1. Weight Encoding Methods in DNA Based Perceptron 2004. 7. 6 임희웅

2. Contents Weight Encoding Criteria Methods –Internal fluorophore –Fluorescence resonance energy transfer –Competitive hybridization of labeled and unlabeled probe Materials

3. Weight Encoding Criteria Output –Fluorescence Signal –Proportional to Concentration of corresponding mRNA (x i ) Corresponding of weight value (t i ) Assumption: Final signal (output) is the sum of each output signal Output i

4. Internal Fluorophore High Absolute Weight Value Low Sign (+) (-) Fluorophore 1 Fluorophore 2 More fluorophoresLess fluorophores

5. Fluorescence Resonance Energy Transfer –Transfer of the excited state energy from the initially excited donor (D) to an acceptor (A) –Distance-dependent interaction between donor and acceptor without emission of a photon

7. High Absolute Weight Value Low Sign (+) (-) Fluorophore 1 Fluorophore 2 Longer probeShorter probe Small weight  short probe length  weak signal Large weight  long probe length  strong signal Quencher

8. FRET Efficiency Förster Distance (R 0 ) –A distance where the efficiency is 50%.

9. In case of R 0 =50

10. DNA Helix Model for FRET Efficiency Clegg et al. PNAS 1993

11. Dietrich et al. Reviews in Molecular Biotechnology 1993

12. Our Dye-Quencher Pairs How Many? –2 pairs for (+) and (-) Condition –Dark quencher No background signal, high S/N ratio –Discrimination between (+) and (-) Large difference in wavelength peak –Multiplexing Small interference between two dyes. How about using the pairs in RT-PCR? –Sensitivity Efficient sensitivity in the concentration of experimental condition. –Enough Förster Distance (R 0 ) Active range of FRET corresponding to probe length 3.3Å for one bp,

13. 6FAMRox (Texas-red) Hex cy5 And BHQ series as dark quencher

14. More to Know Fluorescence intensity vs. concentration Multiplexing

15. Competitive hybridization of labeled and unlabeled probe mRNA Labeled Probe Unlabeled Probe Competitive hybridization Excess Probe Small weight  low ratio of labeled probe  weak signal Large weight  high ratio of labeled probe  strong signal

16. probe mRNA Positive weight Negative weight Sign Encoding with One Fluorophore High Conc. Low Conc.

17. Reference Clegg et al. PNAS vol. 90, p 2994~2998, 1993 –Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer Dietrich et al. Reviews in Molecular Biotechnology, vol. 82, p 221~231, 2002 –Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

18. Materials

19. Dual Labeled Probe (1) http://www.bioneer.co.kr/biomall/mall_oligo.jsp Bioneer

20. Dual Labeled Probe (2) 5’/3’ 1. FAM/BHQ-1 Fluorogenic Probe 2. HEX/BHQ-1 Fluorogenic Probe 3. TET/BHQ-1 Fluorogenic Probe 4. MAX/BHQ-1 Fluorogenic Probe 5. Cy5/BHQ-3 Fluorogenic Probe 6. Cy5/BHQ-2 Fluorogenic Probe 7. Cy3/BHQ-2 Fluorogenic Probe 8. TAMRA/BHQ-2 Fluorogenic Probe 9. ROX/BHQ-2 Fluorogenic Probe http://www.synthegen.com/ Synthegen

21. Dual Labeled Probe (3) http://www.idtdna.com/program/catalog /Dual_Labeled_Fluorescent_Probes.asp IDT

25. Förster Distance (R 0 ) HiLyte Biosciences, Inc.

26. http://www.nlv.ch/Molbiology/sites/Fluorescence4.htm

27. Dark Quenchers

29. This chart shows the absorption spectra of all three BHQ dyes (conjugated to poly-T 9-mers and normalized to the T9 absorbance at 260 nm) with the emission maximum of many commonly used reporter groups indicated. http://www.biosearchtech.com/

30. Multiplexing Instrument