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Indian Journal of Biotechnology Vol 11, April 2012, pp 176-181 Detection of genetically modified cotton seeds using PCR and real-time PCR S Shree Vidhya, P H Ramanjini Gowda*, K N Yogendra, T M Ningaraju and T Salome Department of Biotechnology, University of Agricultural Sciences, Bangalore 560 065, India Received 3 June 2010; revised 8 April 2011; accepted 10 August 2011 Detection of genetically modified (GM) crops and products are necessary to comply with international labeling regulations and to avoid spurious and unapproved GM planting. DNA based analytical tools involving PCR and real-time PCR were used to detect GM cotton seeds. Four pairs of primers specific for CaMV 35S, Nos, nptII and cry1Ac genes were used for the DNA detection of GM crop. The limit of detection in real-time PCR was found to be 0.1%. Evidently, these two PCR techniques were successful in detecting transgenes in the DNA of GM cotton seeds. Keywords: Detection method, GM cotton seeds, labeling regulations, PCR, real-time PCR Introduction The development of new technologies and inventions in genetic engineering has given rise to genetically modified organisms (GMOs). These organisms carry genetic material that has been altered by the insertion or deletion of genes in order to confer pest resistance, herbicide tolerance or to improve the quality of their produce. A number of these GMO products and seeds are available in the markets today after going through rigorous testing and labeling procedures. Yet, there is concern amongst the general public regarding the safety of consumption of products derived from these GMOs. And environmentalists are worried about the cross contaminations of these GMOs with the local or indigenous varieties of crops when both are being cultivated simultaneously. The infiltration of unapproved and spurious varieties of genetically modified (GM) crop seeds into the market has been the reason for controversies regarding their acceptance amongst farmers because planting of these varieties leads to crop failures and huge commercial losses. Hence, there is a need for GM testing and detection, which will help to identify GMOs and stop the infiltration of spurious GM varieties into the markets. GMO analysis is usually carried out using protein and nucleic acid based techniques, such as, enzyme linked immunosorbant _________ *Author for correspondence: Tel: +91-80-2330153 (Ext 263) E-mail: [email protected] assay (ELISA), lateral flow strip ELISA, polymerase chain reaction (PCR), microarray, etc1-5. The analytical techniques are often affected by the quality and the quantity of the target analyte and, hence, conventional PCR and real-time PCR methods are widely accepted for this purpose because of their specificity, sensitivity and reliability. The foreign DNA insert in GMOs usually consists of a promoter, a coding sequence and a terminator. The target sequence is the most important factor that controls the specificity of the PCR method. Screening of GMOs using PCR is based on sequences for control elements used in a majority of GM plants, such as, the cauliflower mosaic virus (CaMV) 35S promoter or terminator (P-35S or T-35S), Agrobacterium tumefaciens nopaline synthase terminator (T-Nos), and cloning vector genes coding for resistance to ampicillin and neomycin/kanamycin (bla or nptII), which are the targets for amplification. Using PCR, construct-specific detection targets the junctions between the adjacent elements of the gene construct, such as, the region between the promoter and the gene of interest. Similarly, event-specific detection targets the junction at the integration locus between the recipient genome and the inserted DNA6. Real-time PCR provides a very convenient technique for rapid and sensitive detection of these transgenics with very limited amount of starting material. In real-time PCR, the detection is based on the fluorescence measurement emitted by the probes or the dyes used during the amplification of the target. VIDHYA et al: DETECTION OF GM COTTON SEEDS Bt-Cotton was among the first GM crops to be commercialized during the 1900’s at the global level and was officially approved for sale in India in 2002. It consists of a gene from the soil bacterium Bacillus thuringensis (Bt), which provides resistance to different bollworm species, a major pest in cotton crops, and helps cotton growers benefit through efficient pest control. India is the second largest producer and consumer of cotton accounting for a little over 21% of the global cotton production in 2008-2009, and is cultivated in an area of about 9.4 m ha7,8. In this study, we have used the P-35S, T-Nos, nptII and cry1Ac specific primers for the screening of transgenic cotton using conventional PCR and realtime PCR (using SYBR Green I chemistry). Materials and Methods Seed Material Three samples of transgenic cotton seeds, RCH2, JK99 and R3, and non-transgenic cotton seeds, MRC 5156, were obtained from Dow AgroSciences India Pvt. Ltd. DNA Extraction and Quantification Genomic DNA from the transgenic and nontransgenic cotton seeds were extracted using a modified hexadecyltrimethylammonium bromide (CTAB)-based method along with Qiagen’s Genomictip 20/G kit (Qiagen, Hilden, Germany) as per the protocol provided. The extracted DNA was dissolved in 1× TE buffer. For sensitivity tests, different per cent transgenic contamination standards (5, 1 & 0.1%) were prepared by mixing appropriate amounts of transgenic seed powder with non-transgenic cotton seed powder. A mixture of 9.5 g non-GM cotton seed powder and 0.5 g GM cotton seed powder, resulting in a 5% (w/w) sample was used as the starting material. 2 g of this 5% (w/w) mixture was added to 8 g of the nonGM cotton seed powder giving a 1% (w/w) sample. A 0.5% (w/w) mixture was prepared using 5 g of the 1% (w/w) sample and 5 g of the non-GM cotton seed powder, while 8 g of non-GM cotton seed powder was mixed with 2 g of 0.5% (w/w) sample giving 0.1% (w/w) sample9. DNA was extracted from these mixtures using the same CTAB-Genomic-tip 20/G method. The real-time PCR was carried out with the DNA extracted from 5, 1 and 0.1% mass fraction mixtures. The concentration of extracted DNA was quantified by measuring the UV absorption at 260 nm 177 using an Eppendorf Biophotometer. DNA was checked for fragmentation by gel electrophoresis. Working stocks of 100 ng/µL were prepared and stored at –20°C for use. PCR Analysis PCR analysis of transgenic and non-transgenic cotton was carried out using the published primers, P-35S, nptII, T-Nos and cry1Ac. The primer sequences and their amplicon size are shown in Table 1. All the primers were synthesized by Sigma Aldrich Chemicals Pvt. Ltd. PCR was carried out in 25 µL final volume reaction mixtures using 10× Taq DNA polymerase buffer with 15 mM MgCl2, Taq DNA polymerase (3 U/µL), dNTP mix (2.5 mM solution) (Bangalore Genei), 0.25 mM of each primer and 100 ng of sample DNA. The thermocycler (TECHNE, Touchgene Gradient, UK) was programmed with initial denaturation at 94oC for 3 min. The cycling conditions for 35 cycles were: denaturing at 94oC for 30 sec, annealing at 55oC (nptII, T-NOS and Cry1Ac) and 54oC (P-35S) for 40 sec, and extension at 72°C for 30 sec. The final extension was at 72°C for 10 min. The PCR amplicons were analyzed on 2% agarose/ethidium bromide gel (Sigma Aldrich Chemicals Pvt. Ltd.) along with 100 bp DNA ladder (New England BioLabs Inc.), visualized by direct observation on a UV transilluminator and the images were recorded using a gel documentation system AlphaImager (Alpha Innotech). Real-time PCR Analysis To find out sensitivity in detection of GMO’s, realtime PCR was carried out using SYBR Green I chemistry [iQ SYBR Green Supermix containing 100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM of Table 1—Details of primers sequences and the respective amplicon size used for qualitative PCR analysis of GM cotton Primer Sequence (5'—3') P-35S F: GCTCCTACAAATGCCATCA R: GATAGTGGGATTGTGCGTCA nptII F: GAGGCTATTCGGCTATGACTG R: ATCGGGAGCGGCGATACCGTA T-Nos F: GAATCCTGTTGCCGGTCTTG R: TTATCCTAGTTTGCGCGCTA cryIAc F: GCCAATGCCTCGTGATTGTTCTCTGC R: GATTTGCGAGGCTGGCCAGCTCCACG F: Forward; R: Reverse [ Amplicon size (bp) 19510,11 70012 18010,11 28013 178 INDIAN J BIOTECHNOL, APRIL 2012 each dNTP, 50 U/mL DNA polymerase (iTaq), and 6 mM MgCl2, SYBR Green I, 20 nM fluorescein, and stabilizers; Bio-Rad, Hercules, CA) and analyzed by an iCycler iQ Real Time Detection System (Bio-Rad). P-35S, nptII and cry1Ac primers were used for this analysis. The reactions were carried out at 95°C for 3 min, followed by 35 cycles of amplification: denaturing at 94°C for 40 sec, annealing at 54°C (P-35S and nptII) and 58oC (cry1Ac) for 30 sec and extension at 72°C for 40 sec. The reactions were further subjected to 91 cycles of 0.5°C increments (30 sec each) beginning at 50°C for melting curve analysis to confirm the specificity of the amplification products. Thermocycling was performed in a final volume of 25 µL (10.5 µL of water, 0.2 µM of each primer, 1 µL of genomic DNA and 12.5 µL of 2X iQ SYBR Green Supermix; Bio-Rad). The real-time PCR analysis was performed with the 5, 1 and 0.1% GMO dilution DNA standards along with the genomic DNA of transgenic and non-transgenic cotton seed samples. Results and Discussion The transgenic construct usually contains the genes of 35S promoter, nptII and Nos terminator. In the present study, these genes were targeted in the DNA samples of transgenic cotton seeds (RCH2, JK99 & R3) and non-transgenic cotton seeds (MRC 5156) for their presence using their specific primers. The presence of cry1Ac gene was also checked with its specific primers. The primers specific to 35S promoter recorded a PCR product of 195 bp (Fig. 1a) in all the three transgenic samples. A study in the semi-quantitative detection of GM grains conducted by Tozzini et al13 also showed similar amplification band size with 35S promoter specific primers. An additional product of about 490 bp was seen as a faint band on the gel with 35S primers. Since the cotton seeds used were from hybrid plants, the parents may have different 35S promoter sequences and, hence, two bands were observed. The PCR with Nos primers recorded 180 bp products (Fig. 1b). Similar results with Nos primers were obtained by Oraby et al10 in their work on screening of food products for CaMV 35S promoter and Nos terminator and also by Hardegger et al14 in their study on quantitative detection of 35S and Nos terminator. The amplification with nptII primers recorded an amplicon size of 700 bp (Fig. 1c); similar to the results observed by Surekha et al12 in their Fig. 1—PCR amplifications using different primers: (a) PCR amplicons of size 195 bp using P-35S primers, (b) amplicons of size 180 bp using T-Nos primers, (c) amplicons of size 700 bp using nptII primers & (d) amplicons of size 280 bp using cryIAc primers. (M, 100 bp ladder; RCH2, JK99 and R3, transgenic cotton; & MRC 5156, Non-transgenic cotton) VIDHYA et al: DETECTION OF GM COTTON SEEDS study on the development of transgenic pigeon pea plants. The primer pair cry1Ac, specific for the Bt gene in cotton, yielded a PCR product of size 280 bp as shown in Fig. 1d. The product was detected only in the transgenic cotton seeds and not in the non-transgenics. Further, the cry1Ac primer was less sensitive and amplified a poor resolution fragment of 280 bp in agarose gel on PCR run of R3 sample DNA. The light amplification bands with cry1Ac specific primers may be because the transgenic cotton had a Bt-event, such as, a truncated cry1Ac, cry1Ab, cry1Ac+cry1Ab or cry1C, which are approved for cultivation in India8. Only the transgenic cotton showed amplifications in all the PCR runs with the primers used and no amplifications were recorded in the non-transgenic seeds as shown in Fig. 1. Therefore, the primers for 35S promoter, Nos terminator, nptII and cry1Ac genes can be used for routine screening and detection of specific traits in GM cotton. 179 Real-time PCR allows highly sensitive detection of the target with very little DNA. In this study, SYBR Green I, a non-specific intercalating fluorescent dye, was used to identify the amplified products. The different percentage of transgenic contamination standards (5, 1 , 0.1 & 100%) were used in each of the real-time PCR runs along with the three transgenic samples (RCH2, JK99 & R3) and one non-transgenic sample (MRC 5156). Real-time PCR read-out is given as the cycle threshold or threshold value (Ct), which is the increasing fluorescence measured above a predetermined set of cycles where the amplification efficiency is constant15,16. The amplification curves for each of the real-time PCR runs with specific primers are indicated in Fig. 2. The real-time PCR with P-35S specific primer showed strong amplification with average Ct value of 21.6 for the 100% transgenic contaminated sample. Similarly, for the 100% transgenic cotton DNA Fig. 2—Real-time PCR amplification curves and melt curve charts for reactions performed using P-35S, nptII and cryIAc specific primers. INDIAN J BIOTECHNOL, APRIL 2012 180 standard sample, average Ct values of 21.58 and 25.62 were obtained with nptII and cry1Ac specific primers, respectively. As evident from Table 2, the Ct value was found inversely proportional to the log of the initial amount of the target molecule. The Ct values of the 5, 1 and 0.1% transgenic contamination standards are higher compared to the average Ct value of the 100% transgenic contaminated sample, indicating that the GM cotton seeds have higher copy number of the transgenes. The non-transgenic cotton seeds (0% transgenic sample), MRC 5156, did not record any Ct values in any of real-time PCR runs, indicating the absence of transgenes in their DNA. Three independent runs were performed with two replicates in each case. The Ct mean values and the respective standard deviation (SD) and the relative standard deviation (RSDr) measured for each run using the three primers are indicated in Table 2. Since SYBR Green I binds non-specifically to double stranded DNA, the measured fluorescence may have been contributed by the non-specific PCR products or by primer-dimers. In order to differentiate such artifacts from the specific PCR products, a melt curve cycle was incorporated into the real-time PCR program. A melt-curve is the relative decrease in fluorescence plotted against the temperature as shown in Fig. 2. The amplified products with each of the specific primers are of the same length and, hence, show melt peaks at about the same temperature. The melting temperature is usually determined by the product’s length, GC content, concentration of the Table 2—The threshold value (Ct) of the transgenic contamination standard samples using the target specific primers a. Real time-PCR with 35S primers Samples Replicates Run 1 Ct Run 2 Run 3 5% 1 2 1 2 1 2 1 2 1 2 23.66 23.83 25.33 25.85 26.6 26.56 21.36 21.77 0 0 23.53 23.76 25.49 25.67 26.78 26.44 21.45 21.85 0 0 23.79 23.34 25.22 25.72 26.86 26.35 21.67 21.55 0 0 28.28 28.43 29.41 29.5 30.41 30.62 21.82 21.37 0 0 28.36 28.67 29.87 29.63 30.39 30.55 21.65 21.58 0 0 28.59 28.34 29.35 29.86 30.75 30.68 21.77 21.34 0 0 c. Real time-PCR with cry1Ac primers 5% 1 27.35 2 27.49 1% 1 28.55 2 28.57 0.10% 1 29.76 2 29.87 100% 1 25.71 2 25.87 0% 1 0 2 0 27.81 27.43 28.65 28.67 29.64 29.65 25.47 25.68 0 0 27.66 27.56 28.32 28.75 29.43 29.83 25.59 25.38 0 0 1% 0.10% 100% 0% b. Real time-PCR with nptII primers 5% 1 2 1% 1 2 0.10% 1 2 100% 1 2 0% 1 2 Mean SD RSDr 23.65 0.18 0.79 25.54 0.24 0.95 26.59 0.19 0.73 21.6 0 0 0.18 0 0 0.87 28.44 0.15 0.54 29.6 0.22 0.75 30.56 0.14 0.47 21.58 0 0 0.19 0 0 0.92 27.55 0.16 0.6 28.59 0.14 0.52 29.7 0.16 0.54 25.62 0 0 0.18 0 0 0.68 0 0 0 The relative standard deviation (RSDr) was calculated from the Ct values obtained from three independent real-time PCR runs under repeatability conditions VIDHYA et al: DETECTION OF GM COTTON SEEDS dye, salt and the specific template in the reaction tubes. Hence, the nonspecific products normally melt at a much lower temperature than the specific products which are longer in size1,17. Conclusion In the present study, GM cotton has been detected for the presence of transgene using PCR and real-time PCR. The protocol developed can be used to detect the transgenes in most of the GM crops since they contain either an antibiotic gene like nptII with Nos terminator or 35S promoter in most of the gene constructs. The real-time PCR could detect the presence of the transgene to an extent of 0.1% as also reported by Hubner et al18. Hence, it is very useful in detecting the transgene contaminations and also to follow the biosafety regulations. Such screening methods will also help to control and identify the illegal/spurious GM seeds in the market and avoid losses to the farmers cultivating them. Acknowledgement The authors are grateful to the Government of Karnataka for its financial support to carry out the experiments and Dow AgroSciences India Pvt. Ltd. for providing the GM cotton seeds for the experiments. References: 1 2 3 4 Ahmed F E, Detection of genetically modified organisms in foods, Trends Biotechnol, 20 (2002) 215-223. 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