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A DNA Computing Readout Operation based on Structure-Specific Cleavage

A DNA Computing Readout Operation based on Structure-Specific Cleavage. Liman Wang, Jeff G. Hall, Manchum Lu, Qinghua Liu, Lloyd M.Smith. Nat. Biotechnol. 19, 1053-1059(2001). 2001. 12. 14 Park, Ji-Yoon. Abstract. Abstract. Previous work

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A DNA Computing Readout Operation based on Structure-Specific Cleavage

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  1. A DNA Computing Readout Operation based on Structure-Specific Cleavage Liman Wang, Jeff G. Hall, Manchum Lu, Qinghua Liu, Lloyd M.Smith Nat. Biotechnol. 19, 1053-1059(2001) 2001. 12. 14 Park, Ji-Yoon

  2. Abstract Abstract Previous work » the solution of a SAT problem using surface-based DNA computing : Nature 403, 175 - 179 (2000) Problem “READOUT” step - identify the DNA molecules present at the end of the computational process - based on PCR » false-positive signal » non-uniform amplification from mixtures of DNA targets Alternative READOUT approach » Structure-specific cleavage-based readout » higher uniformity of detection of the DNA computing products compared to that obtained with PCR amplification » simple, accurate, compatible with multiple-word DNA computing

  3. Surface-based DNA SAT Calculation Introduction How?

  4. Introduction Structure-specific Cleavage = Design of Two Oligonucleotide • Two sequence-specific oligonucleotides • Upstream oligonucleotide • Probe oligonucleotide • »analyte-specific region: forms a duplex with the target • » non-complementary 5’-arm • Hybridized region of the probe sequence • » have a melting temperature near the temperature of the • reaction • » rapid turnover of the probe oligonucleotides • » amplifying the signal ~ 1000 probe oligonucleotides / target

  5. Result- (I) Invasive Cleavage Reaction ( B ) ( A )

  6. Result- (II) Invasive Cleavage Readout Strategy

  7. Result- (III) DNA oligonucleotides in the READOUT

  8. Result- (IV):A DNA oligonucleotides in the READOUT

  9. Result- (IV):B DNA oligonucleotides in the READOUT

  10. Result- (IV):C DNA oligonucleotides in the READOUT

  11. R Two Principal differences Substantially less signal variability »invasive cleavage- based: » PCR- based: Much less false-positive signals

  12. Readout for Multiple-Word DNA Computing To adapt this approach to multiple-word DNA computing » upstream oligonucleotides seq : 13 nts(total)

  13. Result- (V) Histogram of Average Background-to-Signal Ratio during the Surface-based DNA Computation

  14. Word Complements/Upstream Oligonucleotides

  15. Structure-specific cleavage- based Requires substantially less time & effort » Collection of the DNA strands from the surface »Invasive cleavage reaction » Fluorescence detection » Take < 2 hr PCR-based READOUT Additional time & effort »Collection of DNA strands from the surface » PCR reaction »Strand separation of the ds product » Hybridization to addressed arrays » Fluorescence detection » Take < 4-5 hr False-positive signals - carryover contamination Variability in signal amplitude - varying efficiencies of PCR amplification for different targets Result & Discussion Comparison with PCR-based READOUT • Si gnal amplification process rather • than target amplification

  16. Experimental Protocol Experiment Oligonucleotide synthesis DNA Surface Attachment Chemistry DNA Computation Surface Hybridization Collection of DNA strands from surface Invasive cleavage reactions

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