1. 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, (2001)
Park, Ji-Yoon

2. Abstract Previous work
» the solution of a SAT problem using surface-based DNA
computing : Nature 403, (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. 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

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

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

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

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

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

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

11. 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. 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

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