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

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

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

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

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

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

8. The Model of Output Reaction

9. The Output Reaction from WPCR Fig 6. The results of 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

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

11. Discussion & Conclusion  Discussion about preliminary experiments Isothermal output reaction(64°C) * Polymerase, ds DNA target  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  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

12. Three successive transitions ( °C for 1min/ 80°C for 1min / 80°C for 5min); 8 cycles * Component: * WPCR : °C for 90min with control reaction - two partical reaction(WPCR & output reaction) are performed separately * N.BstNBI & template s4-s5 poured into the reaction mixture before 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 Output reaction from WPCR

13. x BAx C B x ab Whiplash PCR

14. x BAx C B

15. x BAxCB x a

16. x BAxCB x a bc