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Keywords:

  • PCR;
  • Bt-toxin gene;
  • maize invertase gene

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A commercially available leaf DNA extraction and amplification kit has been adapted for the detection of genetically modified material in common food products containing maize. Amplification using published primer pairs specific for the Bacillus thuringiensis delta-endotoxin and maize invertase genes results in a 226-bp invertase PCR product in all samples (an internal positive control) plus a 184-bp product in samples that are genetically modified with the endotoxin gene. The ease and rapidity of DNA extraction and PCR make this exercise especially suitable for advanced-placement high school or lower division college biology students.

An undergraduate molecular biology curriculum usually includes one or more exercises involving PCR. Often, the first exposure to PCR occurs in an upper division laboratory course designed for molecular biology majors. However, the trend is to introduce PCR into lower division core biology courses (and even into high school courses) where students are less technically skilled and have varying degrees of interest in molecular biology. At this level, a PCR experiment should be simple enough to ensure a reasonable degree of success, but it should also demonstrate the power and application of genetic technology. Such positive laboratory experiences can encourage students to continue their education in molecular biology or, at the very least, give them an appreciation for how the technology applies to their daily lives. The laboratory exercise we have developed achieves both goals by using simplified DNA extraction and amplification procedures to detect genetically modified (GM) 11 maize in off-the-shelf food products.

In 2002, U.S. farmers planted an estimated 90 million acres of GM soybeans, cotton, and maize, representing 74, 71, and 32%, respectively, of the total acreage for each crop [1]. One of the most popular genes used to engineer these crops encodes the Bacillus thuringiensis (Bt) endotoxin (Bt toxin) that is toxic to Lepidopterans (butterflies and moths), some species of which are very destructive agricultural pests in their larval stages. Studies have shown that two to four times less insecticide is required to control pests when cotton is engineered to express Bt toxin [2].

Application of this technology, however, is not without controversy. There are concerns over the effects of Bt toxin on nonpest insects, such as the Monarch butterfly [3], and on the potential transmission of GM pollen from crops to their wild relatives [4, 5]. Other studies argue that these threats are minimal [68]. Although it has never been proven that ingesting food from GM crops poses health risks to humans [9, 10], some feel that there are still unresolved safety issues [11], including the possibility of unforeseen allergic reactions to foreign proteins [12].

Because of trade disputes between the United States and the European Union over shipments of genetically engineered crops [13] and the discovery of unapproved GM maize in food products [14], analytical methods are needed for the rapid and sensitive detection of GM material in raw and processed foods. The following laboratory exercise, based on a published PCR detection method [15], was developed for lower division biology students in an introductory cell biology class to demonstrate how PCR can be used to detect a Bt-toxin gene in common maize-containing foods. A commercially available DNA extraction and PCR kit was adapted for use with this method to make it simple and robust enough for technically inexperienced students. In addition to teaching students PCR and electrophoresis, the exercise can also serve as a basis for a more general discussion on the applications and ethics of genetic engineering.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials—

The REDExtract-N-Amp Plant PCR Kit, which included extraction solution, dilution solution, and 2 × REDExtract Ready Mix, was purchased from Sigma (St. Louis, MO). Primers specific for the Bt Cry1A(b) (delta-endotoxin) gene and the maize invertase (Ivr) gene [15] were synthesized by Qiagen-Operon (Alameda, CA). Primer sequences were: Cry1Ab (5′-ACCATCAACAGCCGCTACAACGACC-3′); Cry1As (5′-TGGGGAACAGGCTCACGATGTCCAG-3′); Ivr1A (5′-CCGCTGTATCACAAGGGCTGGTACC-3′); and Ivr1B (5′-GGAGCCCGTGTAGAGCATGACGATC-3′). DNA markers (25-bp ladder) were from Promega (Madison, WI). Dry, maize-containing foods (corn bran cereal, corn meal, grits, polenta, taco shells, and tortilla chips) were purchased from local supermarkets.

Laboratory Setup—

Students worked in groups of three or four, with each group performing two DNA extractions and three PCR (including a negative control). For a class of 18–24 students (six groups), the following equipment and materials were required for the first 3-h laboratory meeting: maize-containing foods (six or more), small mortars and pestles (12), nickel spatulas and small weigh boats (12), analytical balance, 10-μl and 100-μl pipettors with sterile tips (6), microcentrifuge racks (6), microcentrifuges (2), heat block or water bath (set to 95 °C), thermal cycler, 1.5-ml microcentrifuge tubes (12), 0.2- or 0.5-ml PCR tubes (18), 320-μl aliquots of extraction solution (6), 320-μl aliquots of dilution solution (6), 10-μl aliquots of 50:50 extraction:dilution solution (6), 35-μl aliquots of 2 × REDExtract Ready Mix (6), and 22-μl aliquots (6) of primer mix containing 1.67 μM each of Cry1Ab, Cry1As, Ivr1A, and Ivr1B primers in 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 (Tris-EDTA buffer). The second 3-h laboratory meeting required horizontal mini-gel electrophoresis units (3), power supplies (3), 10-μl pipettors and tips (6), latex gloves, microcentrifuge racks (6), large staining tray, 500 ml of 1-mg/l ethidium bromide, a transilluminator, UV-resistant goggles, a Polaroid (Waltham, MA) camera with Type 667 film or a digital gel documentation unit, 1l of cold 1 mM EDTA, 40 mM Tris acetate (Tris-acetate-EDTA (TAE)) buffer, 15-μl aliquots (3) of 300 ng/μl 25-bp DNA ladder in loading dye, 30-ml bottles (3) of molten 2.5% agarose (kept at 65 °C) in TAE buffer.

Laboratory 1 Procedure—

A small quantity of each food product was ground with a mortar and pestle, and 18–22 mg were transferred to a 1.5-ml microcentrifuge tube. Extraction solution (100 μl) was added (some of the more finely ground samples absorbed all of the liquid) followed by a 10-min incubation at 95 °C in a heat block or water bath. After the incubation, dilution solution (100 μl) was added, and the samples were resuspended. Due to congealing of starch, most samples could only be resuspended effectively by mixing with a pipettor tip. The samples were then microcentrifuged at high speed for 2 min, and the supernatants were used as the source of DNA for PCR. These crude DNA extracts (4 μl) were transferred to PCR tubes (0.2 or 0.5 ml). As a negative control, 4 μl of 1:1 extraction solution:dilution solution were used. To the samples were added 10 μl of 2 × REDExtract Ready Mix and 6 μl of Primer Mix. The final concentration of each primer in the 20-μl reactions was 0.5 μM. Amplification was performed in an Eppendorf Gradient Mastercycler (Eppendorf Scientific, Westbury, NY) with an initial denaturation of 3 min at 94 °C, 45 cycles of 45 s at 94 °C, 45 s at 60 °C, 30 s at 72 °C, and a final extension for 5 min at 72 °C. After amplification, samples were stored frozen until Laboratory 2.

Laboratory 2 Procedure—

Two groups (6–8 students) shared a 2.5% agarose mini-gel. After pouring the premelted agarose, each group loaded 5 μl of their two amplified maize DNA extracts, a negative control, and the 25-bp DNA ladder. The inclusion of a red dye and dense material in the REDExtract Ready Mix precludes the need to add a loading dye to the PCR products prior to electrophoresis. Electrophoresis was performed at 125 V for 45 min or until the red dye had traveled a minimum of 4.5 cm through the gel. The dye should not be allowed to run off the gel. Gels were stained for 15 min in 1-mg/l ethidium bromide, then destained in water for 10 min. Bands were detected on a transilluminator and recorded by Polaroid or digital photography. For reasons of safety, especially with beginning laboratory students, it is recommended that the instructor check the electrophoresis units prior to turning on the power supplies to ensure proper connections. The instructor should also stain and photograph the gels. Used ethidium bromide (a mutagen) should be transferred to a chemical waste container for proper disposal.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Expected Results—

The multiplex PCR reaction used in this exercise is expected to produce one of two results: 1) a single 226-bp product (maize invertase gene) from non-Bt samples; or 2) the 226-bp product plus a 184-bp product (B. thuringiensis delta-endotoxin gene) from samples containg Bt-modified maize. PCR results from six randomly chosen maize-containing food products are shown in Fig. 1. Four of six products were found to contain GM maize. The amplifications produced high yields with well resolved banding patterns upon electrophoresis. An occasional primer-dimer artifact was observed near 50 bp. In some GM products, minor nonspecific products were observed near 125 bp (Fig. 1, lanes 2 and 7). The only food tested that consistently failed to amplify was corn flakes (not shown), possibly due to greater heat-induced chemical damage to the DNA during processing compared with the other foods.

Student Results—

To demonstrate the range of outcomes, results from two student groups are shown in Fig. 2. Group A produced the expected results (lanes 2 and 3) whereas Group B obtained ambiguous results (lanes 5 and 6), likely due to nonspecific priming caused by inaccurate pipetting and/or incomplete mixing of PCR reagents. Of six groups, three successfully amplified both samples, two successfully amplified one of their two samples, and one group (Fig. 2, Group B) had poor results with both samples. The overall success rate was 75% (9 of 12 samples attempted). The results reported here for one laboratory section of 20 students were very similar to results produced by two other sections.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Summary and Analysis—

This simple protocol was created by applying and slightly modifying a commercially available kit (Sigma's REDExtract-N-Amp Plant PCR Kit) to a published amplification protocol. The goal was to introduce inexperienced students to PCR and DNA electrophoresis in a way that would pique their interest in biotechnology while maximizing the probability of a successful outcome. The key elements in the experimental design were the use of dry food samples and a hot extraction protocol to minimize DNA degradation from endogenous DNases, the adaptation of the DNA extraction and PCR kit to reduce sample handling and pipetting errors, and the selection of a PCR target (Bt-toxin gene) that allowed students to detect the presence of genetically modified maize in their chosen food products. The inclusion of a PCR primer pair for a maize gene acted as an internal positive control that allowed students to successfully produce a PCR product even with non-GM samples.

This lower division biology introductory cell biology course was the first “wet” biology laboratory taken by the majority of these students, although all had taken one or two semesters of general chemistry laboratory. Students had only limited experience with the use of variable volume micropipettors in a previous laboratory exercise, and most had never performed agarose gel electrophoresis. By using a crude DNA extraction procedure, adjusting the concentrations of primers in the Primer Mix, and using the PCR master mix provided in the commercial kit, only two pipettings were required for DNA extraction, three for PCR, and one for electrophoresis (gel loading). This protocol is simple and robust enough that it could also be used in high school advanced biology courses if the appropriate equipment is available. Another laboratory exercise has been described [16] that uses PCR to detect GM material in soy powder, but that protocol is designed for advanced college students and requires significantly more manipulations, especially in the DNA extraction steps.

In the present exercise, the results obtained by the majority of students were similar to those of the instructor, and even occasional poor results (no amplification products or nonspecific products) can be used to make points about the sensitivity of PCR to inaccurate pipetting and incomplete mixing. The findings that over half of all products tested were Bt-positive were consistent with an estimate that ∼70% of processed foods in U.S. supermarkets contain some ingredients derived from genetically engineered crops [17].

Cost and Organizational Considerations—

The total cost of the extraction/PCR kit, four primers and DNA ladder is approximately $300. These reagents are adequate for the analysis of 100 samples and can supply up to five laboratory sections if students work in groups as described. Properly stored, the reagents should be stable for more than 1 year. The cost of the exercise can be reduced significantly by using the rapid extraction and PCR method of Xin et al. [18]; however, their protocol significantly increases the number of pipetting steps and the chance of failed reactions in the hands of technically inexperienced students.

The exercise is easily completed in two 3-h laboratory periods. The DNA extractions and setup of PCR reactions take only 1 h, leaving ample time to demonstrate the proper use of pipettors and the thermal cycler, and also to review PCR theory if not previously covered. Sample amplification can begin in class and will be finished in time to free the cycler for a subsequent laboratory section. Sample loading, gel electrophoresis, staining, and detection can be completed in the second laboratory period within 2 h, allowing time to demonstrate the proper use of electrophoretic equipment and summarize group results.

Study Questions and Reading Assignments—

After distributing copies of the gel images, students were assigned the following questions.

  • Compared with the DNA markers (300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, and 25 bp), estimate the sizes of the bands seen in your two maize sample PCR products and compare them to the expected sizes of invertase and Bt-toxin gene products. Which one, if any, of these products contains GM maize?

  • If two primers are partially complementary to each other they can hybridize and be amplified. This can result in the formation of “primer-dimers” in the PCR products. How large might such a product be? Did you see any evidence of primer-dimer artifacts in your samples?

  • There should be no PCR products containing just the 184-bp band. Why?

  • Some groups may not have seen any bands in their amplified maize DNA samples. What are three possible reasons for this?

  • Does eating GM food bother you? Explain your answer.

In response to this last question, 80% of the students said that they were not bothered by the prospect of eating GM foods. The two major reasons given were: 1) the lack of evidence that eating GM foods is harmful, and 2) they were resigned to the fact that the technology is in place, so why worry about it? Only a few indicated that they would prefer GM foods to be labeled, and only one mentioned a concern about future ecological impacts. These attitudes are in direct contrast with the current European view on this topic [13]. To stimulate a postlaboratory discussion on the pros and cons of GM foods, prelaboratory readings can be assigned from easily understandable articles covering the food safety issues [19] and ecological considerations [20] related to agricultural biotechnology. An excellent general introduction to agricultural biotechnology is also available online [21].

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Figure FIGURE 1.. PCR products from maize invertase and Bt-toxin genes in maize-containing foods (expected results).Lane 1, 25-bp DNA ladder; lane 2, corn bran cereal; lane 3, grits; lane 4, polenta; lane 5, corn meal; lane 6, taco shells; lane 7, tortilla chips; lane 8, negative control. Arrows indicate the 226-bp maize invertase PCR product (ivr), the 184-bp Bacillus thuringiensis endotoxin PCR product (cry), and primer-dimer artifacts (p-d).

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Figure FIGURE 2.. PCR products from maize invertase and Bt-toxin genes in maize-containing foods (range of student results). Student Group A: lane 1, negative control; lane 2, corn bran cereal; lane 3, grits. Student Group B: lane 5, tortilla chips; lane 6, polenta; lane 7, negative control. Lane 4, 25-bp DNA ladder. Arrows indicate the 226-bp maize invertase PCR product (ivr), the 184-bp Bacillus thuringiensis (Bt) endotoxin PCR product (cry), and primer-dimer artifacts (p-d).

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Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank the Biology 3 students at San Jose State University, who took part in this laboratory exercise in the Spring 2003 semester, the Department of Biological Sciences for financial support, and Dr. William Murray for photographic assistance.

  • 1

    The abbreviations used are: GM, genetically modified; Bt, Bacillus thuringiensis.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES