Sex determination using PCR

Authors

  • Peter E. Kima,

    Corresponding author
    1. Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
    • Microbiology and Cell Science Department, University of Florida, P.O. Box 110700, Gainesville, FL 32611-0700
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  • Madeline E. Rasche

    1. Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611
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  • This work was supported in part by National Science Foundation Grant MCB-9876212 and the Florida Agricultural Experiment Station. This article is Florida Agricultural Experiment Station Journal Series Number R-09889.

Abstract

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.

PCR has revolutionized many aspects of molecular biology research with widespread applications that include DNA cloning, identification of organisms in medical and environmental samples, studies of gene regulation, evolutionary biology, and forensic science [14]. In PCR, large quantities of DNA can be synthesized from small quantities of starting material (template DNA) using two oligonucleotide primers and a thermostable DNA polymerase [57]. 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 electrophoresis by making the techniques directly relevant to the students.

PCR requires picogram to nanogram amounts of template DNA plus two oligonucleotide primers that are complementary to the ends of the DNA region to be amplified [58]. The first primer is complementary to the beginning of the target DNA segment. The second primer is complementary 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 aquaticus. 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 anneal 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 repeated 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 chromosome and a 788-bp fragment from the Y chromosome. After separating the PCR products on agarose gels, students 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 microbiology. Ideally, students should have background in the theory of DNA replication and PCR from an introductory biochemistry, genetics, or microbiology lecture course. The principles can be reinforced through an introductory 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 potentially 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 electrophoresis 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 microcentrifuge, 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 microcentrifuge (Eppendorf, Hamburg, Germany), Mini-Sub Cell GT PowerPac 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/μl) was obtained from Eppendorf. DNA sample loading buffer can be purchased (catalog no. 161-0767; Bio-Rad) or prepared according to standard protocols [9]. Agarose (SeaKem GTG) was obtained 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 bromide solutions using gloves with warnings about its mutagenic properties. Other DNA dyes with reportedly less harmful properties are available (Gelstar nucleic acid stain; FMC, Philadelphia, PA).

Laboratory Supplies and Set-up—

For each group, the following supplies are required: gloves for handling solutions with ethidium bromide, pipettes and sterile pipette tips for volumes from 2 to 1000 μl, 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 electrophoresis 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 μl of Taq polymerase per 30 μl 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 μl 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 μl), 10-fold-diluted human genomic DNA (15 μl), and PCR buffer/polymerase mix (120 μl). For agarose gel electrophoresis (day 3), provide 5 × nucleic acid sample buffer (15 μl) and Tris-acetate-EDTA (TAE)11 electrophoresis buffer (300 ml of 40 mM Tris acetate, 1 mM EDTA). Also, premix 20 μl of sample buffer with 100 μl of 100-bp DNA ladder, and aliquot 6 μl 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 concentrated 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 material and saliva should be visible in the pipette tip. Mix the sample thoroughly with 0.6 ml of cold BD-3 solution (lysis solution) in a sterile 1.5-ml microcentrifuge tube. The lysis solution contains detergent and a strong base to disrupt the cell membrane 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 μl 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 microcentrifuge 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 precipitate the DNA. Store the centrifuge tubes at 4 °C until the next laboratory period.

Completion of DNA Isolation and Preparation of the PCR Mixtures—

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 μl of the X&Y chromosome primer mix. Then add 30 μl of PCR buffer/polymerase mix. Immediately before placing the samples in the thermocycler, add 10 μl of female DNA to the first tube, 10 μl of male DNA to the second tube, and 10 μl 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 immediately after PCR).

Agarose Gel Electrophoresis—

Provide instructions and materials 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 μl of 5 × sample buffer to each tube. Using a fresh tip for each tube, add 15 μl of each PCR to the appropriately labeled microcentrifuge tube. Mix briefly. Load 15 μl of each mixture into a separate lane on the gel. In the fourth lane, add 5 μl 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 chromosomes, and therefore a single band of 977 bp is anticipated, 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 amplification 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 relative 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 isolating DNA (such as discarding the BD-4 supernatant that contains the DNA). Another student pitfall involves inaccurate pipetting technique, particularly while drawing up liquid solutions. Because PCR is highly sensitive to the concentrations 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 common warning sign of pipetting inaccuracy is that a group may run out of their solution aliquots during preparation 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 formats for student presentation of data. The first is a worksheet 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 following questions as a guide. Did you obtain PCR products 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 (referencing the manual with technical changes noted), results (including 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 explanations for discrepancies (using theoretical knowledge of trouble-shooting strategies), and describe how the exercise might be improved in the future. An interesting observation 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 principles and techniques of PCR and agarose gel electrophoresis in the context of a problem that is directly applicable to the student (determining the gender of a DNA donor). The techniques have broad applications for careers in scientific research, biotechnology, and medical laboratories. The principles of PCR and agarose gel electrophoresis can be discussed prior to the exercise, and references to additional theoretical and practical topics can be provided [59]. 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 contribute 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), application 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. Examination topics include practical knowledge of PCR and electrophoresis conditions, theoretical understanding of the techniques, trouble-shooting strategies, and application of the techniques to new situations. With the formal laboratory 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 increasingly positive based on responses to the standard Course and Instructor Evaluation surveys given by the University of Florida College of Agricultural and Life Sciences at the end of each semester (Table I).

Sample Examination and Study Questions

  • In PCR, the term that best describes the DNA sample to be amplified is the

    • template

    • primer

    • enzyme

    • Taq polymerase

    • antigen

  • What was the purpose of running the PCR products on an agarose gel?

  • Using your knowledge obtained from the PCR experiment, what is the gender of Individual A in Fig. 3?

  • While purifying the DNA for the PCR, a student breaks the cells using solution BD-3, adds ribonuclease, adds BD-4 solution, centrifuges the mixture, and discards the supernatant. The student 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?

  • Design and describe a protocol that uses PCR to detect anthrax in sheep

Figure FIGURE 1..

Position of the primers in the PCR. Primer 1 is complementary 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.

Figure FIGURE 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.

Figure FIGURE 3..

Diagram provided to students for sample test question 3. Using your knowledge obtained from the PCR experiment, what is the gender of Individual A in Fig. 3?

Table Table I. Summary of student responses to standard course and instructor evaluation at the University of Florida
SemesteraAverage overall rating of course (for section including PCR exercise)Average overall rating of instructor (for section including PCR exercise)
  • a

    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.

Fall 20013.603.90
Spring 20024.024.28
Fall 20024.104.25
Spring 20034.164.79

Acknowledgements

We are grateful to Lawrence Flowers and Johnny Davis for assistance in preparing the laboratory manual for this exercise.

Footnotes

  1. 1

    The abbreviation used is: TAE, Tris-acetate-EDTA.

Ancillary