Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system.
A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.
Department of Chemistry, University of Patras, Patras, Greece 26500.
Pierre J
PJ
Obeid
Theodore K
TK
Christopoulos
Penelope C
PC
Ioannou
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D018744:DNA, Plant / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005898:Glass / Q000737:chemistry; D007834:Lasers; D037102:Lectins / Q000235:genetics; D008904:Miniaturization; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D013025:Glycine max / Q000235:genetics; D013728:Terminator Regions, Genetic / Q000235:genetics
D018744:DNA, Plant; D037102:Lectins; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase