1. Initial Pool Generation Based on DNA Shuffling Method Eun-jeong Lee

2. 26 City TSP Graph (1)

3. 4 12 10 1721 14 1 20 6 3 15 1 3 21 2 1 7 8 2 3 2 4 7 11 3 4 1 0 26 City TSP Graph (2)

4. 4 1 20 6 3 1 7 8 2 7 11 3 1 0 26 City TSP Graph (3)

5. Initial Pool Generation Method  Based on DNA shuffling PCR without primers, with oligomers coding cities and roads  Begining with one set city and road, then expanding

6. Description of Elongation Steps

7. One City, One Road Case (1)  Material vertex19, road19/18 (lower cost) vertex3, road3/0 (higher cost)  PCR mixture contents total volume : 20 μl oligomers concentration : 0.2/0.5/1.0 μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 44.9/51.2/55.1 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 30 times

8. One City, One Road Case (2) lane 1 : M (50 bp) 2 : 0.2 μM 3 : 0.4 μM 4 : 1.0 μM 5 : 0.2 μM 6 : 0.4 μM 7 : 1.0 μM 8 : 0.2 μM 9 : 0.4 μM 10 : 1.0 μM 44.9 ℃ 51.2 ℃ 55.1 ℃ 100 bp 50 bp 1L 1H 100 bp ※ 20% PAGE sample vol. : 9 μl marker vol. : 2 μl annealing T

9. Two Cities, Two Roads Case (1)  Material vertex 19, vertex 18, road 19/18, road 18/23 (Σcost=2) vertex 3, vertex 0, road 3/0, road 0/1 (Σcost=18)  PCR mixture contents total volume : 20 μl oligomers concentration : 0.5/1.0/2.0 μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 44.9/51.2/55.1 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 30 times

10. Two Cities, Two Roads Case (2) 100 bp ※ 20%(2L), 15%(2H) PAGE sample vol. : 9 μl marker vol. : 2 μl lane 1 : M (50 bp) 2 : 0.5 μM 3 : 1.0 μM 4 : 2.0 μM 5 : 0.5 μM 6 : 1.0 μM 7 : 2.0 μM 8 : 0.5 μM 9 : 1.0 μM 10 : 2.0 μM 44.9 ℃ 51.2 ℃ 55.1 ℃ annealing T 100 bp 2L 2H 300 bp 200 bp 123456789 10 123456789

11. Two Cities, Two Roads Case -with Cost (1)  Material vertex 19, vertex 18, road 19/18, road 18/23 + cost 1 (Σcost=2) vertex 3, vertex 0, road 3/0, road 0/1 + cost 11, cost 7 (Σcost=18)  PCR mixture contents total volume : 20 μl oligomers concentration : 1.0 (including the cost oligos) /1.5 (1.0 when excluding the cost oligos ) μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 51.2/55.1 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 30 times

12. Two Cities, Two Roads Case -with Cost (2) 100 bp 2L 100 bp 2LW 300 bp 200 bp ※ 15%(2LW), 20%(2L) PAGE sample vol. : 9 μl marker vol. : 2 μl w/ cost lane 1 : M (50 bp) 2 : 0.5 μM 3 : 0.5 μM 4 : 1.0 μM 5 : 1.5 μM 6 : 1.0 μM 7 : 1.5 μM 51.2 ℃ 55.1 ℃ w/o cost annealing T 1234567

13. Two Cities, Two Roads Case -with Cost (3) 100 bp 1234 5 67 8 9 10 100 bp 300 bp 200 bp ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl lane 1 : M (50 bp) 2 : 1.0 μM 3 : 1.5 μM 4 : 3.0 μM 5 : 1.0 μM 6 : 1.0 μM 7 : 1.5 μM 8 : 3.0 μM 9 : 1.0 μM 10 : M (50 bp) 2L2H w/o cost

14. Two Cities, Two Roads Case -additional case (1)  Material vertex 19, vertex 18, road 19/18, road 18/9 + cost 1 (Σcost=3)  PCR mixture contents total volume : 20 μl oligomers concentration : 0.5/1.0/2.0 (excluding the cost oligos) /1.5 (1.0 when excluding the cost oligos ) μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 51.2/55.1 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 30 times  19->18->23 의 경우, 같은 cost 1 을 두 개 포함. 이 경우는 1 과 2 포함

15. Two Cities, Two Roads Case -additional case (3) 100 bp ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl 2M & 2MW 100 bp 300 bp 200 bp lane 1 : M (50 bp) 2 : 0.5 μM 3 : 1.0 μM 4 : 1.5 μM 5 : 2.0 μM 6 : 1.0 μM 7 : 1.5 μM w/ cost 1234567

16. 3 Cities, 3 Roads Case (1)  Material city 19, city 18, city 23, road 19/18, road 18/23, road 23/5 (Σcost=1+1+1=3) city 4, city 12, city 10, road 4/12, road 12/10, road 10/17 (Σcost=1+2+3=6) city 3, city 0, city 1, road 3/0, road 0/1, road 1/14(Σcost=11+7+4=22)  PCR mixture contents total volume : 20 μl oligomers concentration : 1.0/2.0 μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 55.1 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 30 times

17. 3 Cities, 3 Roads Case (2) 100 bp ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl lane 1 : M (50 bp) 2 : 1.0 μM 3 : 2.0 μM 4 : 1.0 μM 5 : 2.0 μM 6 : 1.0 μM 7 : 2.0 μM 3 L 3 M 3 H 100 bp 300 bp 200 bp

18. 6 Cities, 6 Roads Case (1) 4 1 20 6 3 1 7 8 2 7 11 3 1 0

19. 6 Cities, 6 Roads Case (2)  Material set #1 : optimal path 를 구성하는 city 와 road 만 넣음 set #2 : 모든 city 와 road 를 넣음  PCR mixture contents total volume : 20 μl oligomers concentration : 1.0/2.0 μM dNTP : 400 μM enzyme : I unit ( Pyrobest ® DNA Polymeraze,TaKaRa )  Reaction profile denaturation : 95 ℃, 20 sec annealing : 55.0 ℃, 30sec extension : 72 ℃, 30 sec cycle number : 35 times

20. 6 Cities, 6 Roads Case (3) 100 bp ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl lane 1 : M (50 bp) 2 : 0.5 μM 3 : 1.0 μM 4 : 2.0 μM 5 : 0.5 μM 6 : 1.0 μM 7 : 2.0 μM set #1 set #2 100 bp 300 bp 200 bp

21. Comparison of the amplification efficiency of various enzymes lane 1 : M (50 bp) 2 : Pyrobest 1U (\211,000 /125U) 3 : Pfu (sample) 4 : TaKaRa taq 1U (\153,000 /250U) 5 : TaKaRa premix (Ex taq) 0.5U (\48,000 /32rxn(=16U)) 6 : Sapphire premix 1U (10 만원 정도 /96rxn(=96U)