Research On Short Segments Of DNA Detection Methods In Genetically Modified Organisms

The genetically modified deep-processing products and processing waste which have the variety of species and comple geometry are always the emphasis and difficulty of transgenic safty management. The DNA of these transgenic products are usually highly degraded after a deep processing, So these DNA fragments are relatively short. Therefore, it is difficult to detect. In China, the research on the extraction and detection technology of the short DNA fragments in GM deep-processing products and processing waste is not too many, which leads to the lack of adequate technical support of the safty management of the transgenic industry. In this research, we attempt to establish the extraction and detection method of short DNA fragments which is highly degraded of the deep-processing products and processing waste.CaMV35S promoter is one of the most commonly used transgenic element. It widespreads in different kinds of commercialized transgenic crops and transgenic vector system used for the study. But after the different types of modificaion, sequences of CaMV35S promoter in different transgentic varieties may have larger difference. After the molecular characteristics of the CaMV35S promoter in different kinds of transgenic varieties is analyzed, we attempt to establish a general CaMV35S promoter detection method to provide a reliable method for the screening detection of transgenic products in this research.The research results in this paper are set up:1. The DNA extraction method research of deep-processing products:Nine different methods used to extract DNA of22kinds of deep-processing products whose raw materials come from different species, including the method of Fermentas DNA extraction kit, Takara DNA extraction kit, Promega DNA extraction kit, ect. And then compare concentration and purity of DNA extracted by different methods and quantitative detect the reference gene, the common exogenous original in these species. Through experiments and analysis, the SDS DNA extraction method which has been improved is most suitable for deep-processing products. Some transgenic ingredient has been detected in some deep-processing products whose raw materials come from different species, including rapeseed, corn, soybean, sunflower, peanut. Some other crop ingredient has been detected in some deep-processing products whose raw materials come from different species, including rice, corn, peanut, sesame. This indicates that the DNA extraction method which we screened out in this research is suitable for the follow transgenic detection and ingredient detection for the deep-processing products whose raw materials come from different species.2. The research of short DNA fragments detection method of genetically modified products:For some highly degraded short DNA fragments whose length are only tens of base pair, two kinds of detection methods of short DNA fragments are designed, including the quantitative PCR of no probe and double primer labeled and interact with fluorescence resonance energy transfer and the Loop-mediated ligase quantitative PCR. In this research, the sensitivity and specificity of six kinds of common reference and exogenous gene have been deeply analyzed to use the quantitative PCR of no probe and double primer labeled and interact with fluorescence resonance energy transfer. The experimental results show that this method has high sensitivity and specificity, and can be widely used to detect the short DNA fragments. Some common reference gene, exogenous gene and screening original have been preliminary analyzed to use the Loop-mediated ligase quantitative PCR. The amplified results show that it can be applied to detect the short DNA fragments. The length of detection target is successfully reduced to35-45bp level. These methods can be used to detect particular short DNA fragments in different sources and samples with different content mixture, and provide a useful reference to detect the short DNA fragments which is highly degraded of the deep-processing products and processing waste.3. The research of CaMV35S promoter detection method:In view of the some present problems existing in the detection system of CaMV35S promoter, such as incomplete matching of the primer/probe of detect methods with the detection target, the existence of multiple amplification products, can not be solved. Comparing the collected sequence of CaMV35S promoter which come from different sources to find a length of about300bp conserved region in enhancer sequences, as the conserved region sequences to design the primer and probe, and to establish a general detection method of CaMV35S promoter. The detection specificity of this method is verified in30different GM crops, the Limit of detection of this method is20copies, the Limit of quantification of this method is50copies, resolving the existent problems in CaMV35S promoter detection system.