1 / 15

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.

riva
Download Presentation

Another Quenching Method -Static Quenching-

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related