Q 2009 by The International Union of Biochemistry and Molecular Biology

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 37, No. 4, pp. 236242, 2009

Laboratory Exercises Analysis of a p53 Mutation Associated with Cancer Susceptibility for Biochemistry and Genetic Laboratory Courses*
Received for publication, December 18, 2008, and in revised form, April 22, 2009 Isabel Soto-Cruz and Martha Legorreta-Herrera From the Molecular Oncology Laboratory, Postgraduate Studies and Research Division, FES Zaragoza, Universidad Nacional Auto noma de Me xico, Mexico Molecular Immunology Laboratory, Postgraduate Studies and Research Division, FES Zaragoza, Universidad Nacional Auto noma de Me xico, Mexico

We have devised and implemented a module for an upper division undergraduate laboratory based on the amplication and analysis of a p53 polymorphism associated with cancer susceptibility. First, students collected a drop of peripheral blood cells using a sterile sting and then used FTA cards to extract the genomic DNA. The p53 region is then PCR amplied, and the PCR products are digested with the BstUI enzyme to detect the 72 codon polymorphism. Polyacrylamide gel electrophoresis is used to resolve the PCR products, and the results are statistically analyzed in the context of human population genetics. Blood samples in FTA cards were also collected from 50 women to detect the mutation in a wide range of ages and assess its relationship to familial cancer susceptibility. This module enables students to use materials and methods that are routinely used by scientic researchers to analyze polymorphisms. Therefore, it can be used for laboratory exercises in traditional biochemistry curricula as well as in the growing eld of genomic science and education. Keywords: p53, mutation, cancer, polymorphism, genomic DNA, FTA cards, PCR, susceptibility.

The p53 gene is a well-known tumor suppressor gene encoding a nuclear protein that induces growth arrest or apoptosis in response to cellular stress [1]. When the p53 pathway is inhibited, cancer progression accelerates and resistance to chemotherapy develops [2]. Indeed, p53 function is compromised in the majority of human cancers [35]. There is increasing evidence that allele variants in some oncogenes and tumor-suppressor genes are candidates for genetic risk factors that may alter the clinical outcome of cancer. The participation of p53 alterations, including somatic mutations and germline polymorphisms, in tumor development has been well documented. A polymorphism in codon 72 (Arg/Pro allele) of p53 is widely distributed in various human populations [6, 7]. One of the striking features of this polymorphism is the efcient degradation of the Arg-type gene product of p53 by the human papillomavirus (HPV) E6 oncoprotein; carriers of the Arg-type gene product are about seven times more susceptible to cervical cancer [8]. The high risk of cervical cancer associated with the Arg-type gene product of p53 has been supported by several epidemiological studies on European individuals [7, 9]. However, studies on British [10], Japanese [11], and Korean [12] populations have not veried this risk. The

This work is supported by UNAM (PAPIME PE204105). To whom correspondence should be addressed. E-mail: marthal@servidor.unam.mx.

distribution of the codon 72 polymorphic genotype varies according to ethnicity [6], and it is rather difcult to generalize conclusions obtained for any given population. Nonetheless, we considered it highly important that our students learn an easy technique to detect this polymorphism since cervical cancer is the main cancer in the Mexican population. Our learning objective was to teach students the biochemical principles that are at the root of detecting DNA polymorphisms and to provide a greater appreciation and understanding of its benets and extensive applications in todays clinical diagnosis. A secondary learning objective was to teach students the importance of proper and careful laboratory techniques that are essential for successful applications of this method and for the correct statistical analysis of the generated data. We believe that the actual materials and protocols utilized by research laboratories could be applied in undergraduate laboratory course work to benet our students. Our approach builds on previously developed protocols followed in our research laboratories; many of these protocols can be completed with reagents prepared by us in the laboratory. With the methods reported here, the polymorphism of codon 72 of p53 was amplied, digested, and analyzed via polyacrylamide gel electrophoresis and standard ethidium bromide staining protocols. Our students followed the same methods and procedures used by research laboratories all over the world to detect a polymorphism associated with cancer susceptibility.
This paper is available on line at http://www.bambed.org

DOI 10.1002/bmb.20304

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This laboratory exercise teaches students how to use sterile methods to collect human blood cells, extract genomic DNA from the cells in a simple way, amplify the locus containing the codon 72 polymorphism of the tumor suppressor gene p53, and perform agarose and polyacrylamide gel electrophoresis and ethidium bromide staining of the gel. Students were instructed how to statistically analyze the results in the context of human population genetics and how to relate them to the susceptibility of developing cancer. It is expected that students work in teams to analyze the results and write a nal report which should include data analysis and discussion.
EXPERIMENTAL PROCEDURES

avoid damage to the skin with potentially toxic chemicals and reagents. Students must wear eye protection to completely block UV radiation when visualizing ethidium-stained acrylamide gels (a UV-rated full-face plastic shield is strongly recommended). Potential hazardsAcrylamide and bisacrylamide in powder and liquid form are neurotoxins and must be handled accordingly. An air-tight face mask must be worn when weighing out acrylamide. Ethidium bromide is a suspected carcinogen and should also be handled carefully by wearing gloves.

Cell Lines and Blood Samples

Cells lines (T47D, MDA231, 293T) were a kind gift from Dr. Alejandro Zentella from the Instituto de Investigaciones Biome dicas, UNAM. The INBL cell line was obtained from cervical samples and established as described [14]. DNA from the tumor cell line MDA231 was used as a positive control. These cells were shown by enzyme digestion and sequencing to harbor the mutation. As a negative control, a PCR reaction was carried out without DNA template. Blood cells were obtained by sticking the nger tip with a sterile sting. Two drops were collected directly onto the FTA cards and allowed to dry at room temperature. Blood samples were also obtained from 49 healthy female students and professors on campus. They were informed about the purpose and experimental procedures of this study and written informed consent was obtained. This study was approved by the Ethics and Academic Committee of FES Zaragoza, UNAM. To assure the safety and privacy of the subjects, we established the following protocol: results are never published together with initials, names, or study number. A bar code number, to which students do not have access, is used to identify each sample. Blood samples are handled by the instructor who cuts the discs and transfers them to Eppendorf tubes labeled with a bar code number. The labeled blood samples are redistributed to the students such that they do not know from whom the sample originated. The condential data obtained in this study are kept in an electronic encrypted computer le.

Introductory Materials
The laboratory requires a minimum of three full laboratory periods to complete, and comprises one lecture before the rst laboratory period, one lecture between laboratory activities, and another at the end for discussion of results and ethical issues. Because of the complexity of the steps, the laboratory must be large enough to accommodate the number of students using the equipment. The lectures cover the basic theory of the following topics: p53 codon 72 polymorphism and its relationship to the development of cancer, gel electrophoresis using acrylamide, PCR, restriction fragment length polymorphism (RFLP), isolation of genomic DNA using Whatman FTA cards and their chemical properties that allow for sample collection and analysis, as well as prevention of sample contamination during collection. To successfully complete this protocol, students must be able to accurately pipette specic volumes, have knowledge of the background information on the p53 gene, and understand the processes of PCR, restriction endonuclease digestion, and acrylamide gel electrophoresis. A proper understanding of the above concepts is necessary; therefore, it is suggested that these modules be reserved for upper genetic or biochemistry courses. In addition, laboratory assistants are especially encouraged to provide appropriate guidance when working with acrylamide and ethidium bromide, in order to assure careful handling and proper disposal of this material.

Cell Culture and Cell Counting

Cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, BRL). Cells were subcultured weekly at 0.5 to 1.0 3 105 cells/mL in 10% FBS and maintained at 37 8C in a humidied atmosphere with 5% CO2. Cells were suspended in medium and a small aliquot was evaluated in a Neubauer chamber under a light microscope (Carl Zeiss, Germany) to determine the cell number and density.

Materials
The following materials are necessary to analyze the p53 mutation: laboratory coats, gloves, Pipetman (model numbers p2, p20, p200, and p1000), sterile and UV-irradiated pipette tips, sterile stings, thermocycler with accompanying PCR tubes, a gel electrophoresis power unit, polyacrylamide gel apparatus consisting of glass plates (20 cm 3 20 cm 3 0.5 mm), clamps, tape, and spacers, Whatman FTA cards, Taq polymerase (Applied Biosystems, USA), dNTPs (Invitrogen, USA), primers: (20 lM oligo p53 50 CCCGGACGATATTGAACA 30 forward; 50 AGAAGCCCAGACGGAAAC 30 reverse [13], 25 mM MgCl2 (Sigma), acrylamide, and bisacrylamide (Sigma).

DNA Extraction Using Whatman FTA Cards

A total of 3 3 106 cells were placed on FTA cards and allowed to dry at room temperature. Ten discs of 1.2 mm were cut from the FTA cards and washed in 400 lL of FTA reagent (Whatman, USA) while stirring at 300 rpm for 5 min. This procedure was repeated twice using sterile distilled water. The discs were allowed to dry at room temperature. Once dried, one disc was used for PCR amplication.

Methods
Notes for StudentsSafety precautions must be strictly adhered to for the safety of the students and the instructors. When working with cells, either cell lines or blood cells, it is essential to work under sterile conditions. Additional appropriate sample precautions must also be followed to prevent external- and cross-contamination after DNA is extracted. For blood handling, students must wear latex gloves during all procedures. Students should wear eye goggles and latex gloves to

PCR
The p53 genetic polymorphism was evaluated using PCR. The primers used were as follows: 50 CCCGGACGATATTG AACA30 forward; 50 AGAAGCCCAGACGGAAAC 30 reverse [13].

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One disc of FTA with the DNA template from each cell line was amplied by PCR in a nal volume of 20 lL, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.2 lM of each primer, and 0.5U Taq polymerase (Applied Biosystems). After an initial denaturation at 94 8C for 5 min, 29 cycles of denaturation (94 8C for 1 min), annealing (55 8C for 1 min), and extension (72 8C for 2 min) were carried out in a Mastercycler Eppendorf PCR system. The nal extension was performed at 72 8C for 7 min. PCR products were visualized by acrylamide gel electrophoresis (4%) in 13 TBE buffer. The gel was stained with ethidium bromide (1 mg/200 mL TBE 13 buffer) and gel images were obtained using a UV-image analyzer (BioRad GelDoc 1000, USA). PCR products should be analyzed in a section of the laboratory separate from where PCR work is performed.

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Polyacrylamide Gel Electrophoresis

Glass plates were assembled according to the manufacturers instructions. Briey, a watertight seal was formed between the plates (16 3 20 cm) and the spacers (0.50.75mm) to prevent the unpolymerized gel solution from leaking. The required volume of acrylamide solution was calculated given the dimensions of the glass plates and the thickness of the spacers. The following volumes are for 100 mL of a 4% acrylamide solution. In an Erlenmeyer ask, 13.3 mL of acrylamide/ bisacrylamide (30%), 76 mL of distilled water, 0.7 mL of 10% ammonium persulfate, and 35 lL of TEMED (N,N,N0 ,N0 -tetramethylethylenediamine) (Sigma, St. Louis, MO) were added. Then the solution was mixed by swirling. The acrylamide solution was poured into the gap between the glass plates. Immediately, the appropriate comb was inserted, being careful not to allow air bubbles to become trapped under the teeth. If necessary, the remaining acrylamide solution was used to ll the gel mold completely. Check that no acrylamide solution is leaking from the gel mold. The acrylamide is allowed to polymerize for 60 min at room temperature. The comb was carefully removed and the wells were immediately rinsed with distilled water; otherwise, small amounts of acrylamide solution trapped by the comb will polymerize in the wells, producing irregularly shaped surfaces that may distort the resolution of DNA bands. The gel was attached to the electrophoresis tank and the reservoirs were lled with 13 TBE. The digested PCR products were mixed with an appropriate amount of 53 gel loading buffer. The mixture was loaded into the wells using a Hamilton syringe (usually 10 lL is loaded into the gel). The electrodes were connected to a power pack and the gel was run at 120 V until the marker dyes migrated half the length of the gel. The electrical power was turned off, the leads were disconnected, and the electrophoresis buffer from the reservoirs was discarded. The glass plates were laid at on the bench top and a corner of the upper glass plate was lifted using a thin spatula. The upper plate was pulled smoothly away and the spacers were removed.

FIG. 1. Presence of the p53 codon 72 polymorphism in different cell lines. PCR products were analyzed on a 4% acrylamide gel and visualized by ethidium bromide staining. Lanes 14 show the p53 PCR products from cell lines: T47D, MDA231, 293T, and INBL, respectively. Lanes 58 show the PCR products after digestion with BstUI: T47D, MDA231, 293T, and INBL, respectively. Lane 9 is the negative control and Lane 10 contains molecular markers (50-bp DNA step ladder from Promega). The enzyme digestion produces two fragments of 125 and 78 bp. The undigested fragment is 203 bp.

PCR Restriction Fragment Length Polymorphism

RFLP analysis of codon 72 of the p53 gene was conducted to determine p53 genotypes. Five microliters of each PCR product were digested with 10 U of the restriction enzyme BstUI (New England Biolabs) at 608C for 6 hour. DNA fragments were resolved on a polyacrylamide gel and stained with ethidium bromide as described earlier. The Arg allele is cleaved by BstUI to yield two small fragments (125 and 78 bp). The Pro allele is not cleaved by BstUI and exists as a single 203-bp band. The heterozygote contains three bands (203, 125, and 78 bp). The instructor should make clear that these results represent only one polymorphism of a gene associated with a multifactorial disease in which several genes need to be altered. Therefore, the result is not the diagnosis of cancer; rather it signies only an increased susceptibility to develop the disease.

RESULTS

Ethidium Bromide Staining

Gently submerge the gel and its attached glass plate in staining solution (0.5 lg/mL ethidium bromide in distilled water). Stain for 5 min at room temperature and wash the gel with 400 mL of distilled water for 2 min. Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn when working with solutions that contain this dye. To photograph the gel, the gel is carefully inverted and placed on the UV transilluminator. The glass plate was removed and the gel was photographed using the Bio-Rad gel documentation system.

Before implementing this laboratory experiment in an upper division genetic and biochemistry laboratory, we wanted to ensure its feasibility. Therefore, we performed two trial runs: one with cancer cell lines using the previously designed primers and another with blood samples from healthy female donors. The objective of the trial runs was to ensure the experiment could be completed in a timely manner and that all associated technical aspects were not too demanding for an undergraduate laboratory. The trials were followed by an actual application in an upper division biochemistry clinical genetic laboratory course taught here at FES Zaragoza, UNAM. This experiment proved to be a highly successful mechanism to accomplish our learning objectives. The rst trial entailed the implementation of the DNA analysis and took place in the laboratory of Dr. Martha Legorreta at FES Zaragoza, UNAM. Whatman FTA cards and purication reagents were used to isolate DNA from different cancer cell lines and detect the p53 codon 72 polymorphism (see Fig. 1). The second trial run entailed the collection of peripheral blood cells using FTA cards from 49 women of a wide range of ages who were not associated with the laboratory. The main objective of the second trial run

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TABLE I Frequency of p53 codon 72 polymorphisms in blood samples Group Genotype Pro/Pro Genotype Pro/Arg Genotype Arg/Arg Students (n 20) 3 10 7 Teachers (n 29) 2 11 16 Frequency (%) 10 44 46

was to examine the feasibility of offsite collection followed by direct analysis of the DNA at the laboratory upon receipt of the samples. We also wanted to determine the feasibility of exploring cancer susceptibility by analyzing one mutation in p53 as a function of age and familial cancer development. The blood samples and the genomic DNA proved to be as viable as those collected directly in the laboratory and gave rise to viable PCR products that could be subjected to BstUI treatment for polymorphism analysis. The appearance of polymorphisms is readily apparent upon viewing the band patterns on the gel (Fig. 1) and in Table I. Of the 49 analyzed samples, the p53-Arg allele was identied in 68% and the p53-Pro allele was identied in 32%; 23 women (46%) were Arg/Arg homozygotes, 5 women (10%) were Pro/Pro homozygotes, and 22 women (44%) were Pro/ Arg heterozygotes (Table I). The genotypic frequencies were calculated using the data retrieved from the 49 healthy women analyzed for the p53 gene (Table II). Setting the frequency of the Pro/ Pro genotype as P, Pro/Arg as H, and Arg/Arg as Q, the frequency of each allele is: Frequency Pro p P 1=2H; and FrequencyArg q Q 1=2H: Using the allelic frequencies, we computed the expected genotypic frequencies in order to test if the sampled population was in Hardy-Weinberg equilibrium (Table II). The Hardy-Weinberg principle states that the allelic and genotypic frequencies in a population will remain constant through time providing the population is large in

size and mating is random. Random mating implies that mates select each other without regard to genotype at the p53 locus. Other conditions are required for the Hardy-Weinberg principle to apply, namely the absence of other evolutionary forces (e.g., natural selection, mutation, and migration) affecting that particular locus. A population in Hardy-Weinberg equilibrium is not evolving for that particular locus (it does not refer to other loci that could be affected by, for instance, selection). The frequency of the mutant p53-Arg allele was found to be 0.6836, whereas the p53-Pro allele was 0.3163. After computing the expected genotypic frequencies for the p53 locus, the results showed almost a perfect match to the observed ones (Table II). In order to probe if a statistical difference between the observed and expected genotypic frequencies exist, a chi-square (v2) test was computed as: v2
k X O E 2 i 1

where O and E are the observed and expected frequencies, respectively, and k is the number of genotypic classes (in this case, three). For the statistical analysis, the expected frequencies must be translated into a value representing the number of individuals (calculating v2 with proportions would give a very small number), by multiplying the expected frequencies by the total sample size (N). The observed value of v2 is compared to the theoretical one obtained from probability tables. Our null hypothesis (Ho) states that the population is in Hardy-Weinberg equilibrium for the p53 locus (this implies that the expected and observed genotypic frequencies are equal). Conversely, our alternative hypothesis (HA) states that the population is not in Hardy-Weinberg equilibrium. In order to reject the Ho, the calculated v2 value must be greater than the value of v2 obtained from statistical tables accompanied by the corresponding degrees of freedom and probability level. Therefore, it is necessary

TABLE II Genotypic and allelic frequencies at the p53 locus of 49 women from Mexico City Genotype Pro/Pro Observed individuals in the sample Observed genotypic frequency P 0.1020 Allelic frequency Expected genotypic frequency Expected number of individuals of each genotype v2 n1 5 1 Pn N H 0.4285 P p53-Proline pP1 2H 0.3163 p2 p2 0.10006 n1 p2 3 N 4.9030 0.00191 Pro/Arg n2 21 2 Hn N Q 0.4693 Arg/Arg n3 23 3 Qn N Q p53-Arginine qQ1 2H 0.6836 q2 q2 0.4674 n3 q2 3 N 22.9030 0.00041 Total N 49 1 1 1 49 0.004100

2pq 2pq 0.4325 n2 2pq 3 N 21.1938 0.00177

Observed and expected values of each cell are given together with the formulae to calculate the values. The value of the Chi-square (v2) statistic was calculated, and is compared with the corresponding value from probability distribution, which is given below. v2 (with one degree of freedom at a 0.05) 3.8414. Hence, the null hypothesis (H0) is accepted; the observed and expected frequencies are statistically similar and the population is said to be in Hardy-Weinberg equilibrium.

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TABLE III Presence of the polymorphism related to age or family background

Students (n 20) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Polymorph Pro/Arg Arg/Arg Arg/Arg Arg/Arg Pro/Arg Arg/Arg Pro/Arg Pro/Arg Pro/Arg Pro/Arg Pro/Arg Arg/Arg Pro/Arg Arg/Arg Pro/Arg Pro/Pro Pro/Pro Arg/Arg Pro/Arg Pro/Pro

Age 26 22 21 22 25 22 41 22 24 27 22 22 26 27 23 22 22 22 21 20

Family background No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No Yes Yes No Yes Yes

Teachers (n 29) 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

Polymorph Arg/Arg Pro/Arg Arg/Arg Arg/Arg Pro/Arg Arg/Arg Arg/Arg Pro/Arg Pro/Arg Pro/Pro Arg/Arg Arg/Arg Pro/Arg Arg/Arg Pro/Arg Arg/Arg Arg/Arg Pro/Arg Pro/Arg Arg/Arg Arg/Arg Arg/Arg Pro/Arg Arg/Arg Pro/Pro Pro/Arg Arg/Arg Pro/Arg Arg/Arg

Age 42 41 53 52 48 64 39 42 41 46 46 40 54 35 40 33 52 49 52 55 51 42 50 46 48 48 47 49 27

Family background No Yes No No No Yes No No Yes No Yes No Yes Yes No No Yes Yes No No No No No Yes Yes No Yes Yes Yes

Pearson v2 2.4454, Probability 0.294, Fishers exact 0.384.

to know the degrees of freedom. Usually k equals the product of (columns 2 1) 3 (rows 2 1), which represents the degrees of freedom. In this case, (3 2 1)(2 2 1) 2. However, we lost a degree of freedom when calculating each parameter. In this case, the frequency of the other allele is p (1 2 q) if p q 1. The degrees of freedom for testing Hardy-Weinberg proportions are the number of genotypes minus the number of alleles (3 genotypes 2 2 alleles 1). There is one degree of freedom and the 5% signicance level for one degree of freedom is 3.84. Since the v2 value is less than this, the null hypothesis is not rejected. The results are consistent with the null hypothesis, that is, the observed frequencies do not depart from those obtained assuming the individuals carry out random mating. Nearly half of the population possessed the Arg-type polymorphism of p53, which could indicate they are more susceptible to developing cervical cancer. There was no correlation between the age or the family background and the presence of any of the two alleles (Table III). In this case, we used the Fishers exact test that can be applied to testing for Hardy-Weinberg proportions. Because the test is conditional on the allele frequencies p and q, the problem can be viewed as testing for the proper number of heterozygotes. In this way, the hypothesis of Hardy-Weinberg proportions is rejected if the number of heterozygotes is too large or too small. This test is used in the analysis of contingency tables where sample sizes are small, and the signicance of any deviation from a null hypothesis can be calculated exactly

rather than by relying on a statistical test having a distribution that approximates a known theoretical distribution. The test is useful for categorical data that result from classifying objects in two different ways; it is used to examine the signicance of the association (contingency) between the two kinds of classication. In our case, one criterion of classication is age while the other is the family background. We wanted to know whether these two classications were associated, that is, whether the age and the family background are related to the presence of the polymorphism. The test involves a 2 3 3 contingency table and the p-value (0.384) from the test was computed using a statistical package (mathworld.wolfram.com). This value indicates that the age and the family background are not related to the presence of the polymorphism. Having successfully met the objectives for the two DNA trials described earlier, we implemented the module in an upper division genetic and biochemistry laboratory, which had a total of 15 undergraduate students. Although the protocol is relatively long, our students were able to complete the protocol and properly analyze the resulting data in three laboratory periods. The rst laboratory period entailed the collection of blood samples, washing and drying the lter papers, and PCR amplication of the p53 gene. During the second laboratory period, the PCR products were digested with the BstUI enzyme and two large polyacrylamide gels were prepared and poured by groups of students. During the third laboratory period, the students resolved the samples on the gel and performed the ethidium bromide

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FIG. 2. Presence of the p53 codon 72 polymorphism in blood samples from students. PCR products were analyzed on a 4% acrylamide gel and visualized by ethidium bromide staining. Lanes 112 show the p53 PCR products after digestion with BstUI. Lanes 13 and 15 are genomic DNA from blood cells and from the MDA231 cell line, respectively. Lane 14 is the negative control and Lane 16 is the positive control. Lane 17 contains molecular markers (50-bp DNA step ladder from Promega). The enzyme digestion produces two fragments of 125 and 78 bp. The undigested fragment is 203 bp. Lanes 1, 3, 9, and 11 are Arg/Arg homozygotes while Lanes 2, 4, 5, 6, 7, 8, 10, and 12 are heterozygotes.

staining immediately afterward. An image of the results was obtained with the Bio-Rad gel documentation system (Fig. 2). For the statistical analysis, the students were instructed to use the v2 formula to calculate the frequency of the mutation as described earlier and students were asked to determine if the population was in HardyWeinberg equilibrium. As a part of the experimental discussion, students were asked to present the statistical analysis and its meaning in terms of population genetics.

DISCUSSION

This project proved to be highly successful in establishing a biochemistry laboratory protocol, appropriate for an undergraduate upper division level course, for detection of a mutation present in cancer. The main learning objectives were achieved: the students succeeded at 1) isolating genomic DNA from peripheral blood, 2) using PCR to amplify the polymorphism at codon 72 of p53 and using BstUI restriction enzyme digestion to conrm the genotype, 3) resolving the samples with polyacrylamide gel electrophoresis and using ethidium bromide staining to visualize the results, and 4) learning how to properly analyze the results in the context of human population genetics and how to relate the presence of the polymorphism with the susceptibility of developing cancer. It was really encouraging to hear students comment that they were motivated by the thought of performing protocols used by real laboratory researchers to determine cancer susceptibility. In addition, the students showed greater interest in the experimental results since they were able to use their own DNA in the protocol and discover the allelic nature of human genes. We managed to incorporate all elements of the procedure in three laboratory periods. Collection of the blood samples was easy and quick with the FTA cards, and DNA isolation was straightforward. The PCR amplication steps were properly coordinated and provided the students with the opportunity to get hands-on experience with this important method which is used extensively in many areas of scientic research. We think that the most challenging aspect of this protocol is the preparation of

the glass plates and the pouring of the large acrylamide gels for PAGE resolution of the PCR products. Therefore, it is very important for the instructor to have prior expertise and experience so that students can be properly instructed in pouring, loading, and running the gel. If the exercise is completed according to the protocol, the p72 codon polymorphisms can be visualized quite clearly (Figs. 1 and 2). A polyacrylamide gel offers signicant resolution of the PCR products, and ethidium bromide staining results in sufcient contrast for rapid visualization of the polymorphic bands. The alleles corresponding to the different p53-codon 72 loci are distinguishable relative to the positive and negative controls. We detected a high percentage of arginine homozygotes in our 49 samples, suggesting that this genotype, which is considered a risk factor for cancer associated with oncogenic HPV, is highly prevalent in Mexico City. The identication of this kind of polymorphism has an important preventive impact since it identies a risk factor for cervical carcinoma associated with HPV infection. One element of complexity regarding genetic susceptibility of cancer is the existence of multiple ethnic groups, some of which can be affected by strong founder effects that may explain the local or regional clustering of some cancers. Thus, the use of molecular biomarkers may be particularly helpful in identifying the respective roles of environmental, biological, lifestyle, and genetic risk factors for developing cancer and to help in its detection and prevention. The successful accomplishment of our three objectives and the subsequent positive feedback from the students demonstrate the feasibility of introducing relevant DNA analysis methods and technologies used by research laboratories into an undergraduate laboratory class. The experience gained and knowledge acquired will assist those students who plan to pursue careers in the growing elds of medical genomics, as well as help all students to better understand polymorphisms and cancer susceptibility.
Acknowledgment The authors acknowledge Dr. Juan Nu n ez-Farfa n, Instituto de Ecolog a, UNAM, for his help with the statistical analysis and helpful discussion about population genetics.

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REFERENCES
[1] S. Jin, A. J. Levin (2001) The p53 functional circuit, J. Cell Sci. 114, 41394140. [2] D. Malkin, K. W. Jolly, N. Barbier, A. T. Look, S. H. Friend, M. C. Gebhardt, T. I. Andersen. A. L. Borresen, F. P. Li, J. Garber (1992) Germline mutations of the p53 tumor-suppressor gene in children and young adults with second malignant neoplasms, N. Engl. J. Med. 326, 13091315. [3] M. Hollstein, D. Sidransky, B. Vogelstein, C. C. Harris (1991) p53 mutations in human cancers, Science 253, 4953. [4] A. J. Levine, J. Momand, C. A. Finlay (1991) The p53 tumour suppressor gene, Nature 351, 453456. [5] B. Vogelstein, D. Lane, A. J. Levine (2000) Surng the p53 network, Nature 408, 307310. [6] A. Helland, A. Langerod, H. Johnsen, A. O. Olsen, E. Skovlund, A. L. Borresen-Dale (1998) p 53 polymorphism and risk of cervical cancer, Nature 396, 530531, author reply 532. [7] I. Zehbe, G. Voglino, F. Wilander, F. Genta, M. Tommasino (1999) Codon 72 polymorphism of p53 and its association with cervical cancer Lancet 354, 218219. [8] A. Storey, M. Thomas, A. Kalita, C. Harwood, D. Gardiol, F. Mantovani, J. Breuer, I. M. Leigh, G. Matlashewski, L. Banks (1998) Role of a p53 polymorphism in the development of human papillomavirus-associated cancer, Nature 393, 229234.

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[9] Y. C. Yang, C. L. Chang, M. L. Chen (2001) Effect of p53 polymorphism on the susceptibility of cervical cancer, Gynecol. Obstet. Invest. 51, 197201. [10] A. N. Rosenthal, A. Ryan, R. M. Al-Jehani, A. Storey, C. A. Harwood, I. J. Jacobs (1998) p53 codon 72 polymorphism and risk of cervical cancer in UK, Lancet 352, 871872. [11] T. Minaguchi, Y. Kanamori, M. Matsushima, H. Yoshikawa, Y. Taketani, Y. Nakamura (1998) No evidence of correlation between polymorphism at codon 72 of p53 and risk of cervical cancer in Japanese patients with human papillomavirus 16/18 infection, Cancer Res. 58, 45854586. [12] J. W. Kim, J. W. Roh, N. H. Park, Y. S. Song, S. B. Kang, H. P. Lee(2001) Polymorphism of TP53 codon 72 and the risk of cervical cancer among Korean women, Am. J. Obstet. Gynecol. 184, 5558. [13] W. Settheetham-Ishida, N. Kanjanavirojkul, C. Kularbkaew, T. Ishida (2005) Human papillomavirus genotypes and the p53 codon 72 polymorphism in cervical cancer Northeastern Thailand, Microbiol. Immunol. 49, 417421. [14] J. Caceres-Cortes, J. A. Alvarado-Moreno, K. Waga, R. Rangel-Corona, A. Monroy-Garc a, L. Rocha-Zavaleta, J. Urdiales-Ramos, B. Weiss-Steider, A. Haman, P. Hugo, R. Brousseau, T. Hong (2001) Implication of tyrosine kinase receptor and steel factor in cell density-dependent growth in cervical cancers and leukemias, Cancer Res. 61, 62816289.