1. Wet (DNA) computing
2001년 9월 20일
이지연

2. Contents 1. Wet computing
Until Now.. & Now..ing & Our Plan
Experimental Results
Discussion
: Trouble shooting/Constraints on the reactions
: New DNA sequence design strategy
2. Artificial base & Wet computing into lab-on-a-chip
Progress

3. Wet Computing : Until Now..
Adleman’s experiment : HPP (Hamiltonian Path Problem)
: Problem size ~ 7 nodes
Lipton : SAT problem
: Array-based
???: Maximal clique problem
: Problem size ~ variable, clause
Make all possible solutions
and… select a correct answer
Algorithm and Tools

4. Wet Computing : Now..ing GMD (Germany) : MCP, SAT Japan/Our team : TSP
: Preparing the experiments
: Realization of LOC
Japan/Our team : TSP
: Realization of weight by DNA sequence (length/GC content)
Toy problem
Japan/Our team : Theorem proving
: Toy problem, Preparing the experiments

5. Wet Computing : Our Plan
TSP
larger problem size
Theorem Proving
larger and complex problem
Version Space
learning problem
Other Candidates
Commercial Device

6. Experimental Results Target problem 4 3 1 6 2 5
nodes :  1  2  3  4  5  6 (18) 260bp
edges :  2  1  3  4  5  6 (24) 250bp
weights : 5 3 4 3 7 1
start
&
end 3 9 3 11 11 3 3 6 5 3 3 2 5

7. reduce template amount
TSP 03 I
Target problem
nodes : 4
edges : 10
weights : 3
Possible paths
0  1  2  3 : 140bp
0  2  3 : 100bp
0  1  3 : 90bp
Effect of weight sequcne
CAGTGGGTCCTCCGTTCCGC (14/20)
AATTGGATCCTCCATTCCTT (8/20) 3 7 1 3 3 3 5 2
reduce template amount

8. TSP 03 II Mixture Result lane 1 : marker 0~6 all
phosphorylation
higher annealing temperature
(higher primer Tm)
Result
Specific amplification
Template copy number
lane 1 : marker
lane 2 : negative ctrl
lane 3,4 : PCR with primer 03

9. 3 4 3 7 1
start
&
end 3 9 3 11 11 3 3 6 5 3 3 2 5

10. TSP 16 Target problem 4 3 1 6 2 5 nodes : 6 edges : 18 weights : 4
lane 1: marker
lane 2: 0.02X template
lane 3: 0.004X
lane 4: X
Target problem
nodes : 6
edges : 18
weights : 4 3 4 3 7 1 9 3 11 11 3 6 3 3
lane 1: marker lane 2: 0.1X template
lane 3: 0.01X lane 4: 0.001X
lane 5: X lane 6: negative ctrl
lane 7: ligation 2 5

11. TSP 06 I Possible problems lane 1 : 06(A) lane 2 : 06(A) elution
lane 3 : negative PCR ctrl
lane 4 : marker
lane 5~7 : previous sample
Possible problems
phosphorylation
ligation : longer strand
non-specific annealing

12. TSP 06 II Phosphorylation Primer design Conditions
: length, uniform melting temperature
primer 1 TACCCCGAAA CAACGCAGAA GC 22mer Tm=61.9 ℃
primer 2 TATGTCCAGC TGTCGCAAAG CAG 23mer Tm=61.1 ℃

13. 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ edge_01 edge_12 vertex_0 weight_3 vertex_1
CAACGCAGAA GCGGAACGGAGGAGCCACTG CTCACCTCTC
CACAGTGCAG GCGGAACGGAGGAGCCACTG CGATGGCCTT
ATGGGGCTTT GTTGCGTCTT
CGCCTTGCCTCCTCGGTGAC
GAGTGGAGAG GTGTCACGTC
CGCCTTGCCTCCTCGGTGAC
GCTACCGGAA CACTTAGGTG 3’
vertex_0
weight_3
vertex_1
weight_3
vertex_2 5’
TACCCCGAAA CAACGCAGAA 5’
TACCCCGAAA CAACGCAGAA GC 5’
GAC GAAACGCTGT CGACCTGTAT 5’
GAAACGCTGT CGACCTGTAT 5’ 5’
edge_45
edge_56 3’
ACGACCGTTT GCGGAACGGAGGAGCCACTG TGTCTACAGG
TGGACTGTGC GCGGAACGGAGGAGCCACTG CTTTGCGACA
AAGTAGTGAA TGCTGGCAAA
CGCCTTGCCTCCTCGGTGAC
ACAGATGTCC ACCTGACACG
CGCCTTGCCTCCTCGGTGAC
GAAACGCTGT CGACCTGTAT 3’
vertex_4
weight_3
vertex_5
weight_3
vertex_6 5’

14. Repetitive Ligation For longer strand formation Before After

15. Discussion Unexpected amplification of primers
Amplification of small size of DNA
Reduction of template concentration
Effect of weight sequcne (TSP 03)
Weight 3 : CAGTGGCTCCTCCGTTCCGC (14/20)
Weight 5 : AATTGGATCCTCCATTCCTT (8/20)
Melting temperature of primers
Unexpected Tm differences between node sequences
Node 0 : TTCTGCGTTG TTTCGGGGTA (10/20) Tm = 57.6 ℃
Node 1 : CTGCACTGTG GAGAGGTGAG (12/20) Tm = 52.6 ℃
Node 2 : GTGGATTCAC AAGGCCATCG (11/20) Tm = 57.3 ℃
Node 3 : ATACGGCGTG GTTTTTCGGG (12/20) Tm = 60.4 ℃
Node 4 : AAACGGTCGT AAGTGATGAA ( 8/20) Tm = 49.4 ℃
Node 5 : GCACAGTCCA CCTGTAGACA (11/20) Tm = 50.2 ℃
Node 6 : TATGTCCAGC TGTCGCAAAG (10/20) Tm = 53.6 ℃

16. Discussion - Trouble Shooting
Oligomer design
: Tm calculation by combining nearest-neighbor model and GC %
Higher melting temperature of PCR primers
Hybridization
: Variable temperature gradient
Ligation
: Formation of longer sequence
Selection of desirable range of DNA by electrophoresis
PCR
: Annealing/denaturation temperature, Cycle variation
Specific amplification of interesting strands
PAGE
: Gel percentage (resolution), Gradient temperature/%T

17. Constraints on the reactions
Oligomer design
: higher Tm of oligomers which used as primers
Hybridization
: initial concentration (amount) of oligomers
variation with weights
Ligation
: blunt/sticky end, slice of specific region
PCR
: annealing/denaturation temperature, cycle variation
specific amplification of interesting strands
PAGE
: gel percentage (resolution), gradient temperature/%T
Others

18. New DNA sequence design strategy
Sequencial increase of denaturation temperature in PCR
 more amplification of relative low Tm strands
Tm decision factor
GC%
stacking energy
Vertex sequence
regular melting temperature
both NN and GC%
Weight sequence in Edge sequence
reflection of weight by GC%
in longer strands, there’s no effect by NN method