Date

2010

Author

Cansız, Sena

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DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried; heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5 Ǻ cross-linker, 10 µM capture and 20 µM signal probe concentrations at 35°C. Finally, in order to evaluate the specificity of the array system a cocktail of target DNA were applied to the system on which different capture probes of those target DNAs were immobilized. The results indicated that the optimized array system is specific enough to detect only the target oligonucleotide yet the unspecific ones.

Subject Keywords

Technique.

URI

http://etd.lib.metu.edu.tr/upload/12612658/index.pdf
https://hdl.handle.net/11511/19942

Collections

Graduate School of Natural and Applied Sciences, Thesis