Initial Pool Generation Based on DNA Shuffling Method

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

26 City TSP Graph (1) 4 1 2 1 1 1 5 24 9 18 19 5 11 1 1 2 25 4 2 8 3 232 22 4 2 3 2 2 1 15 5 4 13 2 16 2 7 2 2 3 7 12 10 17 21 14 3 1 1 2 4 2 2 11 8 4 20 1 7 1 3 6 0

26 City TSP Graph (2) 4 15 12 10 17 3 1 2 8 4 7 21 1 2 20 1 6 3 2 14 4 1 1 3 0 7 11

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

Initial Pool Generation Method p Based on DNA shuffling n p 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) p Material n n p PCR mixture contents n n p vertex 19, road 19/18 (lower cost) vertex 3, road 3/0 (higher cost) total volume : 20 μl oligomers concentration : 0. 2/0. 5/1. 0 μM d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 44. 9/51. 2/55. 1℃, 30 sec extension : 72℃, 30 sec cycle number : 30 times

One City, One Road Case (2) 100 bp 50 bp 1 L 1 H lane 1 : M (50 bp) annealing T 2 : 0. 2 μM 3 : 0. 4 μM 44. 9℃ 4 : 1. 0 μM 5 : 0. 2 μM 6 : 0. 4 μM 51. 2℃ 7 : 1. 0 μM 8 : 0. 2 μM 9 : 0. 4 μM 55. 1℃ 10 : 1. 0 μM ※ 20% PAGE sample vol. : 9 μl marker vol. : 2 μl

Two Cities, Two Roads Case (1) p Material n n p PCR mixture contents n n p vertex 19, vertex 18, road 19/18, road 18/23 (Σcost=2) vertex 3, vertex 0, road 3/0, road 0/1 (Σcost=18) total volume : 20 μl oligomers concentration : 0. 5/1. 0/2. 0 μM d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 44. 9/51. 2/55. 1℃, 30 sec extension : 72℃, 30 sec cycle number : 30 times

Two Cities, Two Roads Case (2) 300 bp 200 bp 100 bp 2 L 2 H 1 2 3 4 5 6 7 8 9 10 lane 1 : M (50 bp) annealing T 2 : 0. 5 μM 3 : 1. 0 μM 44. 9℃ 4 : 2. 0 μM 5 : 0. 5 μM 6 : 1. 0 μM 51. 2℃ 7 : 2. 0 μM 8 : 0. 5 μM 9 : 1. 0 μM 55. 1℃ 10 : 2. 0 μM ※ 20%(2 L), 15%(2 H) PAGE sample vol. : 9 μl marker vol. : 2 μl

Two Cities, Two Roads Case -with Cost (1) p Material vertex 19, vertex 18, road 19/18, road 18/23 + cost 1 (Σcost=2) n vertex 3, vertex 0, road 3/0, road 0/1 + cost 11, cost 7 (Σcost=18) n p PCR mixture contents n n p total volume : 20 μl oligomers concentration : 1. 0 (including the cost oligos) /1. 5 (1. 0 when excluding the cost oligos ) μM d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 51. 2/55. 1℃, 30 sec extension : 72℃, 30 sec cycle number : 30 times

Two Cities, Two Roads Case -with Cost (2) 300 bp 200 bp 100 bp 2 LW 2 L 1 2 3 4 5 6 7 lane 1 : M (50 bp) annealing T 2 : 0. 5 μM 51. 2℃ w/o cost 3 : 0. 5 μM 4 : 1. 0 μM 5 : 1. 5 μM w/ cost 6 : 1. 0 μM 55. 1℃ 7 : 1. 5 μM ※ 15%(2 LW), 20%(2 L) PAGE sample vol. : 9 μl marker vol. : 2 μl

Two Cities, Two Roads Case -with Cost (3) lane 1 : M (50 bp) 2 : 1. 0 μM 3 : 1. 5 μM 2 L 4 : 3. 0 μM 5 : 1. 0 μM 6 : 1. 0 μM w/o cost 7 : 1. 5 μM 2 H 8 : 3. 0 μM 9 : 1. 0 μM 10 : M (50 bp) 300 bp 200 bp 1 2 3 4 5 6 7 8 9 10 ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

Two Cities, Two Roads Case -additional case (1) p Material vertex 19, vertex 18, road 19/18, road 18/9 n vertex 19, vertex 18, road 19/18, road 18/9 + cost 1 (Σcost=3) n p PCR mixture contents n n p 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 d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 51. 2/55. 1℃, 30 sec extension : 72℃, 30 sec cycle number : 30 times 19 ->18 ->23 의 경우, 같은 cost 1을 두 개 포함. 이 경우는 1과 2 포함 p

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 w/ cost 6 : 1. 0 μM 7 : 1. 5 μM 300 bp 200 bp 1 2 3 4 5 2 M & 2 MW 6 7 ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

3 Cities, 3 Roads Case (1) p Material n n n p PCR mixture contents n n p 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) total volume : 20 μl oligomers concentration : 1. 0/2. 0 μM d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 55. 1℃, 30 sec extension : 72℃, 30 sec cycle number : 30 times

3 Cities, 3 Roads Case (2) 300 bp 200 bp 100 bp lane 1 : M (50 bp) 2 : 1. 0 μM 3 L 3 : 2. 0 μM 4 : 1. 0 μM 3 M 5 : 2. 0 μM 6 : 1. 0 μM 3 H 7 : 2. 0 μM ※ 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 1 U (211, 000 /125 U) 3 : Pfu (sample) 4 : Ta. Ka. Ra taq 1 U (153, 000 /250 U) 5 : Ta. Ka. Ra premix (Ex taq) 0. 5 U (48, 000 /32 rxn(=16 U)) 6 : Sapphire premix 1 U (10만원 정도/96 rxn(=96 U)

Graph Problem with 6 Cities 2003. 10. 21 Eun Jeong Lee

Graph ( a part of the 26 city TSP graph) 0 7 4 8 7 20 1 6 2 3 1 1 3 11

Materials and Method p Material n n p PCR mixture contents n n p set #1 : optimal path를 구성하는 city와 road만 넣음 set #2 : 모든 city와 road를 넣음 total volume : 20 μl oligomers concentration : 0. 5/1. 0/2. 0 μM d. NTP : 400 μM enzyme : I unit ( Pyrobest®DNA Polymeraze, Ta. Ka. Ra ) Reaction profile n n denaturation : 95℃, 20 sec annealing : 55. 0℃, 30 sec extension : 72℃, 30 sec cycle number : 35 times

PCR without primers (with city & road strands) 300 bp 200 bp 100 bp 240 bp lane 1 : M (50 bp) 2 : 0. 5 μM 3 : 1. 0 μM set #1 4 : 2. 0 μM 5 : 0. 5 μM set #2 6 : 1. 0 μM 7 : 2. 0 μM ※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl

PCR with primers - city 0, road 20/4 p Template n p set#1 & set#2를 agarose gel에 걸어서 240 bp근처를 elution하여 준비 Primers n 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) 7 4 8 0 1 1 1 6 3 3 20 * lane 1 : M (50 bp) 2 : primers- city 1, road 6/20 3 : primers- city 3, road 20/4 148 bp ->length of city 1~city 20

PCR with primers - city 0, complementary city 4 ( for set #2 ) 1. 2. 300 bp 200 bp 3. PCR with city strands and road strands Elute the gel slice near the 240 bp PCR again with primers – city 0, complementary city 4 100 bp 4. Results above my head. . . ( lane : 302/352/402/M)

列外) Orthogonality Test lane 1 : 1 L+1 H 2 : M (50 bp) 3 : 1. 0 μM set #1 4 : 2. 0 μM 5 : 1. 0 μM set #2 6 : 2. 0 μM • 1 L : city 19, road 19/18 • 1 H : city 3, road 3/0