initial pool generation based on dna shuffling method eun-jeong lee
TRANSCRIPT
Initial Pool Generation Based on DNA Shuffling
Method
Eun-jeong Lee
4
12
16
25
18 19924
2
10 17 21
7
8 23
13
14
120
6 3
15
5 11
22
1
2
24
2
32
52
4
1
2
2
2
2
178 2
3
5
21
1
11
31
1
21
1
22
23 4
7
11
3
2
1 1
4
4
1
0
26 City TSP Graph (1)
4
12 10 17 21 14
120
6 3
15
1 32 1 2
178 2
3 2
4
7
11
3
4
1
0
26 City TSP Graph (2)
4 120
6 317
8 2
7
11
3 1
0
26 City TSP Graph (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)
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 μM10 : 1.0 μM
44.9℃
51.2℃
55.1℃
50 bp
1L
1H
100 bp
※ 20% PAGE sample vol. : 9 μl marker vol. : 2 μl
annealing T
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)
※ 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 μM10 : 2.0 μM
44.9℃
51.2℃
55.1℃
annealing T
100 bp
2L
2H
300 bp
200 bp
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
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)
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
1 2 3 4 5 6 7
Two Cities, Two Roads Case -with Cost (3)
1 2 3 4 5 6 7 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)
2L
2Hw/o cost
Two Cities, Two Roads Case -additional case (1) Material
vertex 19, vertex 18, road 19/18, road 18/9 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 t
he 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
Two Cities, Two Roads Case -additional case (3)
※ 12% PAGE sample vol. : 9 μl marker vol. : 2 μl2M &
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
1 2 3 4 5 6 7
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
3 Cities, 3 Roads Case (2)
※ 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 H100 bp
300 bp
200 bp
6 Cities, 6 Roads Case (1)
4 120
6 317
8 2
7
11
3 1
0
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
6 Cities, 6 Roads Case (3)
※ 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
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)