PCR based Testing of Living Modified Organisms (LMOs) Gurinder Jit Randhawa Senior Scientist NRC on DNA Fingerprinting NBPGR New Delhi The first commercially available transgenic crop was the FLAVR-SAVR tomato marketed by Calgene in 1994. Today, the largest cultivation area is under two major crops i.e. maize and soybean. Transgenic plants usually contain three essential elements: a transgene (the new genetic material introduced to give the plant the desired new characteristics), a promoter (which controls when the transgene is expressed in the transgenic plant) and a terminator (which makes sure that the transgene ends properly and does not create chimeric proteins with those from the plant). Although many potential promoters and terminator elements have been identified, the most commonly used are the CaMV 35S promoter, from the 35S RNA of the phytopathogenic Cauliflower mosaic Caulimovirus, and the NOS terminator, derived from the nopaline synthase gene from the Agrobacterium tumefaciens Ti plasmid. Almost all commercially developed transgenic plants contain either the CaMV 35S promoter or the NOS terminator. The ability to detect these elements will allow detection of the vast majority of transgenic planting material. In an interlinking study conducted in 1997 concluded that either the NOS terminator or the CaMV 35S promoter sequences were present in 27 of 28 EU-approved transgenic crops. The principle of direct detection of recombinant DNA in transgenic by the polymerase chain reaction (PCR) constitutes three main steps: (1) DNA-extraction (2) Amplification by PCR (3) Verification of PCR products If a nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be designed and later synthesized and the desired segment of rdna can be amplified. Detection limits are in the range 20pg-10ng target DNA and 0.0001-1% of mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker. Principle of the PCR Segments of genomic DNA can be amplified in vitro under conditions in which short oligonucleotides primers (having the complementary sequence to the target DNA) synthesis that DNA by a thermostable enzyme (e.g. Taq DNA polymerase) and the addition of deoxynucleotides. Repeated cycling of this reaction enables amplification of specific sequences from very few target DNA molecules. At least two types of sequence of a target gene or stretch of DNA is already known specific primers may be synthesized.
The template genomic DNA is then subjected to the amplification procedure (PCR) and the amplification products are analyzed by agarose gel electrophoresis. The detection of genetically modified plant material is based on the basic principle of PCR which follows three steps: 1) Heat denaturating the double stranded DNA 2) Annealing the primers on the sequences by cooling 3) Elongation of the DNA by extending the primers in opposite direction. These three steps constitute a cycle, and the PCR comprises repetition of the cycle. The amount of DNA is exponentially amplified. DNA yield therefore increases as a function of 2 n, 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 10 9 - fold and can easily be detected on electrophoresis gel. 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. This way we can 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 35S 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 these elements. The identification of new promoter and terminator sequences in transgenic constructs is becoming more challenging. The PCR primers are designed to amplify products of different sizes to facilitate their identification. Material and reagents 1. Taq DNA polymerase 11. Sterile distilled water 2. Genomic DNA (5ng/μl) 12. Gel electrophoresis tank, gel mould and slot former 3. DNTP mix (2mM each of datp, 13. Electrophoresis grade agarose dctp, dgtp and dttp) 4. MgCl 2 (25mM) 14. 0.5xTBE buffer 5. Buffer 15. Ethidium bromide solution (5μg/μl) 6. Primers 16. DNA length marker 7. Pipetteman and tips 17. Loading buffer 8. Thin walled PCR tubes 18. 100V power supply 9. 500μl micro centrifuge tubes 19. UV transilluminator 10. PCR machine (Perkin Elmer 9600) 20. Polaroid Camera
Protocol Amplification of transgenes: 1. Each 25 μl of reaction contains 10x buffer 2.5 μl (Final Concentration 1x) dntps (each) 2.5 μl (Final Concentration 200 μm) Primers (R, F) 2.5 μl x 2 (Final Concentration 1 μm) Taq Polymerase 1.0 μl (Final Concentration 2.0U) Template DNA Variable volume (Final Concentration 1 μg to 100pg) Distilled Water Add to makeup the volume 25 μl Prepare a master mix (for all samples and control) that contains all the above components except the DNA. Aliquot the master mix in the PCR tubes and add template DNA. 2. Place the tubes in the PCR machine and carry out an initial denaturation step at 94 0 C for 5 minutes followed by 40 cycles with following parameters: Step 1 94 0 C for 30 seconds Step 2 62 0 C for 1.0 minutes Step 3 72 0 C for 1.0 minutes Extend the 72 0 C for 7.0 minutes and finally hold the sample at 4 0 C 3. when amplification is finished, add 3μl of loading dye and load on to 1.6% agarose gel in 1.0 X TBE buffer with ethidium bromide. Run the gel in 1.0 X TAE buffer at 55v for 6 hrs. 4. Visualise the gel on UV trans illuminator. Photograph the gel by using Polaroid 667 film. If no product specific tests are available, or in addition to them, detection of at least three genetic elements (CaMV-35S, nos 3' and nptii) may be performed. Verification of PCR products Several methods are used to verify PCR results, which vary in reliability, precision and cost. They include (i) (ii) (iii) (iv) Specific cleavage of the amplification products by restriction endonucleases is the simplest method to identify the amplification products. The more time-consuming, but also more specific, transfer of separated PCRproducts onto membranes (Southern Blot) followed by hybridization with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. This is the most accurate proof of amplified DNA. However, this opportunity is not available in all laboratories and is not the method of choice for routine analysis. Nested-PCR assays combine high specificity and sensitive. Use of two pairs of primers spanning the boundary of two or three genetic elements is regarded as sufficiently specific for a LMO-product. In general, the increased
sensitivity of nested-pcr systems allows low levels of LMOs to be detected in raw material and finished products. The applicability of PCR methods to detect GMOs in derivatives (starch, oil, lecithin) and processed products is limited by the quality of the DNA present. Any physical or chemical treatment of food samples, such as heat, ph or shear forces results in a decrease in the average DNA fragment size due to random cleavage of these macromolecules and could make detection impossible. Meyer et al (1999) reported that DNA is no longer detectable in derivatives of starch (glucose syrup, maltodextrin etc.), purified lecithin, refined vegetable oil, soya sauce and powder and highly heat-treated finished products. 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 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. Experimental Procedures based on detection Kit The laboratory equipment used includes the following: powdered soybeans (transgenic and non-transgenic) (SoyBean 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 TM plant kit), a PCR kit (Taq PCR core kit) from Qiagen, synthetic oligonucleotides (Isoprim), PCR Eppendorf thermocyclers (Polylabo), agarose electrophoresis systems with generators, agarose, 10X TBE buffer, a 100-bp DNA ladder (Promega), a microwave to melt the agarose, a concentrated ethidium bromide (EB) solution, 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. Extraction and Purification of Plant DNA The isolation of DNA from soybean with the DNeasy TM plant kit is carried out by the manufacturers (Qiagen) briefly the instructions consist of the following 1) Switch on the heater at 65 0 C to keep the AE buffer hot 2) Weigh 0.1 g of soy powder in a 2ml eppendorf tube (maximum capacity of the kit) 3) Add 400 μl of AP-I buffer (The lysis buffer is constituted of detergents, Proteases and salts) and 4μl of RNase (100mg per ml) (To avoid the purification of RNA and permit the liquifaction of the solution and mix gently. Solution AP-I contains detergents, so avoid contact with skin. 4) Incubate 10-15min at 65 0 C (to keep endogenous DNases inactive, denature the proteins and break the cell walls) 5) Add 130μl of AP-II 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 causing misfolding of other ones and inactivates the DNases. Solution AP-II contains acidic acid so avoid contact with skin. 6) Centrifuge the mix for 2 min. at 12000 G on a QIA shredder 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. 7) The solution is transferred to a fresh 2ml eppendorf tube and the pellet is discarded. By this way they can evaluate the volume of solution they have and thus can calculate how much solution they will add afterwards. At this step it is useful to show them how to measure a volume used in pipetteman. 8) Add 0.5 volume of AP-III solution and vortex the mixture (to constitute the DNA salts). To avoid chloride production, do not mix AP-III solution with Sodium hypo chlorite. 9) Add one volume of absolute ethanol and vortex the mix. The volume is the same as the volume used to add the AP-III solution. 10) Put the solution on the DNeasy minispin column and centrifuge for one minute at 10,000 X 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. Discard the solution at the bottom of the eppendorf tube after each step of centrifugation to avoid the overflow. 11) Place the column on a new 2 ml eppendorf tube. 12) Pipette 500 μl of AW buffer on the column and centrifuge for 1 min at 10,000 X g (to desalt and wash the DNA) 13) Pipette 500 μl of AW buffer on the column and centrifuge for 2 min at 10,000 X g (to be sure to eliminate all the buffer). Remove the AE buffer from the heater 2 min before use. 14) Place the column on a new 2 ml eppendorf tube. 15) Pipette 100 μl of AE buffer on the column and centrifuge for 1 min at 10000 X g (to elute the DNA). 16) Repeat this operation once. 17) Put the eppendorf tube containing the DNA solution on ice (The column can be thrown away) PCR The PCR kit used is the core kit from Qiagen. Different Taq polymerases have been tested and all are efficient on these primers. This choice is because this product is inexpensive and because of the presence of premix of four dntp in one solution. To each tube add 5 μl of the appropriate primer mix solution (initial concentration of 0.1μg / μl). Mix gently and then return to ice. To each tube add 0.6μl of dntp (a mixture of the four dntp at 10mM) and 0.5 μl (1 unit) of 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. Step1: 94 0 C, 10 min for DNA denaturation; Step2: 94 0 C, 1 min; 63 0 C, 1 min (annealing); 72 0 C, 1 min (elongation); Step3: 72 0 C, 10 min for final elongation; 4 0 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. Gel Electrophoresis: During the PCR, prepare a 3% w/v agarose gel in 0.5X TBE (0.09M Tris, 0.09M Borate, 1mM EDTA). Boil the solution gently until it become clear. Pay attention to the overflow during boiling, which can alter the concentration. Then allow it to cool to around 55 0 C and pour a gel. Once the gel has solidified, the gel plate is placed on the electrophoresis apparatus and covered with 0.5 X 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 8μl of each amplified solution and 1μl of 6X loading buffer (0.3 M EDTA containing 10% glycerol to increase density, 0.25% bromophenol blue, 0.25% xylene cyanol, and 0.25% Orange G as migration indicator). In a fifth, mix 3μl of 100-bp DNA ladder and 0.5μl of 6X 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 1500bp), 8μl of the chloroplast amplified DNA solution with dye and then 8μl of lectin-amplified mix, 8μl of the NOS terminator-amplified mix. Run the gel at 200V until the orange G (which migrates at approximately 50bp) is at the bottom of the gel (approximately 30 min). Stain the gel in ethidium bromide (to prepare the stain, put five drops of stock solution (10mg/ml) in 200 ml of distilled water). (Be careful in handling ethidium bromide; Use gloves and decontaminate the area used for this area 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 care of the exposure of the skin and particularly eyes to the high intensity UV light. Use a Plexi glass screen).