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doi:10.1016/j.bios.2005.12.001 
Copyright © 2005 Elsevier B.V. All rights reserved.
Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray
Jia Xua, 1, Haizhen Miaoc, 1, Houfei Wua, Wensheng Huangb, Rong Tanga, Minyan Qiuc, Jianguo Wena, Shuifang Zhub, 
and Yao Lia, , 
Received 14 June 2005;
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Abstract
In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.
Keywords: Oligonucleotide microarray; GMO screening; Multiplex-PCR
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Samples and reference materials
- 2.2. DNA extraction
- 2.3. Primers design
- 2.4. Oligonucleotide probes design
- 2.5. PCR and labeling
- 2.6. Construction of DNA microarrays
- 2.7. Classification of GMO chip
- 2.8. Hybridization and signal detection
- 2.9. Direct sequencing analysis of PCR products
- 3. Results
- 3.1. Design of object probes
- 3.2. Design of control samples
- 3.3. Threshold determination of the positives and negatives
- 3.4. Optimization of the experimental condition and validation of the method
- 4. Discussion
- 5. Conclusion
- Acknowledgements
- References
