Wet (DNA) computing 2001년 9월 20일 이지연 2001-09-20

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 2001-09-20

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 2001-09-20

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 2001-09-20

Wet Computing : Our Plan TSP larger problem size Theorem Proving larger and complex problem Version Space learning problem Other Candidates Commercial Device 2001-09-20

Experimental Results Target problem 4 3 1 6 2 5 nodes : 7 0  1  2  3  4  5  6 (18) 260bp edges : 24 0  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 2001-09-20

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 2 3 4 5 6 7 1 3 3 3 5 2 reduce template amount 2001-09-20

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 2001-09-20

3 4 3 7 1 start & end 3 9 3 11 11 3 3 6 5 3 3 2 5 2001-09-20

TSP 16 Target problem 4 3 1 6 2 5 nodes : 6 edges : 18 weights : 4 1 2 3 4 lane 1: marker lane 2: 0.02X template lane 3: 0.004X lane 4: 0.0008X Target problem nodes : 6 edges : 18 weights : 4 3 4 3 7 1 2 3 4 5 6 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: 0.0001X lane 6: negative ctrl lane 7: ligation 2 5 2001-09-20

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 2001-09-20

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 ℃ 2001-09-20

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’ 2001-09-20

Repetitive Ligation For longer strand formation Before After 2001-09-20

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 ℃ 2001-09-20

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 2001-09-20

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 2001-09-20

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 2001-09-20

2001-09-20