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Henry Jakubowski, Biochemistry


The focus of our research is the denaturation of double-stranded DNA (dsDNA) using fluorescence spectroscopy. The dsDNA, which contains 12 base pairs, has one 5’strand labeled with fluorescein (FAM) – 5’-/56-FAM/TCC ACC TTC CCT-3’ – and a complementary 3’strand labeled with Black Hole Quencher I (BHQ I) – 5’-AGG GAA GGT GGA/BHQ1-3’. The dsDNA, if annealed correctly, should emit little fluorescence given the proximity of the quencher to the fluorophore. As the DNA denatures on the addition of urea, a fluorescence emission is recorded. We are attempting to determine the thermodynamic stability of the 12mer and possible derivatives. We used fluorescence spectroscopy to analyze the conditions necessary to denature a 12-mer of DNA as a function of [urea]. These data were then used to calculate the Keq and the ΔG0 for the denaturation of the 12mer. This research could be applied to DNA of differing base pairs, lengths, backbone strength and composition and solvent composition.