Development Of New DNA-based Methods For Analysis Of Genetically Modified Plants And Their Derived Products

Although the controversies surrounding genetically modified organisms (GMOs) in the past fifteen years, genetically modified (GM) crops has been used worldwide. According to the statistics from ISAAA, the global area of GM crops has consistently increased, reaching 148 million hectares by the end of 2010, and which was approximately 87-fold more than that in 1996. So many countries and regions have implemented the issues and regulations for the labelling and traceability of GMO because of the consumers'concern regarding the safety of GMOs. The implementation of these regulations necessitates the availability of endogenous reference gene and certified reference materials and the development of GMO analytical methodologies. Strategies for automatic, rapid, and high-throughput methods are the major trend for GMO analysis. Accordingly, the endogeneous reference gene and event-specific detection method for new GM crops, and novel high-throughput NDA-based amplification methods were investigated in this study.1. To develop the detection of GM papaya Huanong No.1 approved in China, we firstly reported one papaya species specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Secondly, we revealed the molecular characterization of the exogenous DNA integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Furthermore, we developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5'integration flanking sequence. The relative LOD of the qualitative PCR assay was about 0.01% in total of 100 ng papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the LOD and LOQ were as low as 12.5 and 25 copies of papaya haploid genome, respectively. Collectively, all of these results showed that the developed method is useful for the identification and quantification of Huanong No. 1 papaya and its derived products.2. To develop new methods for screening GMOs, we developed a quadruplex degenerate PCR screening assay on basis of the similar DNA sequences in different GM crops. This assay consists of four PCR systems targeting on 9 DNA sequences from 8 trait genes (Cry1Ab, Cry1Ac, Cry1A(b/c), mCry3A, Cry3Bb1, cp4-EPSPS, bar, and/or pat) widely introduced into GMOs. The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute LOD of approximate 80 target copies. The results showed that this established quadruplex PCR assay is a time- and cost-efficient as well as sensitive approach for GMO detection and monitoring the presence of GMO in food.3. Furthermore, we developed a novel combined approach for high-throughput analysis of multiple DNA targets based on the multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (MPIC), a two-step PCR amplification strategy. In the first step, the multiple target DNAs are pre-amplified using bipartite primers attached with universal tail sequences on their 5'-ends. Then, the pre-amplified templates are compartmentalized individually in the microdroplet of PCR system, and multiple targets can be amplified in parallel employing primers targeting their universal sequences. Subsequently, the resulting multiple products are analyzed by capillary gel electrophoresis (CGE). Here, we optimized the system and procedure, evaluated the specificity and sensitivity of MPIC approach. Using GMO analysis as a model, twenty-four DNA targets can be simultaneously detected with a absolute limit of detection of 39 target DNA copies. The described system provides a promising alternative for high-throughput analysis of multiple DNA targets.