Hide/Show Apps

A sandwich-type DNA array platform for detection of GM targets in multiplex assay

2012-09-01
Cansiz, Sena
Özen, Can
Bayrac, Ceren
Bayrac, A. Tahir
Gül, Fatma
Kavruk, Murat
Yilmaz, Remziye
EYİDOĞAN, FÜSUN
Öktem, Hüseyin Avni
Over the last decade, array detection has been developed to qualitatively assess the presence of genetically modified organisms (GMOs). To date, DNA array systems have the highest capabilities as a result of GMOs analysis. We describe the construction of an array platform in the sandwich hybridization format for the detection of transgenic promoter of Cauliflower mosaic virus (CaMV; p35S). Sequence-specific signal development has been achieved by a sandwich complex composed of a surface immobilized capture probe and a fluorescein-tagged signal probe, which are partially complementary to the p35S oligonucleotide. We used poly-l-lysine-coated glass slides as support material, on which capture probes were immobilized by a heterobifunctional cross-linker. The comparative results of optimization studies including cross-linker types probe concentrations and hybridization conditions (sequence, temperature and duration) were reported. An optimum hybridization signal was obtained with a 32.5 C-0 cross-linker, 10 mu M capture and 20 mu M signal probe concentrations, respectively. A relatively short hybridization time (2.5 h) provided reproducible array signals. No significant effect of hybridization sequence on the fluorescence intensity was observed. The described platform can specifically detect label-free transgenic sequences with a target of 0.01 mu M concentration, while the optimized system exhibits great potential for the application of different GMO target sequences (p35S, tNOS, bar and cry) to multiplex array formats.