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DNA computing using single-molecule hybridization detection. K.A.Schmidt, C.V.Henkel, G.Rozenberg, and H.P.Spaink, NAR , vol. 32, no. 17, Sep. 2004, pp. 4962-4968. Summarized by Byoung-Hee Kim 2004.11.20. Overview. Problem: 3-SAT Algorithm: blocking algorithm Experiment Synthesis - PowerPoint PPT Presentation
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DNA computing using DNA computing using single-molecule hybridization single-molecule hybridization
detectiondetection
K.A.Schmidt, C.V.Henkel, G.Rozenberg, and H.P.Spaink, NAR , vol. 32, no. 17, Sep. 2004, pp. 4962-4968.
Summarized by Byoung-Hee Kim2004.11.20
2
Overview
Problem: 3-SAT
Algorithm: blocking algorithm
Experiment Synthesis Hybridization Detection
Destroy Filtering, marking Blocking
(Direct inactivation of those molecules that are not a solution)
Single molecule detection byFCS, dual-color FCCS
3
Problem
4-variable 3-SAT problem 24 possible solutions
Falsifying one clause is sufficient for falsifying the complete formula
Blocker: falsifying case
).~(~)~()~(~)~( dcadbacbadcbF clauseformula
4
Sequence design
General structure (5’)start – a b c d – stop(3’) Start : CTTGCA Stop : TTGCAC Bit=0 : ATCACC Bit=1 : GTCTGA
Library:16(24) different oligonucleotides of 36bp each Blockers: designed to be complementary to the library oli
gos
).~(~)~()~(~)~( dcadbacbadcbF clauseformula
5
Overall encoding scheme
).~(~)~()~(~)~( dcadbacbadcbF clause A clause B clause C clause D
6
Blocking algorithm
(Rozenberg and Spaink, 2003) Basic idea : Direct inactivation of those molecules that are
not a solution
blocker
PCR
Detection
Build library
Add blockers
Library molecule
•Special prefix and suffix •Use PNA (peptide nucleic acid) blockers to prevent blocked items being amplified
•IF (PCR success) THEN (there are solutions)•Detection by visual inspection (E.g. DNA-specific dyes)
(Blocker construction can be automated)
7
Fluorescent labeling
5’ 3’
3’ 5’
Library molecule
blocker
Cy5
Rhodamine green
Blocking: detect hybridized molecule, or dsDNA
FRETHeteroduplex migration assayEnzymatic mismatch recognitionFCS
μM
nM
DNA 사용량
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FCS
FCS (fluorescence-correlation spectroscopy) Studies fluorescence fluctuations caused by a single
molecule diffusing in the focal detection volume
Detection volume, <0.5 fℓ
(figures are from http://www.probes.com/handbook/boxes/1571.html)
9
FCCS
FCCS (fluorescence cross-correlation spectroscopy) the number of doubly labeled particles(Ncc) can be calculated
fluorescent signal
Laser1488 nm
Laser2633 nm
Beam splitter
Avalanche photodiode
Correlator
Fitted curve
10
Hybridization detection at the level of single DNA molecules
L04 B0
Perfect match
L05 B0
4 bp mismatch
a
•All oligonucleotide concentrations : 10 nM
a: single blocker with two library molecules
b: addition of two types of blocker
c: addition of three types of blocker
※ when using four different blocker, the amplitudes were too low
b
c
L11 A0,A1
Perfect match
L03 A0,A1mismatch
L04 B0,B1,C1
Perfect match
L05 B0,B1,C1mismatch
11
Complete computation
FIND-SAT Find out satisfiable instances for 3-SAT problem
).~(~)~()~(~)~( dcadbacbadcbF clause A clause B clause C clause D
Blocker A Blocker B Blocker C Blocker D
•Hybridize with each library molecule (L00~L15)
•Total trial # : 24 × (#clauses) × (3~4 repeat)
12
Complete computation - Result
a b
c
13
Overview revisited
No PCR
Solved problem:3-SAT
Blocking algorithm
Paradigm
Single molecular detection by FCS
Experiment
DiscussionDiscussion
Bloking algorithm &Silgne molecule
detection
meaning Experimental meaning promise
Future works Vs gel electrophoresis Evolutionary DNA computing
•The first example for the utilization of single-molecule techniques for DNA computing
•Fluorescence cross-correlation spectroscopy was employed
•Considerable improvement compared to the FRET assay
•Less prone to experimental errors
•In contrast to all previous approaches, requires
•-neither large quantities of DNA for detection
•-nor destroy the output molecules
•Promise for extending the size of the libraries
•Combination with LOC (Lab-on-a-chip) => enhance the parallelism of the computation
•Combination with microfluidics systems => high-throughput hybridization detection
•Utilization of universal nucleotides => may decrease the number of blocker molecules
•The amount of DNA is much reduced => by four orders of magnitude => the search space may be increased 10,000 times
•Much faster
•Easily combined with high-throughput screening
•attractive for implementing evolutionary algorithms with DNA
•The selection step is difficult, very high background of ‘wrong’ molecules
•‘Single molecule technique’ and ‘non-destructive character’ has the potential to overcome these problem
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References – blocking algorithm
[1] Rozenberg,G. and Spaink,H. (2003) DNAcomputing by blocking. Theor.Comp. Sci., 292, 653–665.
[2] Schmidt,K.A., Henkel,C.V., Rozenberg,G. and Spaink,H.P. (2002) Experimental aspects of DNA computing by blocking: use of fluorescence techniques for detection. In Kraayenhof,R., Visser,A.J.W.G. and Gerritsen,H.C. (eds), Fluorescence Spectroscopy, Imaging and Probes—New Tools in Chemical, Physical and Life Science. Springer-Verlag, Heidelberg, Germany, pp. 123–128.
[3] Henkel,C.V., Rozenberg,G. and Spaink,H.P. (2004) Application of mismatch detection methods in DNA computing. In Ferreti,C., Mauri,G. and Zandron,C. (eds), DNA10, 10th International Meeting on DNA Computing, Preliminary Proceedings. Universita di Milano-Bicocca, pp. 183–192.
17
References - FCS
[1]Madge,D., Webb,W.W. and Elson,E. (1972) Thermodynamic fluctuations in a reacting system – measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett., 29, 705–708.
[2]Eigen,M. and Rigler,R. (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl Acad. Sci. USA, 91, 5740–5747.