The Application Of Immobilized Peptide Nucleic Acids Probe In Transgenic Detection

It has been legislated world widely on managing genetically modified crops, foods and raw materials. It was absolutely essential to establish reliable and sensitive technologies and methods for detecting GMOs and their derivatives.The peptide nucleic acid (PNA) is a kind of DNA analog, which replaced the DNA pentose phosphate skeleton with N-(2-aminoethyl) glycine structural units. It has been widely used in molecular biology and related fields since it was found. It is considered as a very prospective probe tool, and has many advantages compared with DNA probes. For example, PNA is uncharged, so PNA/DNA complex has higher thermal stability. PNA probes are more selective and affinitive, binding to DNAs independent of salt concentration and so on. Superparamagnetism microsphere is a new type of magnetic material. It is superparamagnetic, and can be manipulated by external magnetic field. It can be modified with a variety of active groups on the surface and can be bound covalently with PNA or DNA. It has been widely used in biotechnology, biochemistry, medicine and other fields. In this research, two immobilized ways of PNA are carried out to detect transgenic rice. Research contents and results are as follows:1. The PCR tube wall immobilized PNA probes for transgenic detection research:bt transgenic insect resistant rice TT51-1is chose as the research object. Gene specific and event specific PNA probes were synthesized and modified with a digoxin at N-terminal. Then PNA probes were immobilized on polycarbonate PCR tube wall, which was coated by anti-digoxin antibodies. Rice genome DNA was denatured, then hybridizated with PNA. After washed off, target DNAs captured by PNA probes are detected by PCR as a template. Optimization of the reaction conditions, including different material of PCR tubes, PNA concentration, temperature of hybridization and elution program et al. By this method, we detected bt gene and event-specific gene in TT51-1material successfully.2. Magnetic beads immobilize PNA probes for transgenic detection research:Amino modified PNA probes covalent binding with carboxyl modified magnetic beads, which form magnetic bead probes to capture target DNAs. And then target DNAs captured by PNA probes are detected by PCR. This study provides a preliminary explore to the basic reaction conditions, and obtain the best dosage of magnetic bead probes. By this method, we also successfully detect TT51-1bt gene specifically and event specifically.3. The ability of PNA probes capturing target DNAs:This article research the ability of capture target of two PNA immobilization methods, the results show that they can capture more than15kb of target fragments. Internal control SPS gene which located on different chromosomes cannot be detected, the results indicate that PNA probe has strong capture ability and specificity. In addition, we study the ability of PNA probe capture target DNA with trace concentrations. The results showed that PNA probe has a good ability of enrichment the trace concentrations target DNA.4. Gene specific PNA probe for detect other genetically modified materials:different bt transgenic materials and be detect by bt gene specific PNA probe. Except for TT51-1, other transgenic events such as KF6、KMD and maize Bt11can also be detected by two immobilized PNA methods.5. PNA probe for rapid detection:DNAs were extracted crudely from seeds of six kinds of bt transgenic materials including TT51-1、KF6、KMD、Bt11、Mon810and Bt176. Then crudely extracted DNA can by captured by two immobilized PNA methods and then be detected by PCR. This process did not require complicated DNA extraction process. The impurities of crude extraction DNA, which include inhibitor and uncombined DNA, can be removed by elution process of PNA capturing. It was meaning for field rapid detection.