A DNA Computing Readout Operation Structure-Specific Cleavage

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Presentation transcript:

A DNA Computing Readout Operation Structure-Specific Cleavage 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 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

Surface-based DNA SAT Calculation Introduction How?

Structure-specific Cleavage = Design of Two Oligonucleotide 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

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

Invasive Cleavage Readout Strategy Result- (II) Invasive Cleavage Readout Strategy

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

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

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

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

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

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

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

Word Complements/Upstream Oligonucleotides

Comparison with PCR-based READOUT Result & Discussion Comparison with PCR-based READOUT 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 Si gnal amplification process rather than target amplification

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