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Another Quenching Method -Static Quenching-

Another Quenching Method -Static Quenching-. 2004. 4. 20 Lim Hee Woong. Quenching. Dynamic Quenching In the excited state Förster quenching Falling off at a rate of 1/R 6 Dexter quenching Depends on the spatial overlap of orbitals Static Quenching In the ground state.

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Another Quenching Method -Static Quenching-

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  1. Another Quenching Method-Static Quenching- 2004. 4. 20 Lim Hee Woong

  2. Quenching • Dynamic Quenching • In the excited state • Förster quenching • Falling off at a rate of 1/R6 • Dexter quenching • Depends on the spatial overlap of orbitals • Static Quenching • In the ground state

  3. Static Quenching vs. Dynamic Quenching

  4. Different from FRET in that… • No excitation and intramolecular dimer • Donor and quencher moieties bind together to form ground state complex • Stemless molecular beacon • Depends on the overlap of absorption spectra • FRET depends on the overlap of absorption and emission spectra

  5. Hypothetical Heterodimer

  6. Experimental Result • Fluorophore and quencher • Cy3.5, FAM • BHQ1 (Black hole quencher 1)

  7. Dual labeld Fluorophore Only BHQ only(unscaled)

  8. Dual labeled Fluorophore Only BHQ only (unscaled) • Absorption spectra of the probes • More overlap of absorption spectra in Cy3.5 • Not equal with the sum of each spectrum

  9. Dual labeled Fluorophore Only • Fluorescence spectra of the probes • Fluorescence spectra changed though BHQ is dark quencher. • More quenching in Cy3.5 in despite of less overlap of absorption and emission spectra • More overlap of absorption spectra

  10. Relation between fluorescence and concentration • Linear relationship • Intra rather intermolecular quenching

  11. Some other results… • See reference

  12. Discussion • Advantage • No need to consider stem loop • No constraint in emission-absorption spectral overlap • Make it easy to design probe • But! • Thermal stability of fluorophore-quencher ground state complex • Most likely to be significant only in room temperature.

  13. Application to Current Project • Probe with intramolecular dimer • In the weighted encoding method with FRET • No need to eliminate unbound probe • Consideration • Annealing temperature? • Appropriate pairs of fluorophore and quencher? • Enough Förster distance?

  14. Reference • M. K. Johansson et al.Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probesJ. Am. Chem. Soc. 2002, 124, 6950 • M. K. Johansson et al.Intramolecular dimers: a new design strategy for fluorescence-quenched probesChem. Eur. J. 2003, 9, 3466 • http://www.biosearchtech.com/ • Black hole quencher

  15. Another Consideration in Fluorimetry • Fluorescence signal • Relation between signal intensity and concentration • Fluorescence signal in case of mixed fluorescence • FRET and distance • Distance dependence of FRET especially in DNA structure

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