A practical class experiment based on the extraction/purification of DNA from soybeans and the identification of exogenous DNA sequences amplified by PCR is described. This is used in a biochemistry and molecular biology course for advanced science undergraduate students. Students are given small amounts of soybean powder, and they practice a DNA extraction, purification, and a PCR with a set of different specific primers followed by an electrophoresis to determine whether the sample is transgenic. A major aim is to provide experience in the use of common techniques such as DNA extraction/purification and PCR and their interpretation.
An important focus of biotechnology training is to gain experience in new fields of biochemistry and molecular biology by using experimental approaches that could also be applied by biotechnological industries. The detection of genetically modified organisms (GMO)11 is a typical example that has broad application in the agriculture and food industries [1–4].
Lay people and even the scientific community have divided opinions concerning GMOs. For example, the use of genetically modified plants can lead to increased yields in agriculture by generating species resistant to diseases, insects, and products (e.g. glyphosate). On the other hand, GMOs may threaten biodiversity by artificial over-resistance, extensive commercialization, and spreading into the wild population. The secondary effects upon the environment and upon mankind are unknown in the long term. This is why, in certain countries, lobby groups have imposed a severe control on the production of a number of GMO plants and are reluctant for their use to be generalized. In European countries, the presence of GMOs in food must be indicated. Thus food manufacturers must be sure that GMOs are not present in their raw materials if they market products claiming to be GMO-free. Government institutions have then been set up to screen for the presence of GMOs in food products.
Bearing in mind that the methods of GMO detection should be reliable and efficient, we have used our laboratory expertise in molecular biology techniques to devise an optimized PCR-based protocol that could be both taught to students and applied in food quality control laboratories. In particular we have carefully monitored the initial step of DNA extraction, which has been shown to be most critical .
OUTLINE AND RATIONALE OF THE METHOD
The extraction kit has been selected on the basis of its efficiency for isolating genomic and plasmid plant DNA that have to be subsequently amplified by the PCR. DNA extraction consists of breaking the cell wall, eliminating the RNA, and removing proteins by precipitation. After this, the DNA is fixed on an affinity column, washed, and eluted. Similar approaches have already been applied to the detection of DNA from other plants  or from viruses .
Note that, in this experiment, PCR is used to determine whether soybean flour contains genetically modified plant material, but the technique can also be used in various contexts such as paleontology and forensic science [8–12]. The PCR proceeds in three phases: 1) heat denaturing the double stranded DNA, 2) annealing the primers on the sequences by cooling, and 3) elongation of the DNA by extending the primers in opposite directions. These three steps are called a cycle, and the PCR comprises a repetition of this cycle. The amount of DNA is exponentially amplified. DNA yield therefore increases as a function of 2n, where n is the number of cycles. This means that after one cycle the amount of DNA is doubled, and after 30 cycles, theoretically the DNA has been amplified over 109-fold and can easily be detected on electrophoresis gel.
For this experiment PCR primers were designed to detect two types of genes. The first primer set amplifies endogenous plant genes, a chloroplast gene and the specific soybean lectin gene. In this way, we verify the presence of the plant DNA and, in particular, soybean DNA. The second primer set amplifies sequences normally not found in plants but introduced by genetic transformation. In this case the two amplified foreign DNAs are the constitutively active 35 S promoter from cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens. If either of these two sequences is amplified, this shows that the target DNA is transgenic . In principle, all the approved genetically engineered agricultural crops have been transformed with constructs containing either or both of these elements. The identification of new promoter and terminator sequences in transgenic constructs is becoming a matter of concern for scientists.
The PCR primers have been designed to create amplified products of different sizes to facilitate their identification. Table I shows the sequences of the synthesized primers. The annealing sites have been chosen to generate only a single DNA fragment to simplify interpretation. These fragments are visualized as bands on agarose gels following PCR (see the table for the sizes of the amplified sequences).
The laboratory equipment used in this class includes the following: powdered soybeans (transgenic and non-transgenic) (Soy Bean Powder SB-Set; Fluka), gloves, Eppendorf tubes (standard and PCR-type), ice boxes, a balance, a heating bath, vortexes, centrifuges, several sets of pipetman (adjustable volume: P10, P200, and P1000) with tips, a DNA plant extraction kit (DNeasy™ plant kit), a PCR kit (Taq PCR core kit) from Qiagen, synthetic oligonucleotides (Isoprim), PCR Eppendorf thermocyclers (PolyLabo), agarose electrophoresis systems with generators (Amersham Biosciences, Inc.), agarose (Euromedex), 10 × TBE buffer (Euromedex), a 100-bp DNA ladder (Promega), a microwave to melt the agarose, concentrated ethidium bromide (EB) solution (Sigma), a plate to stock the EB, a spatula to move the gels in and out of the EB bath, an EB-dedicated dustbin, a UV table, and a system to photograph the gels.
The students are encouraged to read the guides for the plant DNA extraction kit (DNeasy™ plant kit) and for the PCR kit (Taq PCR core kit) from Qiagen (France) provided before the practical experiment. They are also given the address of a comprehensive web site, ohioline.ag.ohio-state.edu/gmo/, which is maintained by scientists from the College of Food, Agricultural, and Environmental Sciences at the Ohio State University. This site is organized as a web link to extensive and unbiased GMO information. Thus the students can visit the sites of industrials such as Monsanto and Novartis but also of opposition campaigners such as Greenpeace. This individual homework should lead to a good understanding of the principles underlying the method to be used. During the practical class itself, the students should use the first 2 h to set up a protocol and to explain to the teacher all the steps to be performed. The rationale for each step should be fully understood.
The students, working in pairs, are then ready to perform a 6-h experiment on two samples, one of transgenic and the other of non-transgenic soy flour, according to the detailed instructions given below. It includes the use of the kits to extract the DNA and amplify it by PCR, as well as the analysis of the results by electrophoresis.
Extraction and Purification of Plant DNA—
The isolation of DNA from soybean with the DNeasy™ plant kit is carried out according to the following instructions and by using AP1, AP2, AP3, AW, and AE solutions and the two types of column provided with the kits by the manufacturer (Qiagen). Briefly, the instructions consist of the following: 1) Switch on the heater at 65 °C to keep the AE buffer hot. 2) Weigh 0.1 g of soy powder in a 2-ml Eppendorf tube (maximum capacity of the kit). 3) Add 400 μl of AP1 buffer (the lysis buffer is constituted of detergents, proteases, and salts) and 4 μl of RNase (100 mg/ml) (to avoid the purification of the RNA and permit the liquefaction of the solution), and mix gently. Solution AP1 contains detergents, so avoid contact with skin. 4) Incubate 10 to 15 min at 65 °C (to keep endogenous DNases inactive, denature the proteins, and break the cell walls). 5) Add 130 μl of AP2 buffer, vortex the solution, and put it on ice for 5 min (to precipitate the proteins and the polysaccharides by acidification of the medium). Ice permits the precipitation of unfolded proteins, causes misfolding of other ones, and inactivates the DNases. Solution AP2 contains acetic acid, so avoid contact with skin (material safety data sheet number 64-19-7). 6) Centrifuge the mix for 2 min at 12000 × g on a QIAshredder spin column (to eliminate the cell walls and the precipitate). This column is a filter. It stops the particles only in function of their size. All the soluble molecules can pass through it. 8) The solution is transferred to a fresh 2-ml Eppendorf tube, and the pellet is discarded. By this way, they can evaluate the volume of solution they have and thus calculate how much solution they will add afterward. At this step it is useful to show them how to measure a volume using a pipetman. 9) Add 0.5 volume of AP3 solution, and vortex the mix (to constitute the DNA salts). To avoid chloride production, do not mix AP3 solution with sodium hypochlorite. 10) Add 1 volume of absolute ethanol, and vortex the mix. The volume is the same as the volume used to add AP3 solution. 11) Put the solution on the DNeasy minispin column, and centrifuge for 1 min at 10000 × g (to fix the DNA on the affinity column). The maximum volume of the column is 650 μl, so this has to be repeated several times. You have to discard the solution at the bottom of the Eppendorf tube after each step of centrifugation to avoid the overflow. 12) Place the column on a new 2-ml Eppendorf tube. 13) Pipette 500 μl of AW buffer on the column, and centrifuge for 1 min at 10000 × g (to desalt and wash the DNA). 14) Again, pipette 500 μl of AW on the column, and centrifuge for 2 min at 10000 × g (to be sure to eliminate all the buffer). Remove the AE buffer from the heater 2 min before use. 15) Place the column on a new 2-ml Eppendorf tube. 16) Pipette 100 μl of AE buffer on the column, and centrifuge for 1 min at 10000 × g (to elute the DNA). 17) Repeat this operation once. 18) Put the Eppendorf tube containing the DNA solution on ice (the column can be thrown away).
Polymerase Chain Reaction—
The PCR kit used is the PCR core kit from Qiagen. We have tested different Taq polymerase, and all are efficient on these primers. This choice is because this product is inexpensive and because of the presence of a premix of the four dNTP in one solution.
To make sure that the PCR will work for all of the teams, it is better to aliquot all of the solutions. This way you avoid contamination and false positives or negatives. To have enough material, put 15% volume in excess in each tube.
Each pair of students will amplify their purified DNA in the presence of the four different primers couples (four different PCR Eppendorf tubes). Tube A contains a mixture of chloroplast DNA-specific primers, tube B contains a mixture of lectin-specific primers, tube C contains a mixture of NOS terminator-specific primers, and tube D contains a mixture of 35 S promoter-specific primers.
To prepare these tubes, students label four PCR Eppendorf tubes. To each tube add, in order, the following: 33.9 μl of milliQ water (up to 50 μl), 5 μl of 10 × PCR buffer (containing 100 mM Tris-HCl buffer, pH 8, 500 mM KCl, 15 mM MgCl2), and 5 μl of the DNA solution prepared before. Mix gently, and place on ice.
To each tube, add 5 μl of the appropriate primer mix solution (initial concentration of 0.1 μg/μl). Mix gently and then return to ice.
To each tube add 0.6 μl of dNTP (a mixture of the four dNTP at 10 mM) and 0.5 μl (1 unit) of Taq polymerase (QIAgen Taq polymerase core kit; Qiagen). Mix gently and then return to ice. Keep the tubes on ice until the rest of the group has finished preparing their tubes.
Start the thermal cycler and verify the program. This program involves the following cycles. Step 1: 94 °C, 10 min for DNA denaturation; Step 2: 94 °C, 1 min; 63 °C, 1 min (annealing); 72 °C, 1 min (elongation); Step 3: 72 °C, 10 min for final elongation; 4 °C (no time limit) for conservation of the PCR amplification products. Place the tubes in the thermal cycler, and start the thermal cycles as indicated. Step 2 is repeated for 34 cycles.
During the PCR, prepare a 3% w/v agarose gel in 0.5 × TBE. For this purpose, the students dilute 3 g of agarose in 100 ml of 0.5 × TBE electrophoresis buffer (0.09 M Tris, 0.09 M Borate, 1 mM EDTA), and boil the solution gently until it becomes clear. Pay attention to the overflow during boiling, which can alter the concentration. Then allow it to cool to ∼55 °C, and pour a gel.
Once the gel has solidified, the gel plate is placed on the electrophoresis apparatus and covered with 0.5 × TBE (to keep the same conductivity everywhere). This precaution avoids the dilution of the samples because of convection movements created by the buffer covering the gel after loading. At the end of the PCR, remove the tubes from the thermal cycler, and place them on ice.
In four fresh Eppendorf tubes, mix 8 μl of each amplified solution and 1 μl of 6 × loading buffer (0.3 M EDTA containing 10% glycerol to increase density, 0.25% bromphenol blue, 0.25% xylene cyanol, and 0.25% Orange G as migration indicator). In a fifth, mix 3 μl of 100-bp DNA ladder and 0.5 μl of 6 × loading buffer.
Onto the agarose gel, load 3 μl of 100-bp DNA ladder Promega solution with dye (which enables them to visualize fragments from 100 to 1500 bp), 8 μl of the chloroplast-amplified DNA solution with dye and then 8 μl of the lectin-amplified mix, 8 μl of the NOS terminator-amplified mix, and 8 μl of the 35 S promoter-amplified mix. Run the gel at 200 V until the Orange G (which migrates at approximately 50 bp) is at the bottom of the gel (∼30 min).
Stain the gel in ethidium bromide (to prepare the stain, put five drops of stock solution (10 mg/ml) in 200 ml of distilled water). (Be careful in handling ethidium bromide; material safety data sheet number 1239–45-8, and use gloves and decontaminate the area used for this every evening.) Using a plastic support, transfer the gel into a box containing the solution of ethidium bromide. Stain for 10 min, destain in water for 1 min, and transfer the gel onto the ultraviolet transilluminator to visualize the bands of purified DNA. Take one photograph for each pair of students (Fig. 1). (Take care of exposure of the skin and particularly your eyes to the high intensity UV light. Use a Plexiglas screen.)
INTERPRETATION OF RESULTS
The photograph of the gel (Fig. 1) is made of the following: In lane M is the DNA marker (100-bp DNA ladder from Promega), which contains fragments from 100 to 1500 bp. Lanes 1–8 show the results of the PCR amplification. Lanes 1 and 7 show the results of the chloroplast amplification, lanes 2 and 8 the lectin amplification, lanes 3 and 5 the 35 S PCR product, and lanes 4 and 6 the NOS amplicons.
This gel represents the two possibilities of results. In the first four lanes, we can see the results from DNA amplification of a non-transgenic soybean, whereas in the last four lanes we observe the DNA amplification obtained from a sample extracted of transgenic soybean.
In lanes 1 and 7, the distinct band at 500 bp corresponds to the amplification of one part of a chloroplast gene. These two lanes indicate the presence of plant DNA in the extract. In lanes 2 and 8, a band at 118 bp is the proof of the presence of the soybean lectin. In this way we demonstrate the presence of soy DNA.
In the other two sets of lanes, if we see bands at the appropriate size (727 bp for the NOS amplification or 199 bp for 35 S amplification), we can determine that the soybean powder analyzed contains a transgenic construct. This would hold true if we saw either one of the two bands, as they are not of plant origin.
The results of a student team are displayed in Fig. 2. The disposition of the lanes is different, but the final result is the same. We can see an amplification of each sequence in the transgenic soybean whereas in the non-transgenic one there is only an amplification of the chloroplast and the lectin sequences.
CRITICAL EVALUATION OF RESULTS
At the end of the experiments, the students are given the following instructions for writing their reports.
“Your report should be submitted to your instructor at the end of the class. It should contain a complete explanation of all the steps of the experiment (it includes the possible constitution of the solution used in all the steps of the kit, not detailed in the kit, and the utility of changing the temperature during the purification), a photograph of the gel appropriately labeled and with a legend noting all the important features, and a brief appraisal of the results with answers to the following questions.”
1) Considering that the smallest visible size markers are 100 and 200 bp, what should you theoretically observe on your gel with the two kinds of samples? Compare with your results, and conclude.
The students should obtain the results observed in Fig. 1.
2) What kind of negative controls can you include to confirm your results?
Students can suggest several controls such as PCR without any extracted DNA to be sure of the purity of their solutions, PCR with only one oligonucleotide to ensure them of their purity, PCR with purified sequences (chloroplasts, lectin, etc.) to verify the length of the transcripts, and so on.
3) With what kind of techniques can you estimate the level of GMO in your sample?
The students can describe the quantitative PCR or the use of PCR with DNA extracted from GMO samples of well known concentration (by mixing GMO and non-GMO soy flour for example).
4) How can you be sure that your specific amplification has worked?
The students can propose to digest the DNA obtained from PCR amplification with restriction enzymes. They will obtain bands on electrophoresis gels. The size of the bands will be compared with predicted sizes calculated from the sequences of the DNA and the corresponding restriction map. Students can also propose to hybridize the transcripts to the complementary specific sequences or to perform nested PCR (PCR with primers hybridizing and amplifying in the amplicon).
5) Is this technique absolutely infallible, and why?
Wrong negatives can sometimes occur, and it is possible to construct new GMO plants without these genes.
Table 1. Sequences of the primers used for the PCR amplification
Size of the predicted band
Sense, LEC 1
Anti-sense, LEC 2
35 S Promoter
Sense, NOS A
Anti-sense, NOS D
The abbreviations used are: GMO, genetically modified organisms; EB, ethidium bromide; NOS, nopaline synthase.