other detection system of theorem proving 2004. 2. 10 park, ji-yoon

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

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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

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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)