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ANALYSIS OF TELOMERIC-DNA THROUGH FLUORESCENCE CORRELATION SPECTROSCOPY

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posted on 2023-08-04, 18:57 authored by Allison Alix

The link between telomerase activity and the development of cancer cells is evident. The modern method for telomerase detection, Telomeric Repeat Amplification Protocol, TRAP, is time consuming and often results in false positives. The purpose of this study is to use Fluorescence Correlation Spectroscopy, FCS, a Single Molecule Detection Method, to monitor the activity of telomerase through telomeric-DNA (TTAGGG)n quantification. Two different assays are tested and presented here: Method A, which utilizes Thioflavin T as a fluorophore, and Method B, which utilizes a molecular beacon, MB, a hairpin shaped, single strand of DNA complementary to telomeric-DNA, containing a fluorophore and quencher on either of its ends. Gold Nanoparticles (AuNPs) and Silica-coated Gold Nanoparticles (Au@SiO2s) are employed as localized reaction sites, to which the telomeric-DNA is tethered to through surface bonds. Fluorescence measurements exhibited an increase in intensity when both the ThT and MB were bound to the telomeric-DNA, compared to when they were not, confirming that both methods are suitable for detection. In the case of the MB assay, FCS measurements were performed to observe intensity fluctuations of the fluorescently tagged molecules. This allowed for the quantification of fluorescent AuNPs in solution at the pM level, with potential to lower the detection limit. The results found here are intended to be used as calibration for samples with unknown amounts of telomeric DNA.

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American University

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Degree awarded: M.S. Chemistry. American University

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http://hdl.handle.net/1961/thesesdissertations:460

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