web-based bioinformatics programs for single and multiplex primer evaluation including OligoAnalyzer 3.1 and PrimerBlast. OligoAnalyzer outputs the melting temperature, primer dimer and hairpin formation, percent GC content, sequence
length, and heterodimer formation. Primer-Blast indicates for
which sequences the primer pairs are specific in a human genome
database (or other selected database). As the goal of this laboratory
is to teach the students the basics of primer and multiplex design,
automated primer design servers are not employed (e.g., Primer 3)
although these are well suited to the research and teaching setting
and are reported for use in a related lab experiment.14
Sample student results from the Web-based analysis for their
designed STR primers, TH01 and D16S539, and sample multiplex
results are given in Table 3. The designed primers indicate that the
students successfully completed the in silico assignment as most of
the data falls within the set criteria. The primers were determined
to be specific for only those specific regions in the human genome
and were 24 base pairs or less in length. The heterodimer pairs for
the TH01 forward primer had a higher than desirable level of six
heterodimer pairs, and the 50 primer multiplex of the opposite loci
had a higher than desirable level of five heterodimer pairs. The
observed hairpins in the primers have sufficiently low melting
temperatures as to be denatured at the annealing temperature
(e.g., 40.8 °C or less). The melting temperatures for the multiplex
primers were within 5.5 °C. Although all students had sufficient lab
time to test a few iterations of their primers and experiment with
shifts in frame and region, the students did not have sufficient lab
time to evaluate all possible primer sequences or to test multiple
multiplexes and make multiple improvements. However, they
were able to consider the challenges in editing the primers to
better the multiplex by comparing to the time already spent to
solve the problem.
All of the student-designed primers from the class are shown in
Table 4. The expected amplicon is shown for each in base pairs
for both the NCBI sequence and the K562 genotype. In a
subsequent laboratory using real-time PCR, students tested the
ability of their primers to amplify human K562 DNA. Five of the
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Journal of Chemical Education
seven primer pairs produced the expected amplicon as assayed by
a post-PCR 2% agarose gel. Additionally, three of the primer pairs
designed by students in the classes of the previous years were
tested, and all produced the expected amplicons. Of the ten
primer sets evaluated, eight successfully amplified DNA (an 80%
success rate). The students also tested a few potential mutliplexes
based on having varied amplicon sizes that could be diffentiated
on the gel. The three multiplexes tested in class (D5S818:
D13S317, D7S820:D5S818, and D7S820:D13S317) provided
only low-level amplification; two of the multiplexes used one of
the primer sets that failed to work in a single PCR reaction
(D13S317). Subsequently four more multiplexes (TH01 with
D16S539, D7S820, D8S1179, or D5S818) were tested using only
primers that amplified the DNA successfully in a single reaction
and that could be differentiated based on size. Three of these
multiplexes produced the predicted DNA amplicons for both
primer sets (including TH01:D16S539, TH01:D7S820, and
TH01:D5S818) although the TH01 amplicon was preferentially
amplified in all cases at the concentrations used. The D8S1179
amplicon is at the upper limits for PCR and too long for real-time
PCR with a two-step reaction.
This follow-up lab is not inexpensive: primer pairs per student
cost approximately $15 each without a dye label (SYBR Green I
was used for detection of the amplicon) and, if desired, $60 80
per primer with a dye label plus shipping for the lot. The primers
were purchased from IDT, and the total cost was $132.01 for the
seven unlabeled primer sets for the current years’ class. The realtime PCR cost was approximately $4.50 per reaction; numerous
reactions were run including one replicate each for each of the
temperatures in the annealing temperature gradient, negative
controls, and multiplexes. (The total cost much exceeded the
collected lab fees collected by the college from the seven students
for the course).
’ ASSESSMENT
In an anonymous survey, student feedback for the laboratory
was positive: students were excited about designing the primers
to better understand how they work and being able to order and
experiment with them in the follow-up real-time PCR laboratory.
Students noted that an understanding of the background through
lecture and the prelab lecture was important. They appreciated
the opportunity to learn how to create primers using the Web
tools and gained an appreciation in multiplex primer design and
felt the concepts would have been confusing without performing
the lab. The majority of the students responded that creating the
primers was more difficult than expected, but predominately that
the data analysis was easier than expected or as easy or difficult as
expected. Students noted that they learned that the process can
be very time-consuming including finding the repeats and pairing
the primers.
’ DISCUSSION
The students follow the instructions contained in the syllabus
for writing concise weekly lab reports; that is, students complete
sections including the title, preparer, partners, date, objective,
method, data, analysis and results, sample calculations, discussion
and answers to lab questions, and a conclusion. Working
individually or in pairs, the students obtained the sequence of a
CODIS STR region and created primers for a single CODIS
locus and analyzed the primers using two free bioinformatics
tools. Subsequently, two students or student groups were merged
LABORATORY EXPERIMENT
and the students compared the primers of the individual unique
loci and evaluated if they could be used simultaneously in a
multiplex, single tube PCR reaction or if changes would be
necessary. Students then suggested and tried alterations to the
first-generation primers to create a suitable multiplex. Multiplexing is an important tool in forensics. This lab is invaluable for
students pursuing molecular biology and forensics as bachelorlevel chemists or biologists and those continuing with postgraduate education.
This lab is compatible with our budgetary constraints as the
fixed costs of the mini-Mac computers had already been paid by
the department and the cost of Internet access is borne by the
students of the college. This laboratory exercise requires no
expensive molecular biology reagents for the in silico portion (in
stark contrast to the commercial multiplex DNA typing kits) and
is highly accessible to instructors at all institutions, regardless of
funding. The PCR primer design multiplexing laboratory experiment could easily be extended to other species and non-CODIS
loci. The follow-up laboratory in which students tested the
designed primers using real-time PCR was expensive, but the
students were enthusiastic about the opportunity to evaluate
their work experimentally. Students were especially pleased to
determine that most of their primers amplified the DNA and that
some of their multiplexes were successful.
’ ASSOCIATED CONTENT
bS Supporting Information
A student handout consisting of background information and
instructions and instructions for the instructor. This material is
available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: kelkins1@mscd.edu.
’ ACKNOWLEDGMENT
ACS Project SEED is gratefully acknowledged for a summer
stipend for Ain Ealey who tested revisions of this lab in the summer
of 2009 and provided useful suggestions. I wish to thank my
students in the spring 2011 CHE 3710 Criminalistics II course for
trying the improved version of the experiment including experimentally testing their primers: Benjamin Bender, Audrionna
Kingsley, Hannah Leger, Megan Owens, Tiffany Stone, and
Francesca Garcia Wheeler. I wish to thank my students in the
spring 2009 CHE 3710 Criminalistics II course for their suggestions for improvement: Patrick Bevins, Sarah Bonsall, Lindsay
Christopherson Powis, Kali Gipson, Natalie Hernandez, Raelynn
Kadunc, Jeffrey Minutillo, Carrie Melanson, Tanya Mokelki,
Michelle Montoya, and Zachary Morin. I wish to thank my
students in the spring 2008 CHE 3710 Criminalistics II course
for their patience, cooperation and input in the inaugural running
of this lab: Katelin Arnold, Jennifer Auger, Lydia Benyam, Megan
Jones McDowell, Jacqueline Keller, Erin Knopka, Gina Mann,
Susan McLaughlin, Andrea Moore, Melanie Newman, Stephanie
Sauter, Kurt Smith, Nia Travers, and Nikole Whitsitt.
’ REFERENCES
(1) Jackson, D. D.; Abbey, C. S.; Nugent, D. J. Chem. Educ. 2006,
83, 774–776.
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LABORATORY EXPERIMENT
(2) Carson, T. M.; Bradley, S. Q.; Fekete, B. L.; Millard, J. T. J. Chem.
Educ. 2009, 86, 376–378.
(3) Millard, J. T.; Pilon, A. M. J. Chem. Educ. 2003, 80, 444–446.
(4) Taylor, A.; Sajan, S. J. Chem. Educ. 2005, 82, 597–598.
(5) Suwanjinda, D.; Eames, C.; Panbangred, W. Biochem. Mol. Biol.
Educ. 2007, 35, 364–369.
(6) Lubin, I. M.; Wilson, B.; Grant, K. B. J. Chem. Educ. 2003,
80, 1289–1291.
(7) Childs-Disney, J. L.; Kauffmann, A. D.; Poplawski, S. G.; Lysiak,
D. R.; Stewart, R. J.; Arcadi, J. K.; Dinan, F. J. J. Chem. Educ. 2010,
87, 1110–1112.
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28, 223–226.
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Biochem. Mol. Biol. Educ. 2002, 30, 51–55.
(10) Brinegar, C.; Levee, D. Biochem. Mol. Biol. Educ. 2004,
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(11) Kim, T. D. Biochem. Mol. Biol. Educ. 2000, 28, 274–276.
(12) Lima, A. O. S.; Garc^es, S. P. S. Biochem. Mol. Biol. Educ. 2006,
34, 332–337.
(13) Bornhorst, J. A.; Deibel, M. A.; Mulnix, A. B. Biochem. Mol. Biol.
Educ. 2004, 32, 173–182.
(14) Thornton, B.; Basu, C. Biochem. Mol. Biol. Educ. 2011, 39,
145–154.
(15) National Center for Biotechnology Initiative. http://www.ncbi.
nlm.nih.gov/ (accessed June 2011).
(16) Integrated DNA Technologies’ OligoAnalyzer 3.1. http://
www.idtdna.com/analyzer/Applications/OligoAnalyzer/ (accessed
June 2011).
(17) NCBI Primer-Blast. http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed June 2011).
(18) Masibay, A.; Mozer, T. J.; Sprecher, C. J. Forensic Sci. 2000, 45,
1360-1362, http://www.cstl.nist.gov/strbase/promega_primers.htm
(accessed June 2011).
(19) Elkins, K. M.; Kadunc, R. E. J. Chem. Educ. 2011, in press.
(20) Butler, J. M. Forensic DNA Typing, 2nd ed.; Elsevier: Burlington,
MA, 2005; pp 1 660.
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Maddox, L. O. J. Forensic Sci. 2006, 51, 758–765.
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Committee on Professional Training
Preparing a Research Report
A research experience provides undergraduates a problem-solving activity unlike anything else in the curriculum.
It provides exposure to research methodology and an opportunity to work closely with a faculty advisor. It usually
requires the use of advanced concepts, a variety of experimental techniques, and state-of-the-art instrumentation.
Ideally, undergraduate research should focus on a well-defined project that stands a reasonable chance of
completion in the time available. A literature survey alone is not a satisfactory research project. Neither is
repetition of established procedures. The Committee on Professional Training (CPT) strongly supports efforts by
departments to establish active and vibrant undergraduate research programs, recognizing the role that research
can play in developing a wide range of student skills. The 2015 guidelines allow for the use of undergraduate
research both as in-depth coursework, as well as a means of meeting 180 of the 400 laboratory hours required for
certification provided that a well-written, comprehensive, and well-documented research report is prepared at the
end of a project (samples of such research reports must be submitted with the periodic reports.) The CPT has a
separate supplement outlining the components of successful research programs and projects.
Preparation of a comprehensive, well-documented and appropriately referenced written research report is an
essential part of a valid research experience, and the student should be aware of this requirement at the outset of
the project. Interim reports may also be required, usually at the termination of the quarter or semester. Sufficient
time should be allowed for satisfactory completion of reports, taking into account that initial drafts should be
critiqued by the faculty advisor and corrected by the student at each stage. It may be expected that concrete
outcomes of any research project would be student presentation of research results at a professional meeting
and/or co-authorship on a journal publication. While desirable outcomes, they are not a substitute for a wellwritten comprehensive report that demonstrates that the student has a full grasp of the scope of the problem, the
techniques/instrumental methods used, and the ramifications of the results generated (much as might be expected
for a capstone paper or a B.S. thesis). The student report should receive substantive critique and correction by
the faculty mentor in its development.
Guidelines on how to prepare a professional-style research report are not always routinely available. For this
reason, the following information on report writing and format is provided to be helpful to undergraduate
researchers and to faculty advisors. Much of what follows is similar to what authors would find in many ‘guidelines
to authors’ instructions for most journal submissions.
The most comprehensive student research reports examined by CPT have been those student reports reviewed
by more faculty than just the supervising research advisor. In some cases, programs require an approval of the
report by several faculty members; in such cases, student research reports are often of high quality.
Organization of the Research Report
Most scientific research reports, irrespective of the field, parallel the method of scientific reasoning. That is: the
problem is defined, a hypothesis is created, experiments are devised to test the hypothesis, experiments are
conducted, and conclusions are drawn. The exact format of scientific reports is often discipline dependent with
variations in order and content. The student is encouraged to adopt the format that is most appropriate to the
discipline of the research. Many journals offer a formatting template to aid the author. One example of such a
framework is as follows:
•
•
•
•
•
•
•
•
Title
Abstract
Introduction
Experimental Details or Theoretical Analysis
Results
Discussion
Conclusions and Summary
References
Title and Title Page
The title should reflect the content and emphasis of the project described in the report. It should be as short as
possible and include essential key words.
The author’s name (e.g., Mary B. Chung) should follow the title on a separate line, followed by the author’s
affiliation (e.g., Department of Chemistry, Central State College, Central, AR 76123), the date, and possibly the
origin of the report (e.g., In partial fulfillment of a Senior Thesis Project under the supervision of Professor Danielle
F. Green, June, 1997).
All of the above could appear on a single cover page. Acknowledgments and a table of contents can be added as
preface pages if desired.
Abstract
The abstract should concisely describe the topic, the scope, the principal findings, and the conclusions. It should
be written last to accurately reflect the content of the report. The length of abstracts varies but seldom exceeds
200 words.
A primary objective of an abstract is to communicate to the reader the essence of the paper. It should provide
sufficient information to describe the important features of the project in the absence of the rest of the document.
The reader will then be the judge of whether to read the full report or not. Were the report to appear in the primary
literature, the abstract would serve as a key source of indexing terms and key words to be used in information
retrieval. Author abstracts are often published verbatim in Chemical Abstracts.
Introduction
“A good introduction is a clear statement of the problem or project and the reasons for studying it.” (The ACS
Style Guide. American Chemical Society, Washington, DC, 2006.)
The nature of the problem and why it is of interest should be conveyed in the opening paragraphs. This section
should describe clearly but briefly the background information on the problem, what has been done before (with
proper literature citations), and the objectives of the current project. A clear relationship between the current
project and the scope and limitations of earlier work should be made so that the reasons for the project and the
approach used will be understood.
Experimental Details, Computation Procedures, or Theoretical Analysis
This section should describe what was actually done. It is a succinct exposition of the laboratory and
computational details, describing procedures, techniques, instrumentation, special precautions, characterization of
compounds and so on. It should be sufficiently detailed that other experienced researchers would be able to
repeat the work and obtain comparable results.
-2-
In theoretical reports, this section would include sufficient theoretical or mathematical analysis to enable
derivations and numerical results to be checked. Computer programs from the public domain should be cited.
New computer programs should be described in outline form.
If the experimental section is lengthy and detailed, as in synthetic work, it can be placed at the end of the report so
that it does not interrupt the conceptual flow of the report. Its placement will depend on the nature of the project
and the discretion of the writer.
Results
In this section, relevant data, observations, and findings are summarized. Tabulation of data, equations, charts,
and figures can be used effectively to present results clearly and concisely. Schemes to show reaction sequences
may be used here or elsewhere in the report.
Discussion
The crux of the report is the analysis and interpretation of the results. What do the results mean? How do they
relate to the objectives of the project? To what extent have they resolved the problem? Because the “Results”
and “Discussion” sections are interrelated, they can often be combined as one section.
Conclusions and Summary
A separate section outlining the main conclusions of the project is appropriate if conclusions have not already
been stated in the “Discussion” section. Directions for future work are also suitably expressed here.
A lengthy report, or one in which the findings are complex, usually benefits from a paragraph summarizing the
main features of the report – the objectives, the findings, and the conclusions.
The last paragraph of text in manuscripts prepared for publication is customarily dedicated to acknowledgments.
However, there is no rule about this, and research reports or senior theses frequently place acknowledgments
following the title page.
References
Thorough, up-to-date literature references acknowledge foundational work, direct the reader to published
procedures, results, and interpretations, and play a critical role in establishing the overall scholarship of the report.
The report should include in-text citations with the citations collated at the end of the report and formatted as
described in The ACS Style Guide or using a standard established by an appropriate journal. The citation process
can be facilitated by using one of several available citation software programs. In a well-documented report, the
majority of the references should come from the primary chemical literature. Because Internet sources are not
archival records, they are generally inappropriate as references for scholarly work. They should be kept to a bare
minimum.
Preparing the Manuscript
The personal computer and word processing have made manuscript preparation and revision a great deal easier
than it used to be. It is assumed that students will have access to word processing and to additional software that
allows spelling to be checked, numerical data to be graphed, chemical structures to be drawn, and mathematical
equations to be represented. These are essential tools of the technical writer. All manuscripts should be carefully
proofread before being submitted. Preliminary drafts should be edited by the faculty advisor (and/or a supervising
committee) before the report is presented in final form.
-3-
Useful Texts
Writing the Laboratory Notebook, Kanare, H.M., American Chemical Society, Washington, DC, 1985.
This book describes among other things the reasons for note keeping, organizing and writing the notebook with
examples, and provides photographs from laboratory notebooks of famous scientists.
rd
ACS Style Guide: Effective Communication of Scientific Information, Coghill, A.M., Garson, L.R.; 3 Edition,
American Chemical Society, Washington, DC, 2006.
This volume is an invaluable writer’s handbook in the field of chemistry. It contains a wealth of data on preparing
any type of scientific report and is useful for both students and professional chemists. Every research laboratory
should have a copy. It gives pointers on the organization of a scientific paper, correct grammar and style, and
accepted formats in citing chemical names, chemical symbols, units, and references.
There are useful suggestions on constructing tables, preparing illustrations, using different fonts, and giving oral
presentations. In addition, there is a brief overview of the chemical literature, the way in which it is organized and
how information is disseminated and retrieved. A selected bibliography of other excellent guides and resources to
technical writing is also provided. See also The Basics of Technical Communicating. Cain, B.E.; ACS
Professional Reference Book American Chemical Society: Washington, DC, 1988.
Write Like a Chemist, Robinson, M.S., Stoller, F.L., Costanza-Robinson, M.S., Jones, J.K., Oxford University
Press, Oxford, 2008.
This book addresses all aspects of scientific writing. The book provides a structured approach to writing a journal
article, conference abstract, scientific poster and research proposal. The approach is designed to turn the
complex process of writing into graduated, achievable tasks.
Last revised in August 2015
-4-
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Ladder
Neg Control multiplex AG/CG
Experimental DNA Multiplex
AC/CG
U937 DNA Multiplex
AC/CG
Extracted DNA multiplex Kate/Ellie
Negative control multiplex Sierra/Hunter/Nicole
U937 multiplex Sierra/Hunter/Nicole
Experimental multiplex Sierra/Hunter/Nicole
Experimetnal DNA multiplex GCW/CC
U937 multiplex ECW
Experimental DNA multiplex ECW
Negative control Multiplex ECW
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Experimental DNA Multiplex Katy/Michael/Madi
Experimental DNA Multiplex Dylan/Josi
Not labeled Exp DNA Multiplex
Not Labeled Exp DNA multiplex
Ladder
Neg Control Multiplex Collin Perry
U937 CSF1Po
Exp DNA multiplex
Exp DNA CSF1PO
U937 multiplex
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Lane 13
Lane 14
Lane 15
U937 CSF1PO
CET
Ladder
Neg Control CSF1PO CET
Exp CSF1PO CET
U937 D75820
ME
Neg Control D75820 ME
Exp DNA D75820 ME
U937 CSF1PO KAL
Neg control CSF1PO KAL
Exp DNA CSF1PO
KAL
Exp DNA HUMTH01 ——– A10
Neg Control HUMTH01 ——— A11
U937 HUMTH01 ———– A12
Neg control Y-Gata-H4
C1
Exp DNA Y-Gata-H4
C2
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Lane 13
Lane 14
Lane 15
Neg Control Y-GATA-H$ —B1
U937 Y-GATAH4 — —- B2
Exp DNA Y-GATA H4 ——- B3
Exp HumTH01 ———- B4
U937 DNA Overflow of Ladder Sorry — B5
Ladder
Negative Control HUMTH01 ———- B6
U937 D7s820—— B7
Neg control D7S820—— B8
Exp DNA D7S820 —– B9
Exp DNA Y-Gata ———- B10
U937 Y-GATA——— B11
Neg Control Y-GATA H4 ——B12
U937 Y-GATAH$———- C3
Purchase answer to see full
attachment
![DescriptionDNA Forensics lab
Week 1
Week 2
Week 3
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: https://doi.org/10.1662/0002-7685(2008)70[29:DFIAFT]2.0.CO;2
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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 fingerprinting, Southern Blot analysis and Restriction Fragment Length
Polymorphism (RFLP), were developed (Rudin, 2002). In 1985,
Alec Jeffreys used RFLP as a way to identify individuals from
their DNA, a process he termed DNA fingerprinting (Jeffreys et
al., 1985). In 1985, Kary Mullis and colleagues from the Cetus
Corporation first published the Polymerase Chain Reaction
(PCR) method of replicating specific regions of DNA utilizing
gene specific primers and specific thermocycler conditions (Saiki
et al., 1985).
The combination of RFLP with PCR led to the possibility
of identifying individual specific regions of DNA from limited
samples, such as a single hair shaft with an intact follicle left at
a crime scene. This technology, termed DNA profiling, would
allow detectives to link a suspect to a crime scene by using
his/her DNA fingerprint. The United Kingdom became the first
country to use DNA profiling to exonerate one suspect of rape
and to convict another for the same crime in 1987 (Canadian
National DNA Database, 2003; Burns, 2005). In 1989, the
United States (U.S.) had its first case overturned because of
DNA evidence and the U.S. federal government began developing regulatory standards for DNA collection and handling procedures. In 1992, the National Research Council deemed DNA
testing a reliable method to identify a criminal suspect, which
prompted the technology to rapidly enter the mainstream court
system. The Federal Bureau of Investigation (FBI) established
the National DNA Index System, enabling city, county, state,
and federal law enforcement agencies to compare DNA profiles
electronically in 1998 (Burns, 2005). This software program is
referred to as CODIS (COmbined DNA Index System) and contains DNA profiles from convicted offenders, missing persons,
and unsolved crimes (FBI, 2006).
In 1994, DNA profiling was further advanced by performing
PCR to evaluate specific loci that are variable between individuals, including monozygotic twins (NIJ, 2002). These regions are
STACY A. WAGONER (swagoner3@cox.net) is a former student, and KIMBERLY
A. CARLSON (carlsonka1@unk.edu) is Associate Professor of Biology, both
in the Department of Biology, University of Nebraska at Kearney, Kearney,
NE 68849.
referred to as short tandem repeats (STRs) and the FBI uses a
standard set of 13 of these for CODIS analysis and databanking
(NIST, 2007). The simplicity of this assay is that the primers for
PCR for the 13 loci can be mixed into one reaction, therefore
giving rise to what is known as multiplex PCR. The specificity of
these 13 loci lies in the fact that the odds that two individuals
share the same DNA profile based on these loci is about one in
one billion (NIJ, 2002).
Today DNA fingerprinting is used in a multitude of ways,
including paternity testing, identifying animal versus human
remains, rape cases, murder trials, historical cases, military dog
tags, missing persons, and disease/health issues (Butler, 2001).
Television programs, such as “CSI,” have pushed forensic science
and DNA fingerprinting into every household. While doing this,
the field has been glamorized, exaggerated, and oversimplified.
This experiment was designed to provide students, in a
classroom laboratory setting, a hands-on demonstration of the
steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analyses of the data. The
PCR parameters were determined by using control human DNA
and first doing the reactions individually. Next, the four forward
and reverse primers were mixed to make a multiplex primer
mix and the parameters tested using control human DNA. Once
the parameters were determined, the experimental DNA was
extracted from pop cans, pop bottles, plastic spoons, cigarette
butts, and cheek swabs and used in multiplex PCR reactions.
The resulting PCR products were analyzed by gel electrophoresis
and visualized utilizing a gel photodocumentation system. From
the gel, the DNA fingerprint of the individual could be determined and analyzed.
Materials
• used pop cans, pop bottles, spoons, and cigarette butts
• BuccalAmp™ DNA Extraction Kit with QuickExtract™
DNA Extraction Solution and Catch-All™ Sample
Collection Swabs (EPICENTRE®)
• Microcon centrifugal filter devices (Amicon)
• GoTaq® Green PCR Master Mix (Promega)
DNA FINGERPRINTING
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29
• 10X TBE (for 1 liter: 108 g Tris base, 55 g boric acid, 40
ml 0.5 M EDTA, pH 8.0) and 1X TBE buffer
• 0.9% saline solution (9 g NaCl in 1 L nanopure water)
• 500 μg/mL ethidium bromide (Fisher) – working concentration
• TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA)
• 5% Chelex solution (Sigma)
• 6X orange/blue loading dye (Promega)
• 90% and 70% ethanol
• Phenol Chloroform Isoamyl alcohol (PCI; Fisher)
• 3 M sodium acetate
• thermocycler
• Mini-PROTEAN® II Gel Electrophoresis Unit and 4-15%
Tris-HCl ready-made polyacrylamide gels (Bio-Rad)
• 1.5 ml microcentrifuge tubes
• pipets and tips
• vortex and centrifuge
• heating blocks or water baths
• sterile razor blade, forceps, 100 base pair ladder (BioRad)
• control human DNA
• gel electrophoresis equipment
• platform shaker
• gel documentation system
• primers (Vermaas & Rhoads, 2004; Invitrogen)
• D7S820
– Forward 5’ GTC ATA GTT TAG AAC GAA CTA
ACG 3’
– Reverse 5’ CTG AGG TAT CAA AAA CTC GA GG
3’
• CSF1PO
– Forward 5’ CTG AGT CTG CCA AGG ACT AGC 3’
– Reverse 5’ CAC ACA CCA CTG GCC ATC TTC 3’
• Y-GATA-H4
– Forward 5’ CCT AAG CAG AGA TGT TGG TTT TC
3’
– Reverse 5’ CTG ATG GTG AAG TAA TGG AAT
TAG 3’
• HUMTH01
– Forward 5’ GTG GGC TGA AAA GCT CCC GAT
3’
– Reverse 5’ CAA AGG GTA TCT GGG CTC TGG 3’
Methods
DNA Extraction from Pop Bottles, Pop Cans,
Plastic Spoons & Cheek Cells
DNA extraction was done in a laminar flow hood to avoid
contamination from other sources, including the experimenter.
The manufacturer’s protocol (EPICENTRE®) was followed for
DNA extraction with the following modifications. A Catch-All
Sample Collection Swab was dampened with 0.9% saline solu-
30 THE AMERICAN BIOLOGY TEACHER, ONLINE PUBLICATION, AUGUST 2008
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tion and the excess solution was squeezed out by pushing the
swab against the inside of a 1.5 ml microcentrifuge tube. For
the cheek sample, an individual swished 0.9% saline solution in
his/her mouth for 10-15 seconds and spit the solution into a beaker to be discarded. The cheek, pop bottle, pop can, and plastic
spoon were brushed with the swab at the locations the DNA was
likely to be found. This was inside the cheek, at the mouth of the
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 temperature for 15 minutes and placed in a microfuge tube containing
Quick Extract™ DNA Extraction Solution, rotated five times, and
pushed against the inside of the microfuge tube to remove excess
liquid. The microfuge tube was vortexed, placed in a heating
block at 65° C for one minute, vortexed again, returned to the
heating block at 98° C for two minutes, vortexed a third time,
and stored at -20° C.
All DNA sample types (pop bottles, pop cans, plastic
spoons, and cheek cells) were further purified by adding
Phenol Chloroform Isoamyl alcohol (PCI) in a 1:1 ratio to each
microfuge tube. PCI is highly toxic. Special care and handling
should be taken when using it. Working in a fume hood is
highly recommended. Each microfuge tube was vortexed and
centrifuged at 13,000 rpm for two minutes. The top aqueous
layer containing the DNA was carefully transferred into new
microcentrifuge tubes and the bottom layer discarded. The DNA
was precipitated by adding 3 M sodium acetate (NaAc) in a 1:10
ratio and 1 ml of 90% ethanol (EtOH) to each microfuge tube.
The microfuge tubes were mixed by vortexing and placed at -20°
C for approximately 24 hours. DNA samples were centrifuged at
16,000 rpm for 20 minutes at 4° C, the supernatant discarded,
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
rpm, the supernatant discarded, and the DNA pellet dried. The
DNA samples were placed with caps open in a 37° C incubator
for 10-15 minutes, the DNA resuspended in 25 μl of TE buffer,
and placed at -20° C for storage.
DNA Extraction from Cigarette Butts
DNA was extracted from cigarette butts following the protocol outlined in Hochmeister et al. (1991). In the laminar flow
hood using a sterile razor blade and forceps, three cross-sections
approximately 3 mm wide were cut from the filter end of a
cigarette butt. The cuttings were placed in a microfuge tube with
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
minutes, vortexed, boiled by heating to 100° C for eight minutes,
vortexed again, and centrifuged at 13,000 rpm for five minutes to
pellet the Chelex. The supernatant was carefully transferred to
a centrifugal filter tube and centrifuged at 4,000 rpm. The retentate was washed with 2 ml of TE buffer and was stored at -20°
C. The cigarette butt DNA was further purified by PCI extraction
and precipitated with NaAc and EtOH as described previously.
PCR Amplification & Gel Electrophoresis
PCR reactions were prepared in the laminar flow hood
with DNA extracted from U937 monocytic cells as a positive
control, without DNA as a negative control, and with DNA from
the cheek sample, cigarette butt, pop can, pop bottle, or plastic
spoons. The positive control and negative control reactions were
prepared with both the individual primer sets and with the
multiplex primer mix containing all four primer sets. The experimental reactions were prepared with only the multiplex primer
mix. These were all prepared by adding 12.5 μl GoTaq® Green
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
of experimental DNA, 2.0 μl of individual primers
sets or multiplex primer mix (final concentration
of each primer is 2.5 μM), and 5.5 μl of water for a
final reaction volume of 25 μl. All reactions were vortexed, centrifuged, and placed in the thermocycler
using the following parameters:
• Initial denaturation: three minutes at 94° C
• Denaturation: one minute at 94° C*
• Annealing: one minute at 58° C*
Figure 1. PCR performed with individual primer sets using U937 DNA. Lane 1
= 100 bp ladder; Lane 2 = CSF1PO + U937; Lane 3 = CSF1PO no-DNA control;
Lane 4 = Y-GATA-H4 + U937; Lane 5 = Y-GATA-H4 no- DNA control; Lane 6 =
HUMTH01 + U937; Lane 7 = HUMTH01 no-DNA control; Lane 8 = D7S820 +
U937; Lane 9 = D7S820 no-DNA control. CSF1PO (Lane 2) shows a PCR product at
~320 bp (arrow), Y-GATA-H4 (Lane 4) shows a PCR product at ~ 390 bp (arrow),
HUMTH01 (Lane 6) shows PCR products at ~180 and 190 bp (arrows), and D7S820
shows a PCR product at ~215 bp (arrow). Bands without arrows are nonspecific
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
100 base pair ladder. Ten microliters of either PCR product or
marker was loaded into the gel and electrophoresed at 100 V
for approximately one hour at room temperature. After the bottom dye band had run through the gel, the gel was placed in
ethidium bromide (final concentration = 0.5 μg/mL) diluted in
water and placed on the shaker for 30 minutes. A picture was
taken using a gel photodocumentation system for comparison
and analysis of the gel.
Results
Each individual primer set was initially tested using control
human DNA extracted from U937 monocytic cells (Figure 1).
Any human genomic DNA would work, but U937 DNA was readily available in the lab. The gel shows:
• a PCR product at ~320 bp for
CSF1PO (Lane 2)
• a ~390 bp product for Y-GATAH4 (Lane 4)
• two products at ~180 and ~190
bp for HUMTH01 (Lane 6)
• a ~215 bp product for D7S820
(Lane 8).
These product sizes are within
the expected ranges for the loci tested
(Table 1). No PCR products were seen
in the no-DNA negative control lanes.
Nonspecific PCR products were detected but were outside the expected range
of sizes for the loci tested.
Once the individual primer sets were tested, the four primer
sets were mixed together to perform a multiplex PCR. This was
first done using control human U937 DNA (Figure 2). The gel
shows successful amplification of three of the four primer sets
at once. A ~390 bp product represents CSF1PO amplification,
two products at ~180 and ~190 bp represent HUMTH01, and a
~215 bp product represents D7S820. Amplification for Y-GATAH4 was not detected. The reaction was attempted multiple times,
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.
There were no bands present in the no-DNA negative controls as
expected. The products for CSF1PO, HUMTH01, and D7S820
on the multiplex PCR gel (Figure 2) coincide with the same size
products from the gel testing the individual primer sets (Figure
Table 1. STR loci utilized in multiplex PCR in this study. All loci are described in Vermaas and
Rhoads (2004) and NIST (2007). In addition, CSF1PO is described in Hammond et al. (1994),
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).
STR
Location on
Chromosome
Repeat Structure
# of
Repeats
PCR Product (bp)
CSF1PO
5q33.3-34
AGAT
6-16
291-331
D7S820
7q
GATA
5-15
194-234
HUMTH01
11p15-15.5
AATG = bottom strand;
TCAT = top strand
3-14
171-215
Y-GATAH4
Y
(GATA)10GAATGGATAGATTA
(GATA)2 AATA(GATA)4
Not defined
~360-400
Not defined
DNA FINGERPRINTING
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31
1). After positive amplification
was seen with control DNA and
three of the four primer sets in
the multiplex primer set mix,
the cigarette butt, the pop can,
the pop bottle, the cheek cells,
and the plastic spoon (Figure 3)
were tested with the multiplex
primer set mix.
Figure 2. PCR performed with multiplexed primers using
U937 DNA. Lane 1 = 100 bp ladder; Lane 2 = multiplexed
primers + U937 DNA; Lane 3 = no DNA control. PCR products
for CSF1PO (~320bp), D7S820 (~215bp), and HUMTH01
(~180 and 190bp) are marked with arrows. The ~390bp PCR
product for Y-GATA-H4 was not apparent. The bands without
arrows are nonspecific PCR products.
The cigarette butt, pop can,
pop bottle, and spoon PCR reactions all show one PCR product
at ~325 bp for CSF1PO, one
product at ~220 bp for D7S820,
and two products at ~200 and
~185 bp for HUMTH01. For the
cheek PCR reaction, two products at ~330 and ~320 bp are
detected for CSF1PO, one product at ~230 bp for D7S820, and
three products (tri-allelic expression) at ~200, ~180, and ~170
bp for HUMTH01. These product sizes are within the expected
ranges for the loci tested (Table
1). No PCR products were seen
in the no-DNA negative control
lanes. Nonspecific PCR products
were detected but were outside
of the expected range of sizes for
the loci tested.
The PCR products for the three loci are the
same size for the cigarette butt, pop can, pop bottle,
and spoon reactions, showing that the DNA originated from one individual, whereas the cheek DNA
was extracted from a sample given by a different
individual. The use of DNA samples from two different individuals was done to demonstrate that differences 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 amplification 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.
In this experiment, positive
amplification was seen as bands
on the gel when testing the individual primer sets and the multiplex
primer mix with or without DNA
in the reaction mix. This showed
positive PCR amplification of target STRs, the specific area of DNA
chosen to be amplified, in three of
the four loci tested. The absence
of PCR products in the no-DNA
negative controls demonstrates
that there was no amplification
occurring in these reactions and
that there was not DNA contamination of the reagents used in
the PCR reactions. This verifies
that amplification of the target
gene sequences was specific and
did not produce spurious PCR
products. The approximate PCR
product sizes for the individual
primer sets coincided with the
PCR products with the same size
from the multiplex PCR when
using the control DNA for all loci
except for Y-GATA-H4. This suggests that more than one specific
area of DNA can be successfully
amplified at a time and that all of
Figure 3. Multiplex PCR for cigarette butt, spoon, pop bottle, pop can, and cheek
DNA samples. Lane 1 = 100 bp ladder; Lane 2 = cigarette butt DNA (Subject 1);
Lane 3 = bottle DNA (Subject 1); Lane 4 = can DNA (Subject 1); Lane 5 = spoon
DNA (Subject 1); Lane 6 = cheek DNA (Subject 2); Lane 7 = negative control. C =
CSF1PO PCR products; D = D7S820 PCR products; H = HUMTH01 PCR products. The
bands without arrows are nonspecific PCR products.
Discussion
Television today oversimplifies much of forensic 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.
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the amplification products can be individually visualized. The
lack of amplification of Y-GATA-H4 does not negate this finding.
It does suggest that a different male specific STR should be used
if deemed necessary.
at Kearney (UNK) Undergraduate Research Council, the UNK
Biology Department, and the UNK College of Natural and Social
Sciences.
The multiplex gels with DNA extracted from a pop bottle,
pop can, cigarette butt, plastic spoon, and cheek swab also show
successful amplification of target STRs as seen by the presence
of PCR products that fall within the expected size range for
the loci as shown in Table 1. Since the PCR product sizes for
CSF1PO, HUMTH01, and D7S820 for the cigarette butt, pop
can, pop bottle, and spoon samples were estimated to be the
same size, we can deduce that they are from one individual. This
is in contrast to the PCR product sizes for these three loci from
the cheek sample, which was from a different individual. The
comparison shows that the multiplex PCR protocol can be used
to distinguish between individuals.
References
DNA could be extracted from more individuals to further
exemplify the differences in the DNA fingerprints. DNA as a
molecular fingerprint for each individual could be discussed, as
well as how DNA fingerprinting is utilized in different areas such
as the criminal justice system, military dog tags, organ donor
compatibility, and species genome projects. Since the DNA
extracted in this experiment was primarily from saliva, DNA
could also be extracted from other items such as blood, hair,
or skin to show that an individual’s DNA is the same regardless
of the source. To differentiate between females and males, a different male specific STR marker could be used and put into the
multiplex PCR primer set.
As a teaching experiment in a laboratory setting, a mock
crime scene could be set up so that students could collect samples for DNA extraction from items such as blood on a shirt, the
rim of a ball cap, or a used cigarette. The DNA could be extracted
from these samples and each student could also extract his/her
own DNA utilizing the cheek swab method. Amplification and
gel electrophoresis could be done comparing the PCR products
to determine if one of the students committed the crime. The
activity teaches students how long the process of DNA fingerprinting takes. Connecting a DNA fingerprint to the perpetrator
of a crime is not something that can be done in minutes or even
hours, as portrayed on television. Many times in real-life crime
scenes, there is no known match to the DNA tested and the
crime becomes a “cold case” until a match in CODIS is made.
This is not popular opinion based on television shows but, in
reality, it is a common occurrence of which students should be
aware. These are a few of the topics that could be discussed. If
the instructor wants to incorporate probability testing into the
laboratory exercise, the DNA profiles can be used to determine
allele frequencies and to perform Hardy-Weinberg analysis. This
laboratory exercise demonstrates how DNA fingerprinting is
performed and how long it takes. It also shows that DNA fingerprinting is a useful tool in forensic science. Lastly, it debunks
the myth portrayed in popular TV shows that obtaining DNA
evidence is a guarantee that the crime will be solved in an hour.
Burns, K.S. (2005). Criminal Investigations, DNA & Forensic Science.
Available online at: http://www.karisable.com/crdna1.htm.
Butler, J.M. (2001). Overview and history of DNA typing. Forensic DNA
Typing: Biology and Technology Behind STR Markers, 1st Edition.
Burlington, MA: Elsevier Academic Press.
Canadian National DNA Database. (2003). Forensic history of DNA:
highlights. Available online at: http://www.nddb-bndg.org/foren_
history_e.htm.
Edwards, A., Civitello, A., Hammond, H.A. & Caskey, C.T. (1991). DNA
typing and genetic mapping with trimeric and tetrameric tandem
repeats. American Journal of Human Genetics, 49, 746-756.
Federal Bureau of Investigation (FBI). (2006). CODIS: Combined DNA
Index System. Available online at: http://www.fbi.gov/hq/lab/
codis/index1.htm.
Hammond, H.A., Jin, L., Zhong, Y., Caskey, C.T. & Chakraborty, R.
(1994). Evaluation of 13 short tandem repeat loci for use in personal identification applications. American Journal of Human Genetics,
55, 175-189.
Hochmeister, M.N., Budowle, B., Jung, J., Borer, U.V., Comey, C.T. &
Dirnhofer, R. (1991). PCR-based typing of DNA extracted from cigarette butts. International Journal of Legal Medicine, 104, 229-233.
Jeffreys, A.J., Wilson, V. & Thein, S.L. (1985). Individual-specific “fingerprints” of human DNA. Nature, 316, 76-9.
Jin, L., Underhill, P.A., Buoncristiani, M.R., & Robertson, J.M. (1997).
Defining microsatellite alleles by genotyping global indigenous
human populations and non-human primates. Journal of Forensic
Science, 42, 496-499.
National Institutes of Justice (NIJ). (2002). Using DNA to solve
cold cases. Available online at: http://www.ncjrs.gov/pdffiles1/
nij/194197.pdf.
National Institute of Standards and Technology (NIST). (2007). Short
tandem repeat DNA internet database. Available online at: http://
www.cstl.nist.gov/div831/strbase/index.htm.
Rudin, N. (2002). A short history of DNA typing. An Introduction to
Forensic DNA Analysis, 2nd Edition. Boca Raton, FL: CRC Press.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A.
& Arnheim, N. (1985). Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for diagnosis of
sickle cell anemia. Science, 230, 1350-1354.
Vermaas, W. & Rhoads, D. (2004). Experiment IV: short tandem repeats
DNA profiling. Available online at: http://photoscience.la.asu.edu/
Photosyn/courses/BIO_343/lab/Experiment-IV.html.
White, P.S., Tatum, O.L., Deaven, L.L. & Longmire, J.L. (1999). New,
male-specific microsatellite markers from the human Y chromosome. Genomics, 57, 433-437.
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
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33
DNA Forensics Lab
Instructions on How to Use OligoAnalyzer
OligoAnalyzer is a free bioinformatics tool used to analyze DNA primers. You will be using it to
analyze the primers you have created in this lab. This tool calculates the physical characteristics
of any oligo sequence, including length, GC content, melting temperature (Tm) and molecular
weight.
1. Access OligoAnalyzer at https://www.idtdna.com/calc/analyzer. You would have to create a
free account to access the tool. The home screen will look as follows:
2. Enter your sequence into the “Sequence” box in the 5’ to 3’ orientation. The following DNA
primer sequence will be used in this tutorial: CGGGAAGGGAACAGGAGTAAG.
Note: to get the correct Tm, it is important to enter the Mg2+ and dNTP concentrations you will use
in your experiment. We will only focus on the understanding the fundamentals of primer design of
this lab and will use SpecSheet settings for all calculations.
1
3. Click on the “Analyze” button (colored in orange) to calculate physical characteristics of DNA
your primer. Sample results of this calculations are shown below.
X
2
4. Click on the “Hairpin” button to calculate thermodynamic parameters of your DNA primer
sequence.
The Gibbs free energy (ΔG) value gives an indication of the strength of the secondary structure.
IDT recommends the ΔG value be more than –9 for self-dimers and hetero-dimers. Sample
thermodynamic values and hte resulting hairpin structure are shown below:
3
5. Click on the “Homo-Dimer Analysis” button to predict all homo-dimers that could possibly
form and calculate their free energies. A sample output of this calculation is shown below:
Note: homo-dimers form between two identical DNA primer sequences.
6. Find another student in the class whose primer has a similar Tm. Create a multiplex of two
primers by checking them against each other via the “Hetero-Dimer Analysis” tool. Using the
remaining time of this lab, make adjustments to make the best multiplex of two primers. Share
the results with the lab section.
4
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Ladder
Neg Control multiplex AG/CG
Experimental DNA Multiplex
AC/CG
U937 DNA Multiplex
AC/CG
Extracted DNA multiplex Kate/Ellie
Negative control multiplex Sierra/Hunter/Nicole
U937 multiplex Sierra/Hunter/Nicole
Experimental multiplex Sierra/Hunter/Nicole
Experimetnal DNA multiplex GCW/CC
U937 multiplex ECW
Experimental DNA multiplex ECW
Negative control Multiplex ECW
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Experimental DNA Multiplex Katy/Michael/Madi
Experimental DNA Multiplex Dylan/Josi
Not labeled Exp DNA Multiplex
Not Labeled Exp DNA multiplex
Ladder
Neg Control Multiplex Collin Perry
U937 CSF1Po
Exp DNA multiplex
Exp DNA CSF1PO
U937 multiplex
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Lane 13
Lane 14
Lane 15
U937 CSF1PO
CET
Ladder
Neg Control CSF1PO CET
Exp CSF1PO CET
U937 D75820
ME
Neg Control D75820 ME
Exp DNA D75820 ME
U937 CSF1PO KAL
Neg control CSF1PO KAL
Exp DNA CSF1PO
KAL
Exp DNA HUMTH01 ——– A10
Neg Control HUMTH01 ——— A11
U937 HUMTH01 ———– A12
Neg control Y-Gata-H4
C1
Exp DNA Y-Gata-H4
C2
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Lane 7
Lane 8
Lane 9
Lane 10
Lane 11
Lane 12
Lane 13
Lane 14
Lane 15
Neg Control Y-GATA-H$ —B1
U937 Y-GATAH4 — —- B2
Exp DNA Y-GATA H4 ——- B3
Exp HumTH01 ———- B4
U937 DNA Overflow of Ladder Sorry — B5
Ladder
Negative Control HUMTH01 ———- B6
U937 D7s820—— B7
Neg control D7S820—— B8
Exp DNA D7S820 —– B9
Exp DNA Y-Gata ———- B10
U937 Y-GATA——— B11
Neg Control Y-GATA H4 ——B12
U937 Y-GATAH$———- C3
LABORATORY EXPERIMENT
pubs.acs.org/jchemeduc
Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the
Forensic Laboratory
Kelly M. Elkins*
Chemistry Department and Criminalistics Program, Metropolitan State College of Denver, Denver, Colorado 80217-3362,
United States
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bS Supporting Information
ABSTRACT: The polymerase chain reaction (PCR) is a common experiment in upper-level
undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and
thermocyclers have become more affordable for institutions. Typically, instructors design PCR
primers to amplify the region of interest and the students prepare their samples for PCR and analyze
the results. However, primers can also be designed in undergraduate laboratories with students at
this level. In a course that focuses on forensic DNA molecular biology for forensic chemistry
students, students have used the Applied Biosystems AmpFlSTR SGM Plus kit that amplifies DNA
at eleven regions in a single test tube. It is important for forensic chemistry students to be able to
design and analyze a single set of primers and, more importantly, create multiplexes of primers. This
enables students to more fully understand how the primers and the kits that are routinely employed
by the crime laboratories function. Creating a single set of primers does not demonstrate the extent
of design and engineering inherent in creating multiplexes or adequately prepare students for
research and careers in the field. The in silico method described herein uses free bioinformatics tools
and results in student-designed multiplexes for Combined DNA Index System (CODIS) loci. Sample student data are shown.
KEYWORDS: Upper-Division Undergraduate, Biochemistry, Interdisciplinary/Multidisciplinary, Laboratory Instruction, Internet/WebBased Learning, Problem Solving/Decision Making, Biotechnology, Forensic Chemistry, Molecular Biology, Nucleic Acids/DNA/RNA
T
he polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry and molecular biology courses as reagents and thermocyclers have
become more affordable for institutions. There are numerous
reports of the use of PCR in undergraduate laboratories with a
forensic application. For example, PCR can be used to amplify a
369 801 base pair D1S80 locus of the human genome,1 to
amplify hypervariable regions of DNA from dog hair and saliva,2
to amplify a 440 base pair hypervariable region of human mtDNA
from a simulated crime scene,3 and to amplify a 192 base pair
DNA segment present in genetically modified foods.4 In the
undergraduate lab, PCR has also been demonstrated to differentiate bacterial species,5 to genotype the normal variation in
human color vision,6 to evaluate a metabolic polymorphism,7 in
diagnostics,8 and to test for genetically modified organisms in
foodstuffs.9,10 In all of these examples, the instructors designed
the experiments and created and secured the primers, and the
students prepared their PCR samples using the procedure as
written.
In the second semester of a two-semester forensic science
course that focuses on DNA molecular biology for forensic
chemistry students, students employ the Applied Biosystems
AmpFlSTR SGM Plus multiplex kit that amplifies DNA at 11
regions (D21S11, FGA, TH01, vWA, D8S1179, D18S51,
D3S1358, D19S433, D16S539, D2S1338, and the gender marker, amelogenin) including nine of the thirteen short tandem
repeat (STR) loci of the U.S. Combined DNA Index System
(CODIS) (all of the above except D19S433 and D2S1338) for
Copyright r 2011 American Chemical Society and
Division of Chemical Education, Inc.
PCR and DNA typing (Table 1). Most U.S. crime laboratories
utilize 16-plex kits for human casework for sample completion so
that the data can be entered into CODIS, as necessary. It is
important for forensic chemistry students to be able to employ a
kit for PCR and mimic crime lab work and, more importantly, to
design and analyze a single set of primers and create primer
multiplexes. This enables students to more fully understand how
the primers and the kits that are routinely employed by the crime
laboratories function and extends their research skills.
Primers can be designed in undergraduate laboratories with
students at this level.11 14 In a single three-hour laboratory
session, students design PCR primers in silico using free bioinformatics tools available on the World Wide Web and collaborate
with a peer to create a multiplex PCR primer set for two loci.
Creating only a single set of primers does not demonstrate the
extent of design and engineering inherent in creating multiplexes
or adequately prepare students for jobs in forensics or even
modern clinical testing in medicine.
’ MATERIALS AND METHODS
This study was approved by Metro State’s Institutional Review
Board (IRB)—Human Subjects Review Committee (HSRC),
and the students consented to participate in the study and to the
release of the anonymous data shown here.
Published: August 10, 2011
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LABORATORY EXPERIMENT
Table 1. CODIS STR Loci by NCBI Accession Number and Repeata
a
Data from ref 20.
Table 2. Sample PCR Primer Results
Figure 1. Genome sequence for TPOX CODIS locus region on human
chromosome 2 thyroid peroxidase (hTPO) reference sequence and
published validated forward and reverse primers (arrows and bold)
producing a 64 base pair product.
This in silico lab does not require specialized materials,
expensive chemicals, or extensive preparation. A reliable Internet
connection and student laptops or computers (via a computer or
department lab) are required. The utilized Web sites include
NCBI,15 IDT’s OligoAnalyzer,16 NCBI Primer-Blast,17 and
NIST’s STRbase.18
An example of the type of data collected and evaluated in this
lab is presented to the students during the prelab lecture. The
primers shown as an example (TPOX locus) are used in the same
course in a subsequent laboratory (real-time PCR amplification)
(Figure 1 and Table 2),19 and amplify a nonvariable portion of
the locus and student-designed primers from this lab. Students
are shown how to navigate to the Web sites and locate a STR
repeat. The differences between the forward and reverse primers
(both 50 to 30 but located on the plus and minus strands,
respectively) and the STR repeat region for a sample locus from
NCBI (Table 1) are highlighted prior to inputting the primers in
the analysis Web sites, collecting the output to be saved in a text
file, and creating multiplexes.
Working in alone or in pairs, the students obtain the DNA
sequence for a CODIS STR locus (Table 1)20 from the National
Center for Biotechnology Initiative (NCBI)15 using mini-Mac
computers in one of the department computer laboratories. After
locating the STR repeat (Figure 2), they initially try to create the
primers from the 50 region directly upstream from the repeats
and the region directly downstream for the 30 complement using
the given guidelines for optimal primers including the following:
40 60% guanine cytosine (GC) content, melting temperatures (Tm) within 5 °C, less than four identical base repeats,
only weak primer dimer and hairpin formation, a length between
18 30 bases, and overall Tm values in the range of 50 72 °C.
The amplicon should not exceed 500 base pairs. The primers are
evaluated in Integrated DNA Technologies’ (IDT) OligoAnalyzer 3.116 by pasting the primer sequence into the sequence box
and clicking on the Analyze, Hairpin: Submit and Self-Dimer
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Journal of Chemical Education
Figure 2. Genome sequence for TH01 CODIS locus region on
human chromosome 11 tyrosine hydroxylase sequence, AATG
(named for 30 strand) STR repeat (underlined), and student designed
forward and reverse primers (arrows and bold) producing an 87
base pair product given the 9 repeats observed in the NCBI
reference sequence (or 90 base pair product using K562 with the 9.3
repeat allele).
LABORATORY EXPERIMENT
boxes on the right. The defaults are used for all calculations. The
Hetero-Dimer: Calculate calculation is used to evaluate the two
primers together. Then the primer sequences are submitted to
NCBI Primer-Blast (Homo sapiens)17 to evaluate specificity by
entering the NCBI accession number in the PCR template box
and the designed primers in the forward and reverse primer boxes
under primer parameters. The students also indicated the primer
region for the NCBI accession number by base number in the
boxes to the right of the PCR template box and the expected
product size below the primer sequence boxes.
After the students completed a reasonable single primer set
(Table 3), the students paired themselves or their groups to
create a multiplex of the two sites by first locating other students
with similar melting temperatures for their primers. The multiplex primers were checked against each other in OligoAnalyzer
using the Hetero-Dimer function. The students made adjustments to the primers to create their best multiplex using the
remaining lab time or out of class while recording all attempts in
the text file (Table 3).
In a follow-up laboratory experiment, the students tested the
primers they designed experimentally using real-time PCR19 in a
Table 3. Sample Student PCR Primer Multiplex Results
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Table 4. Student Designed STR PCR Primers
a
Size based upon NCBI sequence. b Size based upon K562 DNA
sequence (both alleles indicated).
25 μL reaction. The students amplified human K562 DNA
(using 1 ng total in the 25 μL reaction) (Promega, Madison,
WI) using the published and validated TPOX primers21 and their
designed primers (Table 4) (purchased from IDT, Coralville, IA)
(1 μL each of 5 μM primers) in a real-time PCR experiment
conducted on a Bio-Rad (Hercules, CA) iQ5 instrument using
the iQ5 software (v. 2.0) and 12.5 μL of 2 iQ SYBR Green
Supermix (Bio-Rad, Hercules, CA) and nuclease-free water to
total 25 μL on a 96-well plate according to the following protocol
for a gradient plate: an initial 3 min denaturation at 95 °C and 40
cycles of denaturation at 95 °C for 15 s using an annealing extension gradient at 50 65 °C for 60 s. A melting curve
was produced by conducting 91 cycles of 30 s of melting every
0.5 °C from 50 to 95 °C. The SYBR Green I excitation
wavelength is 497 nm, and the emission wavelength is 522 nm.
Three primer sets from classes of previous years were also tested.
A total of ten primer sets were evaluated. The production of the
single reaction and multiplex PCR products was evaluated by the
shape of the melt peaks from real-time PCR and post-PCR
agarose gel electrophoresis using a 2% gel run in 1 TAE
(40 mM Tris-acetate, 1 mM EDTA, pH 8.0) (National Diagnostics, Atlanta, GA) using the DNA Logic (Lambda Biotech, St.
Louis, MO) and Lonza (Basel, Switzerland) 20 bp (base pair)
ladders. Upon completion, digital photographs were recorded of
each gel upon illumination under UV light using a digital
photography system equipped with a SYBR filter.
Hazards
No chemicals are needed, and there are no intrinsic chemical
hazards associated with this laboratory experiment as it is
performed entirely on a computer. For the follow-up lab, SYBR
Green I is a weak mutagen and may be an intercalating agent and
should be handled with care. Gloves, goggles, long pants, and
closed-toed shoes should be worn at all times in the laboratory.
The digital camera should be set to UV vis safety at all times,
and gel pictures should be recorded with the illuminator door in
the closed position.
’ RESULTS
Sample results for the example TPOX primers are shown in
Table 2. The TPOX PCR primers amplify a nonvariable, 64 base
LABORATORY EXPERIMENT
pair amplicon of the Homo sapiens thyroid peroxidase on
chromosome 2 (NCBI Accession number: NG_011581) from
bases numbered 81262 81325 (inclusive) (Figure 1). An analysis of these primers yields that the primer length varies by one
base pair, the GC content varies by 7.1%, the Tm vary by 0.2 °C,
and the best self-dimers and hairpins were 2 3 base pairs. The 30
primer Tm had the lower GC content, but the overall Tm was
almost identical to that of the 50 primer due to the one base
increase in length. The hairpins and self-dimers exhibited sufficiently low melting temperatures as compared to the annealing
temperature of the primer to the template. The best heterodimer
formed is four base pairs, which is more than optimal (three or
less). The primers were determined to be specific for only this
region in the human genome.
Students work individually or in pairs in the lab. Each student or
group of students chooses a different locus from the thirteen
CODIS loci (Table 1). After inputting the accession number and
locating the STR repeats from the NCBI nucleotide file for a
CODIS locus (Table 1), the students evaluate the 50 region
directly upstream from the repeats as a potential 50 primer and
the complementary region directly downstream for the 30 complement. This is advantageous for forensic use as the recovered DNA
is often degraded by environmental exposure, arson, or mass
disaster events and shorter DNA segments are more easily
amplified (e.g., mini-STRs). Then the students use currently
available Web-based bioinformatics programs for single and multiplex primer evaluation including OligoAnalyzer 3.1 and PrimerBlast. OligoAnalyzer outputs the melting temperature, primer dimer and hairpin formation, percent GC content, sequence
length, and heterodimer formation. Primer-Blast indicates for
which sequences the primer pairs are specific in a human genome
database (or other selected database). As the goal of this laboratory
is to teach the students the basics of primer and multiplex design,
automated primer design servers are not employed (e.g., Primer 3)
although these are well suited to the research and teaching setting
and are reported for use in a related lab experiment.14
Sample student results from the Web-based analysis for their
designed STR primers, TH01 and D16S539, and sample multiplex
results are given in Table 3. The designed primers indicate that the
students successfully completed the in silico assignment as most of
the data falls within the set criteria. The primers were determined
to be specific for only those specific regions in the human genome
and were 24 base pairs or less in length. The heterodimer pairs for
the TH01 forward primer had a higher than desirable level of six
heterodimer pairs, and the 50 primer multiplex of the opposite loci
had a higher than desirable level of five heterodimer pairs. The
observed hairpins in the primers have sufficiently low melting
temperatures as to be denatured at the annealing temperature
(e.g., 40.8 °C or less). The melting temperatures for the multiplex
primers were within 5.5 °C. Although all students had sufficient lab
time to test a few iterations of their primers and experiment with
shifts in frame and region, the students did not have sufficient lab
time to evaluate all possible primer sequences or to test multiple
multiplexes and make multiple improvements. However, they
were able to consider the challenges in editing the primers to
better the multiplex by comparing to the time already spent to
solve the problem.
All of the student-designed primers from the class are shown in
Table 4. The expected amplicon is shown for each in base pairs
for both the NCBI sequence and the K562 genotype. In a
subsequent laboratory using real-time PCR, students tested the
ability of their primers to amplify human K562 DNA. Five of the
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seven primer pairs produced the expected amplicon as assayed by
a post-PCR 2% agarose gel. Additionally, three of the primer pairs
designed by students in the classes of the previous years were
tested, and all produced the expected amplicons. Of the ten
primer sets evaluated, eight successfully amplified DNA (an 80%
success rate). The students also tested a few potential mutliplexes
based on having varied amplicon sizes that could be diffentiated
on the gel. The three multiplexes tested in class (D5S818:
D13S317, D7S820:D5S818, and D7S820:D13S317) provided
only low-level amplification; two of the multiplexes used one of
the primer sets that failed to work in a single PCR reaction
(D13S317). Subsequently four more multiplexes (TH01 with
D16S539, D7S820, D8S1179, or D5S818) were tested using only
primers that amplified the DNA successfully in a single reaction
and that could be differentiated based on size. Three of these
multiplexes produced the predicted DNA amplicons for both
primer sets (including TH01:D16S539, TH01:D7S820, and
TH01:D5S818) although the TH01 amplicon was preferentially
amplified in all cases at the concentrations used. The D8S1179
amplicon is at the upper limits for PCR and too long for real-time
PCR with a two-step reaction.
This follow-up lab is not inexpensive: primer pairs per student
cost approximately $15 each without a dye label (SYBR Green I
was used for detection of the amplicon) and, if desired, $60 80
per primer with a dye label plus shipping for the lot. The primers
were purchased from IDT, and the total cost was $132.01 for the
seven unlabeled primer sets for the current years’ class. The realtime PCR cost was approximately $4.50 per reaction; numerous
reactions were run including one replicate each for each of the
temperatures in the annealing temperature gradient, negative
controls, and multiplexes. (The total cost much exceeded the
collected lab fees collected by the college from the seven students
for the course).
’ ASSESSMENT
In an anonymous survey, student feedback for the laboratory
was positive: students were excited about designing the primers
to better understand how they work and being able to order and
experiment with them in the follow-up real-time PCR laboratory.
Students noted that an understanding of the background through
lecture and the prelab lecture was important. They appreciated
the opportunity to learn how to create primers using the Web
tools and gained an appreciation in multiplex primer design and
felt the concepts would have been confusing without performing
the lab. The majority of the students responded that creating the
primers was more difficult than expected, but predominately that
the data analysis was easier than expected or as easy or difficult as
expected. Students noted that they learned that the process can
be very time-consuming including finding the repeats and pairing
the primers.
’ DISCUSSION
The students follow the instructions contained in the syllabus
for writing concise weekly lab reports; that is, students complete
sections including the title, preparer, partners, date, objective,
method, data, analysis and results, sample calculations, discussion
and answers to lab questions, and a conclusion. Working
individually or in pairs, the students obtained the sequence of a
CODIS STR region and created primers for a single CODIS
locus and analyzed the primers using two free bioinformatics
tools. Subsequently, two students or student groups were merged
LABORATORY EXPERIMENT
and the students compared the primers of the individual unique
loci and evaluated if they could be used simultaneously in a
multiplex, single tube PCR reaction or if changes would be
necessary. Students then suggested and tried alterations to the
first-generation primers to create a suitable multiplex. Multiplexing is an important tool in forensics. This lab is invaluable for
students pursuing molecular biology and forensics as bachelorlevel chemists or biologists and those continuing with postgraduate education.
This lab is compatible with our budgetary constraints as the
fixed costs of the mini-Mac computers had already been paid by
the department and the cost of Internet access is borne by the
students of the college. This laboratory exercise requires no
expensive molecular biology reagents for the in silico portion (in
stark contrast to the commercial multiplex DNA typing kits) and
is highly accessible to instructors at all institutions, regardless of
funding. The PCR primer design multiplexing laboratory experiment could easily be extended to other species and non-CODIS
loci. The follow-up laboratory in which students tested the
designed primers using real-time PCR was expensive, but the
students were enthusiastic about the opportunity to evaluate
their work experimentally. Students were especially pleased to
determine that most of their primers amplified the DNA and that
some of their multiplexes were successful.
’ ASSOCIATED CONTENT
bS Supporting Information
A student handout consisting of background information and
instructions and instructions for the instructor. This material is
available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: kelkins1@mscd.edu.
’ ACKNOWLEDGMENT
ACS Project SEED is gratefully acknowledged for a summer
stipend for Ain Ealey who tested revisions of this lab in the summer
of 2009 and provided useful suggestions. I wish to thank my
students in the spring 2011 CHE 3710 Criminalistics II course for
trying the improved version of the experiment including experimentally testing their primers: Benjamin Bender, Audrionna
Kingsley, Hannah Leger, Megan Owens, Tiffany Stone, and
Francesca Garcia Wheeler. I wish to thank my students in the
spring 2009 CHE 3710 Criminalistics II course for their suggestions for improvement: Patrick Bevins, Sarah Bonsall, Lindsay
Christopherson Powis, Kali Gipson, Natalie Hernandez, Raelynn
Kadunc, Jeffrey Minutillo, Carrie Melanson, Tanya Mokelki,
Michelle Montoya, and Zachary Morin. I wish to thank my
students in the spring 2008 CHE 3710 Criminalistics II course
for their patience, cooperation and input in the inaugural running
of this lab: Katelin Arnold, Jennifer Auger, Lydia Benyam, Megan
Jones McDowell, Jacqueline Keller, Erin Knopka, Gina Mann,
Susan McLaughlin, Andrea Moore, Melanie Newman, Stephanie
Sauter, Kurt Smith, Nia Travers, and Nikole Whitsitt.
’ REFERENCES
(1) Jackson, D. D.; Abbey, C. S.; Nugent, D. J. Chem. Educ. 2006,
83, 774–776.
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Journal of Chemical Education
LABORATORY EXPERIMENT
(2) Carson, T. M.; Bradley, S. Q.; Fekete, B. L.; Millard, J. T. J. Chem.
Educ. 2009, 86, 376–378.
(3) Millard, J. T.; Pilon, A. M. J. Chem. Educ. 2003, 80, 444–446.
(4) Taylor, A.; Sajan, S. J. Chem. Educ. 2005, 82, 597–598.
(5) Suwanjinda, D.; Eames, C.; Panbangred, W. Biochem. Mol. Biol.
Educ. 2007, 35, 364–369.
(6) Lubin, I. M.; Wilson, B.; Grant, K. B. J. Chem. Educ. 2003,
80, 1289–1291.
(7) Childs-Disney, J. L.; Kauffmann, A. D.; Poplawski, S. G.; Lysiak,
D. R.; Stewart, R. J.; Arcadi, J. K.; Dinan, F. J. J. Chem. Educ. 2010,
87, 1110–1112.
(8) Claros, M. G.; Quesada, A. R. Biochem. Mol. Biol. Educ. 2000,
28, 223–226.
(9) Thion, L.; Vossen, C.; Couderc, B.; Erard, M.; Clemenc-on, B.
Biochem. Mol. Biol. Educ. 2002, 30, 51–55.
(10) Brinegar, C.; Levee, D. Biochem. Mol. Biol. Educ. 2004,
32, 35–38.
(11) Kim, T. D. Biochem. Mol. Biol. Educ. 2000, 28, 274–276.
(12) Lima, A. O. S.; Garc^es, S. P. S. Biochem. Mol. Biol. Educ. 2006,
34, 332–337.
(13) Bornhorst, J. A.; Deibel, M. A.; Mulnix, A. B. Biochem. Mol. Biol.
Educ. 2004, 32, 173–182.
(14) Thornton, B.; Basu, C. Biochem. Mol. Biol. Educ. 2011, 39,
145–154.
(15) National Center for Biotechnology Initiative. http://www.ncbi.
nlm.nih.gov/ (accessed June 2011).
(16) Integrated DNA Technologies’ OligoAnalyzer 3.1. http://
www.idtdna.com/analyzer/Applications/OligoAnalyzer/ (accessed
June 2011).
(17) NCBI Primer-Blast. http://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed June 2011).
(18) Masibay, A.; Mozer, T. J.; Sprecher, C. J. Forensic Sci. 2000, 45,
1360-1362, http://www.cstl.nist.gov/strbase/promega_primers.htm
(accessed June 2011).
(19) Elkins, K. M.; Kadunc, R. E. J. Chem. Educ. 2011, in press.
(20) Butler, J. M. Forensic DNA Typing, 2nd ed.; Elsevier: Burlington,
MA, 2005; pp 1 660.
(21) Horsman, K. M.; Hickey, J. A.; Cotton, R. W.; Landers, J. P.;
Maddox, L. O. J. Forensic Sci. 2006, 51, 758–765.
1427
dx.doi.org/10.1021/ed100676b |J. Chem. Educ. 2011, 88, 1422–1427
Committee on Professional Training
Preparing a Research Report
A research experience provides undergraduates a problem-solving activity unlike anything else in the curriculum.
It provides exposure to research methodology and an opportunity to work closely with a faculty advisor. It usually
requires the use of advanced concepts, a variety of experimental techniques, and state-of-the-art instrumentation.
Ideally, undergraduate research should focus on a well-defined project that stands a reasonable chance of
completion in the time available. A literature survey alone is not a satisfactory research project. Neither is
repetition of established procedures. The Committee on Professional Training (CPT) strongly supports efforts by
departments to establish active and vibrant undergraduate research programs, recognizing the role that research
can play in developing a wide range of student skills. The 2015 guidelines allow for the use of undergraduate
research both as in-depth coursework, as well as a means of meeting 180 of the 400 laboratory hours required for
certification provided that a well-written, comprehensive, and well-documented research report is prepared at the
end of a project (samples of such research reports must be submitted with the periodic reports.) The CPT has a
separate supplement outlining the components of successful research programs and projects.
Preparation of a comprehensive, well-documented and appropriately referenced written research report is an
essential part of a valid research experience, and the student should be aware of this requirement at the outset of
the project. Interim reports may also be required, usually at the termination of the quarter or semester. Sufficient
time should be allowed for satisfactory completion of reports, taking into account that initial drafts should be
critiqued by the faculty advisor and corrected by the student at each stage. It may be expected that concrete
outcomes of any research project would be student presentation of research results at a professional meeting
and/or co-authorship on a journal publication. While desirable outcomes, they are not a substitute for a wellwritten comprehensive report that demonstrates that the student has a full grasp of the scope of the problem, the
techniques/instrumental methods used, and the ramifications of the results generated (much as might be expected
for a capstone paper or a B.S. thesis). The student report should receive substantive critique and correction by
the faculty mentor in its development.
Guidelines on how to prepare a professional-style research report are not always routinely available. For this
reason, the following information on report writing and format is provided to be helpful to undergraduate
researchers and to faculty advisors. Much of what follows is similar to what authors would find in many ‘guidelines
to authors’ instructions for most journal submissions.
The most comprehensive student research reports examined by CPT have been those student reports reviewed
by more faculty than just the supervising research advisor. In some cases, programs require an approval of the
report by several faculty members; in such cases, student research reports are often of high quality.
Organization of the Research Report
Most scientific research reports, irrespective of the field, parallel the method of scientific reasoning. That is: the
problem is defined, a hypothesis is created, experiments are devised to test the hypothesis, experiments are
conducted, and conclusions are drawn. The exact format of scientific reports is often discipline dependent with
variations in order and content. The student is encouraged to adopt the format that is most appropriate to the
discipline of the research. Many journals offer a formatting template to aid the author. One example of such a
framework is as follows:
•
•
•
•
•
•
•
•
Title
Abstract
Introduction
Experimental Details or Theoretical Analysis
Results
Discussion
Conclusions and Summary
References
Title and Title Page
The title should reflect the content and emphasis of the project described in the report. It should be as short as
possible and include essential key words.
The author’s name (e.g., Mary B. Chung) should follow the title on a separate line, followed by the author’s
affiliation (e.g., Department of Chemistry, Central State College, Central, AR 76123), the date, and possibly the
origin of the report (e.g., In partial fulfillment of a Senior Thesis Project under the supervision of Professor Danielle
F. Green, June, 1997).
All of the above could appear on a single cover page. Acknowledgments and a table of contents can be added as
preface pages if desired.
Abstract
The abstract should concisely describe the topic, the scope, the principal findings, and the conclusions. It should
be written last to accurately reflect the content of the report. The length of abstracts varies but seldom exceeds
200 words.
A primary objective of an abstract is to communicate to the reader the essence of the paper. It should provide
sufficient information to describe the important features of the project in the absence of the rest of the document.
The reader will then be the judge of whether to read the full report or not. Were the report to appear in the primary
literature, the abstract would serve as a key source of indexing terms and key words to be used in information
retrieval. Author abstracts are often published verbatim in Chemical Abstracts.
Introduction
“A good introduction is a clear statement of the problem or project and the reasons for studying it.” (The ACS
Style Guide. American Chemical Society, Washington, DC, 2006.)
The nature of the problem and why it is of interest should be conveyed in the opening paragraphs. This section
should describe clearly but briefly the background information on the problem, what has been done before (with
proper literature citations), and the objectives of the current project. A clear relationship between the current
project and the scope and limitations of earlier work should be made so that the reasons for the project and the
approach used will be understood.
Experimental Details, Computation Procedures, or Theoretical Analysis
This section should describe what was actually done. It is a succinct exposition of the laboratory and
computational details, describing procedures, techniques, instrumentation, special precautions, characterization of
compounds and so on. It should be sufficiently detailed that other experienced researchers would be able to
repeat the work and obtain comparable results.
-2-
In theoretical reports, this section would include sufficient theoretical or mathematical analysis to enable
derivations and numerical results to be checked. Computer programs from the public domain should be cited.
New computer programs should be described in outline form.
If the experimental section is lengthy and detailed, as in synthetic work, it can be placed at the end of the report so
that it does not interrupt the conceptual flow of the report. Its placement will depend on the nature of the project
and the discretion of the writer.
Results
In this section, relevant data, observations, and findings are summarized. Tabulation of data, equations, charts,
and figures can be used effectively to present results clearly and concisely. Schemes to show reaction sequences
may be used here or elsewhere in the report.
Discussion
The crux of the report is the analysis and interpretation of the results. What do the results mean? How do they
relate to the objectives of the project? To what extent have they resolved the problem? Because the “Results”
and “Discussion” sections are interrelated, they can often be combined as one section.
Conclusions and Summary
A separate section outlining the main conclusions of the project is appropriate if conclusions have not already
been stated in the “Discussion” section. Directions for future work are also suitably expressed here.
A lengthy report, or one in which the findings are complex, usually benefits from a paragraph summarizing the
main features of the report – the objectives, the findings, and the conclusions.
The last paragraph of text in manuscripts prepared for publication is customarily dedicated to acknowledgments.
However, there is no rule about this, and research reports or senior theses frequently place acknowledgments
following the title page.
References
Thorough, up-to-date literature references acknowledge foundational work, direct the reader to published
procedures, results, and interpretations, and play a critical role in establishing the overall scholarship of the report.
The report should include in-text citations with the citations collated at the end of the report and formatted as
described in The ACS Style Guide or using a standard established by an appropriate journal. The citation process
can be facilitated by using one of several available citation software programs. In a well-documented report, the
majority of the references should come from the primary chemical literature. Because Internet sources are not
archival records, they are generally inappropriate as references for scholarly work. They should be kept to a bare
minimum.
Preparing the Manuscript
The personal computer and word processing have made manuscript preparation and revision a great deal easier
than it used to be. It is assumed that students will have access to word processing and to additional software that
allows spelling to be checked, numerical data to be graphed, chemical structures to be drawn, and mathematical
equations to be represented. These are essential tools of the technical writer. All manuscripts should be carefully
proofread before being submitted. Preliminary drafts should be edited by the faculty advisor (and/or a supervising
committee) before the report is presented in final form.
-3-
Useful Texts
Writing the Laboratory Notebook, Kanare, H.M., American Chemical Society, Washington, DC, 1985.
This book describes among other things the reasons for note keeping, organizing and writing the notebook with
examples, and provides photographs from laboratory notebooks of famous scientists.
rd
ACS Style Guide: Effective Communication of Scientific Information, Coghill, A.M., Garson, L.R.; 3 Edition,
American Chemical Society, Washington, DC, 2006.
This volume is an invaluable writer’s handbook in the field of chemistry. It contains a wealth of data on preparing
any type of scientific report and is useful for both students and professional chemists. Every research laboratory
should have a copy. It gives pointers on the organization of a scientific paper, correct grammar and style, and
accepted formats in citing chemical names, chemical symbols, units, and references.
There are useful suggestions on constructing tables, preparing illustrations, using different fonts, and giving oral
presentations. In addition, there is a brief overview of the chemical literature, the way in which it is organized and
how information is disseminated and retrieved. A selected bibliography of other excellent guides and resources to
technical writing is also provided. See also The Basics of Technical Communicating. Cain, B.E.; ACS
Professional Reference Book American Chemical Society: Washington, DC, 1988.
Write Like a Chemist, Robinson, M.S., Stoller, F.L., Costanza-Robinson, M.S., Jones, J.K., Oxford University
Press, Oxford, 2008.
This book addresses all aspects of scientific writing. The book provides a structured approach to writing a journal
article, conference abstract, scientific poster and research proposal. The approach is designed to turn the
complex process of writing into graduated, achievable tasks.
Last revised in August 2015
-4-
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Lane 12
Ladder
Neg Control multiplex AG/CG
Experimental DNA Multiplex
AC/CG
U937 DNA Multiplex
AC/CG
Extracted DNA multiplex Kate/Ellie
Negative control multiplex Sierra/Hunter/Nicole
U937 multiplex Sierra/Hunter/Nicole
Experimental multiplex Sierra/Hunter/Nicole
Experimetnal DNA multiplex GCW/CC
U937 multiplex ECW
Experimental DNA multiplex ECW
Negative control Multiplex ECW
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Lane 8
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Lane 10
Experimental DNA Multiplex Katy/Michael/Madi
Experimental DNA Multiplex Dylan/Josi
Not labeled Exp DNA Multiplex
Not Labeled Exp DNA multiplex
Ladder
Neg Control Multiplex Collin Perry
U937 CSF1Po
Exp DNA multiplex
Exp DNA CSF1PO
U937 multiplex
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Lane 15
U937 CSF1PO
CET
Ladder
Neg Control CSF1PO CET
Exp CSF1PO CET
U937 D75820
ME
Neg Control D75820 ME
Exp DNA D75820 ME
U937 CSF1PO KAL
Neg control CSF1PO KAL
Exp DNA CSF1PO
KAL
Exp DNA HUMTH01 ——– A10
Neg Control HUMTH01 ——— A11
U937 HUMTH01 ———– A12
Neg control Y-Gata-H4
C1
Exp DNA Y-Gata-H4
C2
Lane 1
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Neg Control Y-GATA-H$ —B1
U937 Y-GATAH4 — —- B2
Exp DNA Y-GATA H4 ——- B3
Exp HumTH01 ———- B4
U937 DNA Overflow of Ladder Sorry — B5
Ladder
Negative Control HUMTH01 ———- B6
U937 D7s820—— B7
Neg control D7S820—— B8
Exp DNA D7S820 —– B9
Exp DNA Y-Gata ———- B10
U937 Y-GATA——— B11
Neg Control Y-GATA H4 ——B12
U937 Y-GATAH$———- C3
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)
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