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

26 City TSP Graph (1) 4 12 16 25 18 19 9 24 2 10 17 21 7 8 23 13 14 1 20 6 3 15 5 11 22

26 City TSP Graph (2) 4 12 10 17 21 14 1 20 6 3 15 2 7 8 11

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

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

Description of Elongation Steps

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

One City, One Road Case (2) 100 bp 50 bp 1L 1H 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℃ annealing T ※ 20% PAGE sample vol. : 9 μl marker vol. : 2 μl

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

Two Cities, Two Roads Case (2) 100 bp Two Cities, Two Roads Case (2) 100 bp 2L 2H 300 bp 200 bp 1 2 3 4 5 6 7 8 9 10 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 ※ 20%(2L), 15%(2H) PAGE sample vol. : 9 μl marker vol. : 2 μl

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

Two Cities, Two Roads Case -with Cost (2) 100 bp Two Cities, Two Roads Case -with Cost (2) 100 bp 2LW 300 bp 200 bp 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 1 2 3 4 5 6 7 ※ 15%(2LW), 20%(2L) PAGE sample vol. : 9 μl marker vol. : 2 μl 2L

Two Cities, Two Roads Case -with Cost (3) 100 bp Two Cities, Two Roads Case -with Cost (3) 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) 1 2 3 4 5 6 7 8 9 10 100 bp 300 bp 200 bp 2L 2H w/o cost ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

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 1을 두 개 포함. 이 경우는 1과 2 포함

Two Cities, Two Roads Case -additional case (3) 100 bp Two Cities, Two Roads Case -additional case (3) 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 300 bp 200 bp 100 bp 1 2 3 4 5 6 7 ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl 2M & 2MW

3 Cities, 3 Roads Case (1) Material PCR mixture contents 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

3 Cities, 3 Roads Case (2) lane 1 : M (50 bp) 2 : 1.0 μM 3 : 2.0 μM 3 H ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

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 (10만원 정도/96rxn(=96U)

Graph Problem with 6 Cities 2003.10.21 Eun Jeong Lee

Graph ( a part of the 26 city TSP graph) 4 1 20 6 3 7 8 2 11

Materials and Method Material PCR mixture contents Reaction profile set #1 : optimal path를 구성하는 city와 road만 넣음 set #2 : 모든 city와 road를 넣음 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 : 55.0℃, 30sec extension : 72℃, 30 sec cycle number : 35 times

PCR without primers (with city & road strands) 100 bp PCR without primers (with city & road strands) 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 #2 300 bp 240 bp 200 bp 100 bp ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

PCR with primers - city 0, road 20/4 Template set#1 & set#2를 agarose gel에 걸어서 240bp근처를 elution하여 준비 Primers city 0 and road 20/4 * lane 1 : M (50 bp) 2 : template from set#1(1) 3 : template from set#1(2) 4 : template from set#2

Tracking the Path (set #1) 4 1 20 6 3 8 7 * lane 1 : M (50 bp) 2 : primers- city1, road 6/20 3 : primers- city3, road 20/4 148 bp ->length of city 1~city 20

PCR with primers - city 0, complementary city 4 ( for set #2 ) 100 bp 300 bp 200 bp PCR with city strands and road strands Elute the gel slice near the 240 bp PCR again with primers –city 0, complementary city 4 Results above my head... ( lane : 302/352/402/M)

列外) Orthogonality Test lane 1 : 1L+1H 2 : M (50 bp) 3 : 1.0 μM 4 : 2.0 μM 5 : 1.0 μM 6 : 2.0 μM set #1 set #2 1L : city19, road 19/18 1H : city3, road3/0