13 PCR to detect GMO foods

Genetic selection of crops 

Humans have been modifying crop plants since the dawn of civilization. Ten thousand years ago human societies began to transition from hunting and gathering to agriculture. As of 4,000 years ago, early civilizations had completed the domestication of all major crop species upon which human survival is now dependent, including rice, wheat, and maize. 

Year after year ancient peoples selected and saved seeds from plants displaying specific traits. Later, with cross breeding and the development of hybrid plants, modern plant breeding emerged. Most humans alive today have never seen and would not recognize the quirky wild plants that were the early progenitors of current crops. 

For example, the ancestor of modern corn, teosinte, had small kernels each inside a tough husk. Teosinte plants had multiple stalks and long branches, while modern cultivated maize has a single stalk. During the domestication of maize, which began thousands of years ago, humans selected for large sheathed cobs containing large kernels without husks. 

Genetic engineering of crops

Today, modern biotechnology and genetic engineering allow scientists and breeders to confer very specific traits rapidly by introducing particular genes directly into plants. Introduced genes (or transgenes) may derive from the same species of plant, from other plant species, or even from animals or bacteria. For example, the gene for the insecticidal toxin in transgenic cotton, potato, and corn plants comes from the bacterium Bacillus thuringiensis (Bt). One of the genes allowing vitamin A production in golden rice is derived from the bacterium Erwinia uredovora, commonly found in soil. 

Genetic engineering of foods usually pursues one or more of these benefits: 

▪ Agricultural: increased yield or tolerance to suboptimal conditions (e.g. drought) 

▪ Environmental: reduced use of herbicides, pesticides, or fertilizers (e.g. Bt cotton) 

▪ Nutritional: higher quality, supplementation of diet deficiencies (e.g. Vitamin A)

For example, newly-engineered GMO bananas can produce ß-carotene, an essential nutrient and the primary dietary source of provitamin A especially needed by children. In this laboratory activity we will examine the genetic makeup of various foods, and weigh the evidence around the genetic engineering of crops. 

Methods for genetically engineering foods 

How do you get a plant to take up a foreign gene? At least three methods can be used to introduce foreign DNA into the host plant: 

• Biological vectors (plasmid from Agrobacterium). The biological vector system is the one most commonly used.
• Physical methods (gene gun or electroporation) 
• Chemical methods (polyethyleneglycol and calcium chloride).

In order for the transgene to work effectively in its new host it needs to be controlled by a promoter sequence and a terminator sequence. This grouping is called a gene cassette, where the promoter and terminator regulatory regions influence where and when a gene will be expressed. The most commonly used promoter in engineered plants is the CaMV35S (35S) promoter derived from the cauliflower mosaic virus (a virus that infects cauliflower plants). The NOS terminator from the Ti plasmid in Agrobacterium tumefaciens is the most common terminator. These regulatory regions enable strong transcription of the transgenes across all tissues of the host plant. 

Today we will isolate DNA from food products containing corn or soy, which have commonly been genetically modified. We will then use PCR and gel electrophoresis to assess the presence of transgenes in commercial food products. We will amplify the 35S transgenic regulatory sequences by PCR to test foods derived from genetically engineered crops as well as “GMO” and “non-GMO” control DNA samples. We will also test for the endogenous plant “housekeeping” gene Tubulin to confirm that we have extracted viable DNA.  The Tubulin gene encodes the tubulin proteins that form microtubules essential for chromosomal division. Since tubulin plays an essential role in all plants, the Tubulin gene is highly conserved across plant species and thus can be amplified by the same primer set.

POLYMERASE CHAIN REACTION (PCR) Introduction

Adapted from work by: John Urbance, Ph.D. by Doug Luckie, Ph.D., and Mike Haenisch

Have you ever wondered how forensic scientists get enough DNA from a single drop of dried blood or from a single hair to conduct investigations? Or how, out of the millions of base pairs that make up an organism’s genome, scientists isolate a particular gene, or set of genes, for analysis?

They can accomplish these things by “amplifying” the targeted region of the genome using a technique called the Polymerase Chain Reaction (PCR). PCR is a method of synthesizing (“amplifying”) large quantities of a targeted region of DNA in vitro (in the test tube). The DNA is synthesized the same way that cells do it—using a DNA polymerase (the enzyme that cells use to replicate their DNA). Once amplified, PCR products can simply be visualized by agarose gel electrophoresis or can be further analyzed by subsequent enzymatic digestion for DNA fingerprinting, by cloning, or by DNA sequencing.

PCR works by using a thermostable DNA polymerase (Taq polymerase) and short DNA fragments, called primers, to direct the synthesis of a specifically-targeted region of genomic DNA. The synthesis reaction is repeated numerous times in a series of PCR ‘cycles’. The products of previous synthesis cycles serve as template for the next cycle. This results in an exponential amplification of the targeted region of DNA—every cycle will double the copy number of the target region. This repeated cycling is made possible by the use of Taq polymerase, a thermostable (heat-tolerant) DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus, originally isolated from a hot spring in Yellowstone National Park (ambient temperature 80 °C!). Because it comes from a heat-adapted bacterium, Taq polymerase can withstand the repeated, high-temperature DNA denaturation steps that are part of the PCR procedure.

The Primers

All DNA polymerases require a short segment of double-stranded nucleic acid to initiate DNA synthesis. During DNA replication, cells use short stretches of complementary RNA—synthesized by enzymes called ‘primases’—to initiate polymerization. In the laboratory, short, complementary single-stranded DNA primers are also used in PCR to initiate DNA synthesis and to designate the specific target region to be amplified. The primers are easily synthesized and can be designed to be complementary to any known DNA sequence.  They typically range from 15 to 30 nucleotides for PCR. The primers determine the target specificity (i.e. which segment of the template DNA will get amplified) of the PCR reaction.

The Cycles

1. Denaturation (HOT). During the denaturation step, the reaction cocktail is exposed to high temperature, usually 95 °C. This high temperature will denature the DNA– meaning the two complementary strands of the DNA molecule unravel, exposing the nucleotide bases.
2. Primer Annealing (COOL). During the second step of each cycle, the temperature is lowered to an annealing temperature, allow annealing of the primers to their complementary targets on the DNA template (one for each DNA strand). These are designed to flank the desired target region of your DNA template and serve as the starting points for DNA synthesis by the Taq polymerase. Each pair of primers will have a particular annealing temperature determined by the length of the primers and their nucleotide content.
3. Extension (MEDIUM). The reaction cocktail is now brought to the optimum reaction temperature for Taq polymerase (68 to 72° C). During this step, the Taq will bind to each DNA strand and “extend” from the priming sites (synthesize a complementary strand of the targeted DNA by linking together nucleotides).

Notice that these three steps are accomplished simply by varying the incubation temperature of the reaction tubes. Typically, PCR reactions are run for 30 to 40 cycles, which are performed by a specialized machine called a thermocycler designed to rapidly heat and cool the reaction tubes to the desired temperatures. Each cycle DOUBLES the number of copies of the target DNA sequence. In reality, PCR is not always 100% efficient, but it is the primary way scientists rapidly obtain large quantities of a desired DNA section that can be used for sequencing, gel electrophoresis, gene cloning, protein production, and more.

What’s in the reaction mixture?

You must create a reaction mixture in your tube that provides everything the enzyme (in this case, Taq polymerase) needs to function as it would in the cell during DNA Replication. Your reaction contains:

• Taq Buffer and MgCl₂: Each cellular enzyme has a specific salt concentration, pH, and temperature required for its optimum performance. At 1X your PCR reaction buffer provides the proper salt concentration and pH (~8.5) for Taq polymerase.
• dNTPs: (i.e. nucleotide bases) DNA is a polymer of these four nucleotides [A,T,G,C]. They are the building blocks polymerase uses to synthesize new DNA.
• Primers:  PCR usually requires two primers, one targeted to each DNA strand so that both strands are copied. Each primer is a short single-stranded DNA molecule so it can bind to a complementary DNA strand.
• DNA Template: In the cell, DNA polymerases use denatured, genomic DNA as a template upon which to synthesize complementary DNA strands.
• Taq polymerase: You can’t carry out an enzymatic reaction without the enzyme.

What reactions will we prepare today?

We will test a “GMO Banana” DNA sample, a non-GMO negative control, along with foods and plants of your choice. We strongly recommend testing one or more CORN or SOY derivatives, as these are some of the most abundant genetically engineered crops. You typically perform many PCR reactions at one time. To set up these reactions efficiently, you will use a master mix, which is a single tube that contains all the reagents that each reaction requires (The buffer, dNTPs, MgCl₂, polymerase, and water).  Your reactions will contain two different primer sets. Your primer mixture contains primers to amplify Tubulin and primers that amplify the 35S DNA sequence.

You will dispense aliquots into individual reaction tubes, and then you can add the unique component (usually the DNA template). This ensures that all of your reactions received the same concentration of reagents. You will perform your PCR reactions in small, 0.2 mL strip tubes (small tubes connected to each other by plastic).

What else do I need to know to successfully perform PCR?

• Accurate pipetting is essential to PCR success. Do not contaminate the PCR reagents. PCR is very sensitive!
• Keep your reactions COLD (on ice) before running them in the PCR machine

Protocol (present in your lab workbook but read over it now so you are prepared)

 DNA Extraction 

Each group of four will select two foods to analyze; each pair of students will process one food sample. Label two 1.7 mL tubes per lab group on the side AND cap of the tube. Complete the table below to indicate your selected foods and predictions for if they are genetically modified.

• 1 tube labeled “F1”and the group’s name. Used for DNA extraction from Food 1
• 1 tube labeled “F2” and the group’s name. Used for DNA extraction from Food 2

1. Prepare test foods for DNA extraction. Put a very small amount of food on a piece of weigh paper, fold the paper over the food, and crush the food into a fine powder.
2. Use clean tweezers to transfer a small amount, approximately 1 mm in diameter or less, into your labeled 1.7 mL tube. Aim for a food fragment the size of a pinhead.
3. Add 50 μL of DNA-EZ™ Lysis Solution to each tube and mix by pipetting up and down. Avoid contact with skin! 
4. Tightly cap the tubes containing Lysis Solution and the test foods. Ensure that food fragments are well mixed into the Lysis Solution.
5. Incubate the food mix in Lysis Solution for 5 minutes at 95 °C.
6. Remove tubes from heat and let them rest in a tube rack at room temperature. Ensure the tubes remain in a vertical position.
7. Add 5 μL of DNA-EZ Neutralization Solution to each tube. Cap and then flick your tube to mix.
8. Spin down the tubes in a microcentrifuge at 10,000 RPM (rotations per minute) for 2 minutes. The debris will sediment to the bottom of the tube. The DNA extract should be used immediately for PCR.
9. Label 4 clean 200 μL PCR tubes per group on the side wall and cap. Your group needs:

• 1 tube labeled “T1”: Test DNA extracted from Food 1
• 1 tube labeled “T2”: Test DNA extracted from Food 2
• 1 tube labeled “G”: ‘GMO Banana’ DNA. This is your positive control.
• 1 tube labeled “W”: ‘non-GMO Banana’ DNA. This is your negative control.

10. Keep your PCR tubes and reagents on ice. Use a P100 micropipette to add 20 μL primer mix to each of your 4 PCR tubes. The primer mixture contains primers to amplify the Tubulin gene (present in all plants) and the 35S promoter, which is present in the majority of genetically engineered crops.
11. Use a P10 pipette to add 5 uL of 5X PCR master mix to each of your 4 PCR tubes. Change pipette tips each time you transfer the master mix.
12. Add DNA samples to each PCR tube, using a clean P10 tip for each sample.

• Tubes T1 and T2 (Food DNA extracts): Use the P10 pipette to add 2 μL of DNA extract. Pipette the liquid near the top of your tube and avoid any food particles.
• Tubes G and W (control DNA samples): Pipette 2 μL of ‘GMO Banana DNA’ and ‘non-GMO Banana’ samples into your control reactions.

13. Cap the PCR tubes firmly and flick to mix. Spin the tubes briefly using a minicentrifuge. Make sure all the liquid volume collects at the bottom of the tubes.
14. Keep your samples on ice until all groups are ready to start the PCR. The PCR machine has been programmed to perform the following steps:

• Initial denaturation: 94 °C for 60 seconds
• 35 cycles of:
  • o Denaturation: 94 °C for 20 seconds
  • o Annealing: 55 °C for 20 seconds
  • o Extension: 72 °C for 20 seconds
• Final extension: 72 °C for 60 seconds
• Hold samples at 12 °C