Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection.
In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively.
The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12612659/index.pdf |
| Date | 01 October 2010 |
| Creators | Gul, Fatma |
| Contributors | Oktem, Huseyin Avni |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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