Fluorescent Hairpin Probes for the Detection of Chemically-Induced DNA Damage
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Abstract
In this thesis, we develop a sensitive, robust, accurate method to detect chemically-induced DNA damage by three therapeutic agents, cisplatin, psoralen and busulfan. The method uses fluorescence spectroscopy and fluorescent hairpin or molecular beacon probes in a hybridization assay for chemically-induced ssDNA damage probed in solution and on a microarray platform.
Damage is confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and UV absorbance spectroscopy. The decrease in fluorescence upon damage scales with the number of mutation sites, drug doses and irradiation time. All these results indicate that the hybridization probes can quantitatively and selectively detect the number of DNA lesions/strand for chemically-induced DNA damage.
Finally, fluorescent hairpin probes have some limitations, particularly with regards to their modifications and costs. However, the development of easy, fast and reliable assays is essential for biological as well as clinical applications, but requires these limitations to be addressed.