Other Detection System of Theorem Proving Park, Ji-Yoon
February 10, 2004 APRL × 3 Matrix Chip (1) 1 × 3 Matrix 3 × 3 Matrix Poly-L-Lysine 45 mer 15 mer 45 mer 15 mer 45 mer 15 mer 45 mer
February 10, 2004 APRL 3 Experimental Steps 1. Poly-L-Lysine Coating 3. Washing & Dry 4. Attachment of Target DNA 6. Washing & Dry 7. Confocal LIF Detection 5. S1 nuclease Digestion 2. Attachment of Probe DNA
February 10, 2004 APRL 4 Amine-Coated Slide glass (Corning) FAM (ST~Q) Cy3 (~P) Cy5 (R) ~S ~T ~RPQ Target DNA Probe DNA
February 10, 2004 APRL 5 Poly-L-Lysine Patterned Slide glass FAMCy5 Overlay
February 10, 2004 APRL 6 Problems & Discussion PDMS Chip Channel Cross-talking Large sample volume require (~ 10 ul/channel) We’ll use fresh PDMS Chip for every experiments Using Thin PDMS Chip Low Intensity of Cy3 Cy 3 → Alexa fluoro dye (Molecular Probe)
February 10, 2004 APRL 7 More Consideration Hybridization (6 strands) Ligation Surface Filtration Nil Detection S, T, Q, ￢ P, R ￢ S, ￢ T, Q, P, ￢ R 5’ ST ￢ Q ￢ P￢ P￢ P￢ PR QP ￢ R 5’ ￢S￢S￢S￢S ￢T￢T￢T￢T
February 10, 2004 APRL 8 Nanoparticle-based Detection (2) Nature 382, (1996) 15 mer 45 mer
February 10, 2004 APRL 9 Experimental Step 1. Au modification with thiol-capped oligonucleotides - Note: Oligonucleotide: non-complementary 3-thiol-TACCGTTG 3’-thiol-TTTGCTGA Au Au 2. Assembly (700 nm: aggregation) - Addition of linking DNA duplex 5’-ATGGCAACTATACGCGCTAG 3’-ATATGCGCGATCTCAGCAAA 3 3. Immediately Color change - with naked eyes
February 10, 2004 APRL 10 Fig 1. (A) The UV-vis spectrum of oligonucleotide- modified 13-nm particles before (red line) and after (blue line) DNA-induced assembly (B) Solutions of 13-nm-diameter Au particles before (cuvette A) and after (cuvette B) DNA-induced assembly (c) After extended periods of time a polymeric precipitate forms and settles to the bottom of the cuvette (cuvette C) Expected Results (1)
February 10, 2004 APRL 11 Fig 2. TEM images of DNA-linked nanoparticle assemblies at early stages in the growth process Expected Results (2)