Preparation Of DNA Microarray For The Detection Of Primary Product

With the research and development of transgenic technology, more and more genetic modified crops (GMCs) were produced and their tillage area increased year on year. However, the GMCs encouraged widespread disputes across the whole world on their uncertain safety. Many countries established corresponding policies and codes to enhance the management of GMCs and products made from them, also carried on the studies on genetic modified organism (GMO) detection technology. Currently, the detection technology of GMCs and their products routed from PCR and the targets were the exogenous genes. Though PCR is easy and sensitive, it can't carry on high throughput detection of GMCs products. The development of DNA chips provides a new approach for the high throughput detection of GMCs products.In this study a new detection method for GMCs and products made from them was tried on the DNA chips technology.1. An improved CTAB method which was used as DNA extraction protocol was tried and high quality DNA was gained from the soybean seeds and dregs. PCR and MPCR detection system were established and the detection efficiency of transgenic soybean were greatly increased by MPCR detection system.2. A home made poly-L-lysine slide was used as the carrier of DNA chip and its practicality was studied with the commercial slide as control. The results showed that the quality of home made poly-L-lysine slide was similar to the commercial slide and it could meet the requirement for DNA chips preparation. The slide cost will be cut off as much as 80 percent if the homemade slides are used instead of the commercial slides, thus the experiment cost was reduced too.3. A serial of experiments were conducted to study the most moderate conditions for the preparation of DNA chip. Optimal choice of spot solutions was made and themost suitable volume for printing array was determined when a 96-well plate is used. The bleaching effect of laser on Cydye was studied carefully and qualitatively. Relationship between signal to noise and PMT value were also studied.4. Many common transgenic elements used in Commercial GMCs were collected and their conservative fragments were confirmed. Then we tired to collect the clone vectors or expression vectors that contain these transgenic elements via all kinds ways. Thus the conservative fragments were amplified and purified as probes. These probes were printed on the poly-L-lysine slide by micoarryer. Then we got the transgenic detection DNA chips.5. Using the transgenic detection DNA chips, we screened the exogenous genes from the transgenic soybean successfully. The results showed that DNA chips could be used for high throughput detection and its efficiency was much higher than that of MPCR. Furthermore, based on the molecular hybridization, the reliability of GMOsdetection by DNA chip was highly increased.