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Cascading Whiplash PCR with a Nicking Enzyme

Cascading Whiplash PCR with a Nicking Enzyme. Daisuke Matsuda and Masayuki Yamamura. MEC Seminar 2002. 9. 27 Park, Ji-Yoon. Introduction. Previous history - Proposed by Hagiya et al. - Wood et al - Yamamura et al - State copy on aqueous computing with PNA

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Cascading Whiplash PCR with a Nicking Enzyme

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  1. Cascading Whiplash PCR with a Nicking Enzyme Daisuke Matsuda and Masayuki Yamamura MEC Seminar 2002. 9. 27Park, Ji-Yoon

  2. Introduction • Previous history - Proposed by Hagiya et al. - Wood et al - Yamamura et al - State copy on aqueous computing with PNA • Disadvantage - Back annealing - Rose et al; scheme to inhibit back annealing with bis-PNA • In this paper… - Scheme to cascade results of WPCR from molecules to molecules

  3. Scheme to Cascade WPCR • Nicking enzyme - cleave only one DNA strand & introduce a nick into the DNA - N.BstNBI(nonpalindromic sequence 5’-GAGTCNNNN^N-3’)

  4. Preliminary Experiments Table 1. DNA sequences used in this paper Nick site Fig 2. The structure of Trans3

  5. The Products of the Transitions Fig 3. The products of the transitionState 1: 1st transition State 2: 2nd transition State 3: 3rd transition

  6. State 3 of WPCR State 2 of WPCR No transition Fig 4. Gel electrophoresis of the transition products Denaturing PAGE

  7. The Model of Output Reaction

  8. The Output Reaction from WPCR Fig 6. The results of output reaction from WPCR Lane M2: Marker output product Lane 1: separated output product Lane 2: thermal output product Lane 3: Isothermal output product

  9. Fig 7. Ideas to realize [IF A AND B THEN C] and [IF ~A AND B THEN C]

  10. Discussion & Conclusion • Discussion about preliminary experiments * Equilibrium between divorced output fragments & back-annealed output fragments→ The concentration of the output products tends to converge * Isothermal output reaction(64°C) * Polymerase, ds DNA target • Further Issues- Plan to implement an expert system for medical diagnosis - Scheme to cascade WPCR - Reliability study to produce output fragments by a nicking enzyme

  11. Output reaction from WPCR • Three successive transitions(64°C for 1min/ 80°C for 1min / 80°C for 5min); 8 cycles * Component:Trans3(7 pmol), dNTP(60 μmol each), Bst DNA polymerase(4 Units), Bst DNA pol buffer/ N.BstNBI buffer * WPCR : 64°C for 90min with N.BstNBI & template s4-s5 * Separated output reaction: control reaction - two partical reaction(WPCR & output reaction) are performed separately* N.BstNBI & template s4-s5 poured into the reaction mixture before WPCR - isothermal & thermal reaction

  12. Whiplash PCR B x b a C x B A x

  13. Whiplash PCR B C x B A x

  14. Whiplash PCR a x x B A x C B

  15. Whiplash PCR a c b x x B A x C B

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