1 C118 Laboratory - Genetically Modified Foods Biotechnology Explorer GMO Foods (BioRad Kit #166-2500EDU) Introduction: Overview: The goal of the lab is to determine if a food sample contains GMO’s. In the first week, you will extract genomic DNA from food samples and run polymerase chain reactions (PCR) to amplify GMO and natural plant sequences from the DNA. In the second week, you will separate and detect certain DNA sequences using gel electrophoresis as shown in Figure 1.2 Fig. 1. Detecting GM foods by PCR. Genomic DNA is extracted from test foods and then two PCR reactions are performed on each test food genomic DNA sample. One PCR reaction uses primers specific to a common plant gene (plant primers) to verify that viable DNA was successfully extracted from the food. No matter whether the food is GM or not, this PCR reaction should always amplify DNA (See lanes 1 and 3 of the gel above). The other PCR reaction uses primers specific to sequences commonly found in GM crops (GMO primers). This PCR reaction will only amplify DNA if the test food is GM (See lane 4). If the test food is non-GM, then the GMO primers will not be complementary to any sequence within the test food genomic DNA and will not anneal, so no DNA will be amplified (see lane 2). To find out whether DNA has been amplified or not, the PCR products are electrophoresed on a gel and stained to visualize DNA as bands. A molecular weight ruler (lane 5) is electrophoresed with the samples to allow the sizes of the DNA bands to be determined. Electrophoresis (Used for Week 2) Gel electrophoresis using a polyacrylamide gel is the method used to separate different size DNA fragments. In general, electrophoresis is the movement of ions in an electric field. This technique is an extraordinarily important method of separating and analyzing biological molecules. The movement of a molecule in an electric field E is a function of net charge q and the frictional coefficient f. The velocity v at which a molecule moves in an electric field is a function of the field strength and net charge as defined by the relationship given below. fEq=ν A molecule’s mobility μ is defined as the ratio of velocity to field strength. By extension, mobility is also equal to the ratio of charge and frictional coefficient as illustrated below: fqE==νμ The frictional coefficient is a measure of the hydrodynamic size of the molecule wherein the larger the molecular size of a molecule, the larger the frictional coefficient. Molecular shape is also an important component of the frictional coefficient. Biological molecules like proteins and polynucleic acids have distinctive shapes. Therefore, the electrophoretic mobility of a biological macromolecule is a function of its size, shape, and net charge.3 Week 2: Gel Electrophoresis, Staining, and Analysis In lab last week, DNA was extracted from non-GMO (Genetically Modified Organism) and test food samples as well as positive control GMO DNA. The DNA was then amplified using the polymerase chain reaction (PCR). In the next part of the experiment, gel electrophoresis and staining will be used to separate and image different size strands of DNA. The presence or absence of certain size DNA fragments will enable you to determine if the test food contains genetically modified organisms. The PCR product bands of interest in this lab are 455 bp for the plant primers and approximately 200 bp for the GMO primers. Specifically, the promoter 35 from the cauliflower mosaic virus (CaMV 35 S) is 203 bp and the terminator from nopaline synthase (NOS) gene of Agrobacterium tumefaciens is 225 bp. Nearly 85% of GMO’s will have one or both of these GMO primers. Materials and Reagents- PCR samples from last week Orange G loading dye PCR molecular weight ruler flip top micro test tubes (2 per group) 20-200 uL adjustable volume micropipette with aerosol barrier tips 2-20 uL adjustable volume micropipette with aerosol barrier tips Criterion electrophoresis units power supply Ready Gel 10% TBE Precast polyacrylamide gels for criterion electrophoresis cells (Biorad: 345-0053) (2 student groups per gel) TBE electrophoresis run buffer (Biorad: 161-0770) fast blast DNA stain (Biorad: 166-1110EDU) gel staining trays (use tray that gel was shipped in) centrifuge orbital shaker gel loading micropipette tips Safety and Waste Disposal- Wear eye protection at all times. Wear gloves when handling gels and using the DNA fast blast stain. Although this substance is non toxic and non carcinogenic, contact with the skin will result in staining. A 10% bleach or 70% alcohol solution will remove Fast Blast from most surfaces. Use caution. Electrophoresis occurs at high voltage (200 V). Do not touch unit while voltage is applied. Dispose of all solutions in the liquid waste container.4 AI preparation: 1. Thaw the Orange G loading dye and PCR molecular weight ruler. Pulse spin the tubes in a centrifuge to bring all the contents to the bottom. 2. Add 40 uL of Orange G loading dye to the vial of PCR molecular weight ruler. Mix well and pulse spin. 3. Prepare TBE electrophoresis run buffer. Dilute the TBE 10x run buffer by a factor of 10. (2 L will be needed for each lab section) 4. Prepare fast blast DNA stain. Fast blast DNA stain is provided as 500x concentrate that must be diluted to 1x prior to use. Dilute 1 mL of 500x Fast Blast with 499 mL of deionized water. This solution stains DNA in polyacrylamide in around 30 minutes. It is a convenient, safe, and nontoxic alternative to ethidium bromide for the detection of DNA. Fast Blast contains a cationic compound that belongs to the thiazin family of dyes. The positively charged dye molecules are attracted to and bind to the negatively charged phosphate groups on DNA. DNA will stain a deep blue color with consistent results. Procedure Preparation of Samples Using a fresh pipet tip for each sample, add 10 uL of Orange G loading dye to each sample and mix well. Samples are 1) Non-GMO food with plant primers 2) Non- GMO food with GM primers 3) Test food with plant primers 4) Test food with GM primers 5) Positive control DNA with plant primers 6) Positive control DNA with GM primers Electrophoresis The Criterion cell is a dedicated electrophoresis cell for running Criterion precast gels. The Criterion cell includes a tank with lower electrodes, a lid including power cables