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Initial Pool Generation Based on DNA Shuffling Method

Published byἈρτεμᾶς Μακρή Modified over 6 years ago

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Presentation on theme: "Initial Pool Generation Based on DNA Shuffling Method"— Presentation transcript:

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

2 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

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

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

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 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

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 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

13 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

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 1을 두 개 포함. 이 경우는 1과 2 포함

15 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

16 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

17 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

18 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)

19 Graph Problem with 6 Cities
Eun Jeong Lee

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

21 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

22 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

23 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

24 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

25 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)

26 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

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