Other Detection System of Theorem Proving
2004. 2. 10
Park, Ji-Yoon
February 10, 2004 February 10, 2004 APRL http://gene.hanyang.ac.krAPRL http://gene.hanyang.ac.kr 22
3 × 3 Matrix Chip (1)
1 × 3 Matrix
3 × 3 Matrix
Poly-L-Lysine
45 mer15 mer
15 mer
45 mer15 mer
15 mer
45 mer15 mer
15 mer
15 m15 merer
15 m15 merer
45 m45 merer
February 10, 2004 February 10, 2004 APRL http://gene.hanyang.ac.krAPRL http://gene.hanyang.ac.kr 33
Experimental Steps
1. Poly-L-Lysine Coating 1. Poly-L-Lysine Coating
3. Washing & Dry 3. Washing & Dry
4. Attachment of Target DNA 4. Attachment of Target DNA
6. Washing & Dry 6. Washing & Dry
7. Confocal LIF Detection 7. Confocal LIF Detection
5. S1 nuclease Digestion 5. S1 nuclease Digestion
2. Attachment of Probe DNA 2. Attachment of Probe DNA
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Amine-Coated Slide glass (Corning)
FAM(ST~Q)
Cy3(~P)
Cy5(R)
~S
~T
~RPQ
Target DNA
Probe DNA
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Poly-L-Lysine Patterned Slide glass
FAM Cy5
Overlay
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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)
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More Consideration
Hybridization(6 strands)
Ligation Surface Filtration
Nil Detection
S, T, Q, ¬ P, R
¬ S, ¬ T, Q, P,
¬ R
5’ ST5’ ST ¬¬ QQ ¬¬ PP RR
QPQP ¬¬ R 5’R 5’¬¬ SS ¬¬ TT
February 10, 2004 February 10, 2004 APRL http://gene.hanyang.ac.krAPRL http://gene.hanyang.ac.kr 88
Nanoparticle-based Detection (2)Nature 382, 607-609 (1996)
15 m15 merer
15 m15 merer
45 m45 merer
February 10, 2004 February 10, 2004 APRL http://gene.hanyang.ac.krAPRL http://gene.hanyang.ac.kr 99
Experimental Step
1. Au modification with thiol-capped oligonucleotides - Note: Oligonucleotide: non-complementary
3-thiol-TACCGTTG 3’-thiol-TTTGCTGA AuAuAuAu
2. Assembly (700 nm: aggregation)
- Addition of linking DNA duplex
5’-ATGGCAACTATACGCGCTAG
3’-ATATGCGCGATCTCAGCAAA 33. Immediately Color change
- with naked eyes
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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)
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Fig 2. TEM images of DNA-linked nanoparticle assemblies at early stages in the growth process
Expected Results (2)