solution of a 20-variable 3-sat problem on a dna computer
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Solution of a 20-Variable 3-SAT Problem on a DNA Computer. Ravinderjit S. Braich, Nickolas Chelyapov, Cliff Johnson, Paul W. K. Rothemund, and Leonard Adleman Science vol. 296 19 April 2002 Summarized by Jiyoun Lee. Introduction. The Boolean formula - PowerPoint PPT PresentationTRANSCRIPT
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Solution of a 20-Variable 3-SAT Problem on a DNA Computer
Ravinderjit S. Braich, Nickolas Chelyapov, Cliff Johnson,Paul W. K. Rothemund, and Leonard Adleman
Science vol. 296 19 April 2002Summarized by Jiyoun Lee
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Introduction The Boolean formula
20 variable with 24-clause 3-conjunctive normal form (3-CNF) formula, was designed to have a unique satisfying truth assignment
Sticker model Mix and split for half-library generation Polymerase extension method for full-length library generation Graduate PCR to read the answer
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Sticker model Sticker model
Library + Sticker Operations
Combine Separation Setting Cleaning
Separation based on subsequence use only Application of stickers
Random access memory that requires no strand extension, uses no enzyme, and (at least in theory) its materials are reusable
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Separation operation Separation using AcryditeTM phosphoamidite for modifying DNA
molecules at 5’-end during chemical synthesis Covalently linking the probes to the gel matrix Gives one benefits of solid-support-based system while still remaining ch
aracteristics of a solution-based system
Separation Oligonucleotide probes immobilized in polyacrylamide gel-filled glass mod
ules Capture with immobilized probes and release at a higher temperature
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Attachment of oligonucleotides to solid Various methods are available to attach oligonucleotides to solid
surfaces such as microarray slides, microtiter plates or magnetic beads, including: Biotin-oligo non-covalently complexed with Streptavidin. SH-oligo covalently linked via a disulfide bond to a SH-surface. Amine-oligo covalently linked to an activated carboxylate or an aldehyde g
roup. Phenylboronic acid (PBA)-oligo complexed with salicylhydroxamic acid (S
HA)
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AcryditeTM
Enables covalent attachment of oligonucleotides and other macromolecules to surfaces via acrylic linkages.
An oligonucleotide derivatized with Acrydite group can polymerize with acrylamide monomer to form polyacrylamide or can react with thiol or silane surfaces. This chemistry is also compatible for attachment to polymer surfaces.
2D 3D immobilization
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An acrylic acid group can be directly attached to t
he 5'-end of an oligonucleotide (with a 6-carbon lin
ker arm) at the time of synthesis using Acrydite, an
acrylic-
phosphoramidite developed by Mosaic Technologie
s
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The library I Xk
T, XkF 15 base value sequences, 2N library strands
Constraints Library sequences contain only A, T, C less secondary structure All library and probe sequences have no occurrence of 5 or more consecutive iden
tical nucleotides Every probe sequence has at least 4 mismatches with all 15 base alignment of any
library sequence Every 15 base subsequence of a library sequence has at least 4 mismatches with
all 15 base alignment of itself or any library sequence No probe sequence has a run of more than 7 matches with any 8 base alignment o
f any library sequence No library sequence has a run of more than 7 matches with any 8 base alignment
of itself or any other library sequence Every probe sequence has 4, 5, or 6 Gs in its sequence Discourage intra- and interlibrary strand hybridization and unintended probe-library
strand hybridization
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The library II Xk
Z, 5’-end Acrydite-modified oligonucleotides Used as probes
Synthesis of long molecules Synthesis of two ‘half-libraries’ x0 through x10 (left half-library), x11 through
x20 (right half-library) Half-libraries: a mix-and split combinatorial synthesis technique was used The 300-oligomer (300-mer) full library was created from the two half-libra
ries using a polymerase extension method
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Mix and split Combinatorial DNA library
construction (half-library)
During synthesis
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Polymerase extension method Assembly PCR method for the synthesis of long DNA sequences
from large numbers of oligonucleotides
Does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process
Derived from DNA shuffling
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Left half (1~10) 150 mer Right half (11~20) 150 mer
X11 X10
5’ 3’
5’3’
5’ 3’
10 pmole2 pmole each
Final volume 20 l in 1X T4 DNA ligase buffer, incubate at RT for 2 hrs
Mixture 0.5 lPrimer: X1
T, X1F, Acrydite-modified ~X20
T, ~X20F
1 ml aliquot, PCR again
Band extraction, creat stock solution
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The computer and the computational protocol Step 1: Insert the library module into the hot chamber of the electrophoresis
box and the first clause module into the cold chamber of the box. Begin electrophoresis.
Step 2: Remove both modules from the box. Discard the module from the hot chamber. Wash the box and add new buffer. Insert the module from the cold chamber into the hot chamber and the module for the next clause into the cold chamber. Begin electrophoresis.
Step 3: Repeat Step 2 for each of the remaining 22 clauses.
Step 4: Extract the answer strands from the final clause module, PCR-amplify, and “read” the answer.
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r 0.5cm thick plexiglass
Probe layer(releasing)
Probe layer(capturing)
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A clause module
4.5 cm
3.2 cm
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Detection of the answer Graduate PCR
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Capture-release efficiency