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Laboratory Exercises
Sex Determination Using PCR*
Received for publication, September 8, 2003, and in revised form, October 24, 2003
Peter E. Kima‡ and Madeline E. Rasche
From the Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following
exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y
chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on
the pattern of PCR products, students can distinguish between male and female samples and determine the
gender of an unknown DNA donor. The exercise is presented for upper division undergraduate majors in
microbiology, biochemistry, and molecular biology, but can be adapted to different academic levels and
disciplines. The use of student samples in the exercise can enhance learning of these techniques by making
PCR and agarose gel electrophoresis directly relevant to the students.
Keywords: Agarose gel electrophoresis, DNA isolation
PCR has revolutionized many aspects of molecular bi-
ology research with widespread applications that include
DNA cloning, identification of organisms in medical and
environmental samples, studies of gene regulation, evolu-
tionary biology, and forensic science [1–4]. In PCR, large
quantities of DNA can be synthesized from small quantities
of starting material (template DNA) using two oligonucleo-
tide primers and a thermostable DNA polymerase [5–7].
The current work describes a laboratory exercise in which
students isolate their own DNA, amplify segments of the X
and Y chromosomes, and separate the PCR products on
horizontal agarose gels. The sizes and patterns of PCR
products enable students to distinguish between male and
female samples and identify the gender of an unknown
DNA donor. The use of student samples in this exercise
can enhance the understanding of PCR and gel electro-
phoresis by making the techniques directly relevant to the
students.
PCR requires picogram to nanogram amounts of tem-
plate DNA plus two oligonucleotide primers that are com-
plementary to the ends of the DNA region to be amplified
[5–8]. The first primer is complementary to the beginning
of the target DNA segment. The second primer is comple-
mentary to the other end of the target region, but on the
opposite strand (Fig. 1). The template and primers are
mixed in a buffer solution that contains magnesium ions,
the four deoxyribonucleotides, and a thermostable DNA
polymerase such as Taq polymerase, which was originally
isolated from the thermophilic bacterium Thermus aquati-
cus. The sample is heated to 94 °C to separate the strands
of template DNA (denaturation step). Although heating at
this temperature destroys the activity of most mesophilic
DNA polymerases, Taq polymerase remains active. The
sample is cooled to �50 °C to enable the primers to an-
neal to the template (annealing step); then the sample is
heated to 72 °C to allow Taq polymerase to synthesize
DNA starting from the two primers (extension step). The
cycles of denaturation, annealing, and extension are re-
peated 35–40 times using an automatic thermocycler,
which results in exponential increases in the amount of
DNA delineated by the primers.
With the availability of commercial kits to facilitate the
isolation of genomic DNA, quantities of DNA sufficient for
amplification by PCR can be obtained from cells scraped
gently from the cheek of a donor. A commercial kit is also
available for the amplification of a segment of the human X
and Y chromosome (X&Y chromosome primer set; Maxim
Biotech, South San Francisco, CA). This kit contains two
primers that amplify a 977-bp fragment from the X chro-
mosome and a 788-bp fragment from the Y chromosome.
After separating the PCR products on agarose gels, stu-
dents use the DNA size information to explain the banding
pattern of the products and determine the gender of the
DNA donors. The exercise can be extended to include an
unknown DNA sample and can be completed in three 2- to
3-h laboratory periods. Students begin DNA isolation in the
first period (2–2.5 h); complete the DNA isolation and set
up the PCR in the second period (2 h); and pour and run
agarose gels during the last period (2 h).
The laboratory exercise as described here is appropriate
for advanced upper division undergraduate laboratories in
biochemistry, molecular biology, molecular genetics, or
* This work was supported in part by National Science Foun-
dation Grant MCB-9876212 and the Florida Agricultural Experi-
ment Station. This article is Florida Agricultural Experiment Sta-
tion Journal Series Number R-09889. The cost of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
‡ To whom correspondence should be addressed: Microbiol-
ogy and Cell Science Department, University of Florida, P.O. Box
110700, Gainesville, FL 32611-0700. E-mail: pkima@ufl.edu.
© 2004 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION
Printed in U.S.A. Vol. 32, No. 2, pp. 115–119, 2004
This paper is available on line at http://www.bambed.org 115
microbiology. Ideally, students should have background in
the theory of DNA replication and PCR from an introduc-
tory biochemistry, genetics, or microbiology lecture
course. The principles can be reinforced through an intro-
ductory lecture prior to the first laboratory period. The
laboratory exercise can also be adapted to lower division
biology courses by simplifying the introductory materials,
laboratory write-ups, and test questions and by providing
prepoured gels that minimize student contact with poten-
tially harmful chemicals such as ethidium bromide.
EXPERIMENTAL PROCEDURES
Prelaboratory Skills—For success in this exercise, the following
technical skills are required: sterile laboratory technique, accurate
pipetting ability, knowledge of techniques for preserving enzyme
activity, and use of microcentrifuges and semi-logarithmic paper.
Knowledge of DNA replication, PCR, and agarose gel electro-
phoresis is helpful, but may be provided in a background lecture
prior to the laboratory exercise. Students work in groups of two or
three; at least one member of the group must be male and one
must be female.
Equipment—The laboratory exercise requires a microcentri-
fuge, two heating water baths, gel electrophoresis equipment
(power supplies, horizontal gel boxes, trays, and combs), and one
or more thermocyclers with sufficient capacity to accommodate
the number of student groups. An ultraviolet light source with an
ultraviolet-protective shield is needed to visualize the DNA bands.
If available, a gel documentation device or camera can be used to
produce a picture of the gel. For the experiments described here,
the Mastercycler Gradient thermocycler and 5415C microcentri-
fuge (Eppendorf, Hamburg, Germany), Mini-Sub Cell GT Power-
Pac 300 gel electrophoresis system (catalog no. 165-4347; Bio-
Rad, Hercules, CA), and IS-1000 Digital Imaging System (Alpha
Innotech Corp., San Leandro, CA) were used.
Kits, Enzymes, and Solutions—The genomic DNA isolation kit
(catalog no. SA-40001) and X&Y chromosome primer set kit
(catalog no. SP-10704) were purchased from Maxim Biotech. The
primer kit provides the two primers, PCR buffer with nucleotides,
100-bp DNA ladder, and human DNA for use as a positive control.
Theoretically, each kit contains enough material for 100 assays
(33 groups); however, because excess solution is provided to
each group, one kit will accommodate 20 to 24 sets of three PCR,
including pretesting the experiment. The amount of 100-bp DNA
ladder in the kit may be limiting, but additional DNA ladder may be
purchased separately from the company. Taq polymerase (5 U/�l)
was obtained fromEppendorf. DNA sample loading buffer can be
purchased (catalog no. 161-0767; Bio-Rad) or prepared accord-
ing to standard protocols [9]. Agarose (SeaKem GTG) was ob-
tained from BioWhittaker Molecular Applications (Rockland, ME).
Ethidium bromide (a mutagen) was purchased as a 10 mg/ml
solution (Bio-Rad), and the concentrated solution was handled
only by the instructor or teaching assistant wearing appropriate
gloves. Students were allowed to handle diluted ethidium bro-
mide solutions using gloves with warnings about its mutagenic
properties. Other DNA dyes with reportedly less harmful proper-
ties are available (Gelstar nucleic acid stain; FMC, Philadelphia,
PA).
Laboratory Supplies and Set-up—For each group, the follow-
ing supplies are required: gloves for handling solutions with
ethidium bromide, pipettes and sterile pipette tips for volumes
from 2 to 1000 �l, four sterile 1.5-ml microcentrifuge tubes for
isolating DNA, two sterile 1-ml pipette tips (or two sterile swabs)
for collecting cheek cell samples, three 0.5-ml PCR tubes, and
three sterile 0.5-ml microcentrifuge tubes for preparing electro-
phoresis samples.
Because students often have limited experience in pipetting
and handling active enzymes, it may be beneficial to provide
individual groups with aliquots of the various solutions used in the
procedure. In our laboratories, it was also advantageous to have
either the teaching assistant or the instructor add the enzymes.
Without these precautions, pipetting errors by students have
occasionally resulted in significant losses of expensive enzymes
from the stock containers. Alternatively, Taq polymerase can be
mixed with the optimized PCR buffer immediately prior to the
laboratory period and provided as a PCR buffer/polymerase mix
(0.2 �l of Taq polymerase per 30 �l of PCR buffer).
Provide each group of students with an ice bucket containing
separate aliquots of labeled components (1.2–2 times the volume
specified in the protocol). The solutions and volumes indicated
below apply for the Maxim Biotech genomic DNA purification kit.
Other kits may also be used. For DNA isolation (day 1), aliquot cell
lysis solution (1.5 ml of BD-3 solution), ribonuclease A (25 �l on
ice), protein precipitating solution (0.5 ml of BD-4 solution on ice),
and 100% ethanol (1.5 ml at room temperature).
For day 2, supply ice-cold 70% ethanol (2.5–3 ml), sterile
distilled, deionized water (0.5 ml), primers from the X&Y primer kit
(40 �l), 10-fold-diluted human genomic DNA (15 �l), and PCR
buffer/polymerase mix (120 �l). For agarose gel electrophoresis
(day 3), provide 5� nucleic acid sample buffer (15 �l) and Tris-
acetate-EDTA (TAE)1 electrophoresis buffer (300 ml of 40 mM Tris
acetate, 1 mM EDTA). Also, premix 20 �l of sample buffer with 100
�l of 100-bp DNA ladder, and aliquot 6 �l to each group. To
minimize exposure of students to concentrated ethidium bromide
solutions, the teaching assistant can melt the agarose (1% in TAE
buffer) immediately before the laboratory period, place 50-ml
aliquots in heat-resistant disposable 50-ml tubes, add ethidium
bromide, and store the tubes in a 65 °C water bath until use.
Safety Concerns—Ethidium bromide and some other DNA
dyes are mutagenic. Wear gloves and avoid contact with these
substances. Properly dispose of pipette tips that contact concen-
trated ethidium bromide. Autoclave all biological samples at the
end of the exercise and dispose according to the Environmental
Health and Safety policy of the individual campus.
Experimental Procedures—The manufacturer’s instructions for
the genomic DNA isolation kit were provided to the students, and
the procedure is summarized below. Prepare separate male and
female DNA samples. Gently scrape the inside of both cheeks 10
times with a sterile 1-ml pipette tip or a sterile swab. Some solid
1 The abbreviation used is: TAE, Tris-acetate-EDTA.
FIG. 1. Position of the primers in the PCR. Primer 1 is com-
plementary to the beginning of the target region to be amplified.
Primer 2 is complementary to the end of the target region, but on
the opposite strand. After 35 cycles of denaturation, annealing,
and extension, large quantities of the DNA region defined by the
primers are produced.
116 BAMBED, Vol. 32, No. 2, pp. 115–119, 2004
material and saliva should be visible in the pipette tip. Mix the
sample thoroughly with 0.6 ml of cold BD-3 solution (lysis solu-
tion) in a sterile 1.5-ml microcentrifuge tube. The lysis solution
contains detergent and a strong base to disrupt the cell mem-
brane and release the cellular contents. Homogenize the sample
by mixing and then incubate at 65 °C for 30 min. Cool the solution
to room temperature and add 10 �l of RNase A solution to digest
the RNA molecules present in the sample. Incubate at 37 °C for
20 min and then place the tube on ice for 5 min. Add 0.2 ml of
BD-4 solution, which contains a salt that causes the proteins to
precipitate through a salting-out effect. Mix well by inverting the
tube several times, and centrifuge for 10 min in a microcentri-
fuge at maximum speed to remove the precipitated proteins.
Collect the supernatant containing DNA and transfer to a fresh
1.5-ml microcentrifuge tube. Add 0.6 ml of 100% ethanol at
room temperature, and invert the tube 40–50 times to precip-
itate the DNA. Store the centrifuge tubes at 4 °C until the next
laboratory period.
Completion of DNA Isolation and Preparation of the PCR Mix-
tures—To complete the DNA isolation, centrifuge the tubes at
maximum speed for 10 min and discard the supernatant. The
DNA should precipitate, but in most cases will not be visible.
Gently pipette 1 ml of ice-cold 70% ethanol into the tube to wash
the DNA. Do not mix the contents of the tube. Centrifuge for 5
min, discard the supernatant, and allow the pellet to air-dry. Add
0.2 ml of sterile distilled deionized water. Heat the tube at 65 °C
for 20 min, inverting the tube several times during the incubation
to dissolve the DNA. If necessary, the DNA sample can be stored
at �20 °C, but should not be repeatedly frozen and thawed.
Label three PCR tubes with the group name and DNA sample
(either “female,” “male,” or “control”). To each tube add 10 �l of
the X&Y chromosome primer mix. Then add 30 �l of PCR buffer/
polymerase mix. Immediately before placing the samples in the
thermocycler, add 10 �l of female DNA to the first tube, 10 �l of
male DNA to the second tube, and 10 �l of 10-fold-diluted
control human DNA from the kit to the third tube. Use a fresh tip
for each tube, and gently mix the solution using the pipette tip.
If necessary, centrifuge the tubes briefly (5 s) to bring all the
liquid down to the bottom of the tube before placing in the
thermocycler. The recommended conditions are 96 °C for 1 min
for initial DNA denaturation (one cycle); 94 °C for 1 min, 55 °C
for 1 min, 72 °C for 1 min (40 cycles); 72 °C for 10 min (one
cycle); and 4–25 °C (hold, if samples cannot be collected im-
mediately after PCR).
Agarose Gel Electrophoresis—Provide instructions and mate-
rials to enable students to prepare horizontal 1% agarose gels
(7 � 10 cm) in TAE buffer. Place the gels in the gel boxes with TAE
buffer. Label three sterile microcentrifuge tubes as female, male,
or control DNA. Add 3 �l of 5� sample buffer to each tube. Using
a fresh tip for each tube, add 15 �l of each PCR to the appropri-
ately labeled microcentrifuge tube. Mix briefly. Load 15 �l of each
mixture into a separate lane on the gel. In the fourth lane, add 5
�l of DNA ladder (molecular size standard) premixed with DNA
sample loading buffer. Secure the lid on the gel box and run the
gel until the darker blue dye reaches one-half to two-thirds the
length of the gel (typically 45 min at 100 V). Turn off the power
supply, remove the gel, and record the banding pattern observed
with ultraviolet light.
RESULTS
Anticipated Results—Females possess only X chromo-
somes, and therefore a single band of 977 bp is antici-
pated, corresponding to the expected size of the PCR
product from the X chromosome(Fig. 2, lane 1). Males
possess both X and Y chromosomes, and therefore am-
plification by PCR will produce two DNA bands of 977 bp
and 788 bp (Fig. 2, lane 2). The control DNA provided with
the kit produces either one band, indicating a female DNA
donor, or two bands, indicating a male (Fig. 2, lane 3).
Typically, the DNA bands representing PCR products from
the control DNA are more intense than the products from
the student-isolated DNA, presumably because there is
less DNA template in the student samples. Faint bands
greater than 1000 bp in size occasionally appear due to
nonspecific binding of the primers.
The control DNA is used to verify that the PCR reagents
are functional, but can also serve as an unknown. The
results for an additional DNA donor (such as the teaching
assistant or instructor) can be provided to the students as
a second unknown (Fig. 2, lane 5). Students can determine
the gender of unknown DNA donors by comparison to
known samples or by the sizes of the PCR products rela-
tive to the DNA standards. Students should estimate the
size of the X and Y chromosome fragments using semi-
logarithmic paper. Plotting the size of the DNA standards
on the logarithmic axis versus the Rf value (or distance
migrated on the gel) on the x-axis typically results in a
straight line.
Common Student Pitfalls—In each of the four semesters
that the exercise has been offered, some of the groups
have obtained the anticipated results. Among the groups
demonstrating only partial success, many obtained visible
bands in the lanes containing control DNA and molecular
size standards but lacked bands in one (or both) of the
student-derived samples. This can result from starting with
a very small sample of cheek cells or from errors in isolat-
ing DNA (such as discarding the BD-4 supernatant that
contains the DNA). Another student pitfall involves inaccu-
rate pipetting technique, particularly while drawing up liq-
uid solutions. Because PCR is highly sensitive to the con-
centrations of its reaction components, pipetting errors
can lead to the appearance of extra bands on the gel or to
complete failure of the PCR, even with the control DNA. A
FIG. 2. Agarose gel of PCR products from the amplification
of regions of the human X and Y chromosomes. PCR products
from female DNA (lane 1), male DNA (lane 2), and human control
DNA (lane 3). The DNA molecular size standard is in lane 4. The
PCR product from an unknown DNA donor (one of the instructors)
is shown in lane 5.
117
common warning sign of pipetting inaccuracy is that a
group may run out of their solution aliquots during prepa-
ration of the DNA samples.
To maximize the probability of student success, it is
recommended that the exercise be prerun with all of the
actual kits, enzymes, and solutions to be used by the
students. Prerunning the exercise will also enable the
teaching assistant to optimize the volumes of PCR product
loaded onto the gel as well as the time period for running
the gels.
Presentation of Data—We have used two different for-
mats for student presentation of data. The first is a work-
sheet or short report in which students respond to the
following instructions. First, attach a copy of the PCR
data to the report; second, generate a standard curve on
semi-logarithmic paper and estimate the sizes of the
PCR products; third, discuss the results using the fol-
lowing questions as a guide. Did you obtain PCR prod-
ucts from each of your samples? How many bands did
you get from amplification of the control DNA? What size
fragments did you obtain from the amplification of your
samples?
The second format is a short formal laboratory report
(two to three typed, double-spaced pages) requiring a title,
statement of objectives, materials and methods (referenc-
ing the manual with technical changes noted), results (in-
cluding labeled figures with titles and legends), discussion,
and references. In the discussion, students should explain
the banding pattern of known samples, identify the gender
of the unknown DNA donor, discuss whether the results
were consistent with anticipated results, propose explana-
tions for discrepancies (using theoretical knowledge of
trouble-shooting strategies), and describe how the exer-
cise might be improved in the future. An interesting obser-
vation in one of our classes has been that many of the
students are puzzled by the appearance of two bands in
the male sample. The answer can be drawn out of students
by asking them to explain the chromosomal composition
of males (XY) versus females (XX).
DISCUSSION
This laboratory exercise introduces students to the prin-
ciples and techniques of PCR and agarose gel electro-
phoresis in the context of a problem that is directly appli-
cable to the student (determining the gender of a DNA
donor). The techniques have broad applications for ca-
reers in scientific research, biotechnology, and medical
laboratories. The principles of PCR and agarose gel elec-
trophoresis can be discussed prior to the exercise, and
references to additional theoretical and practical topics
can be provided [5–9]. Examples of background topics
that have been presented are principles of primer design,
optimization of PCR conditions, and trouble-shooting
strategies.
We have found that the use of student samples as the
DNA template can assist in capturing and maintaining
student attention throughout the exercise, which can con-
tribute strongly to effective learning [10]. It is likely that the
focus on sex, albeit obliquely, also helps to maintain the
students’ interest. Bloom’s taxonomy of cognitive learning
describes a hierarchy of six levels of cognition: knowledge
(memorization), comprehension (understanding), applica-
tion of principles to new situations, analysis, synthesis,
and evaluation [11]. Our examinations and reports typically
address the first three levels of this pedagogical model.
For example, use of the worksheet format assists students
in understanding and interpreting their results. Examina-
tion topics include practical knowledge of PCR and elec-
trophoresis conditions, theoretical understanding of the
techniques, trouble-shooting strategies, and application of
the techniques to new situations. With the formal labora-
tory report, students are further challenged to analyze their
data and account for discrepancies from the anticipated
results (analysis, the fourth level of cognitive learning).
Student response to the PCR exercise has been in-
creasingly positive based on responses to the standard
Course and Instructor Evaluation surveys given by the
University of Florida College of Agricultural and Life Sci-
ences at the end of each semester (Table I).
Sample Examination and Study Questions
1. In PCR, the term that best describes the DNA
sample to be amplified is the
A. template
FIG. 3. Diagram provided to students for sample test ques-
tion 3. Using your knowledge obtained from the PCR experiment,
what is the gender of Individual A in Fig. 3?
TABLE I
Summary of student responses to standard course and instructor
evaluation at the University of Florida
Semestera
Average overall rating
of course (for section
including PCR exercise)
Average overall rating of
instructor (for section
including PCR exercise)
Fall 2001 3.60 3.90
Spring 2002 4.02 4.28
Fall 2002 4.10 4.25
Spring 2003 4.16 4.79
a Each semester, 72 students were enrolled in the course. The
number of respondents to the survey ranged from 53 to 59 students
each semester. In the scale used, 5 � excellent, 4 � above average,
3 � average, 2 � below average, and 1 � poor.
118 BAMBED, Vol. 32, No. 2, pp. 115–119, 2004
B. primer
C. enzyme
D. Taq polymerase
E. antigen
2. What was the purpose of running the PCR prod-
ucts on an agarose gel?
3. Using your knowledge obtained from the PCR
experiment, what is the gender of Individual A in
Fig. 3?
4. While purifying the DNA for the PCR, a student
breaks the cells using solution BD-3, adds ribo-
nuclease, adds BD-4 solution, centrifuges the
mixture, and discardsthe supernatant. The stu-
dent then adds 100% ethanol, centrifuges the
mixture, discards the supernatant, and washes
the pellet with 70% ethanol. The PCR shows no
DNA product. What is the most likely source of
error?
5. Design and describe a protocol that uses PCR to
detect anthrax in sheep
Acknowledgments—We are grateful to Lawrence Flowers and
Johnny Davis for assistance in preparing the laboratory manual
for this exercise.
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