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Hierarchical DNA Memory bas Hierarchical DNA Memory bas ed on Nested PCR ed on Nested PCR Satoshi Kashiwamura, Masahito Yama Satoshi Kashiwamura, Masahito Yama moto, Atsushi Kameda, moto, Atsushi Kameda, Toshikazu Shiba, Azuma Ohuchi Toshikazu Shiba, Azuma Ohuchi

Hierarchical DNA Memory based on Nested PCR

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Hierarchical DNA Memory based on Nested PCR. Satoshi Kashiwamura, Masahito Yamamoto, Atsushi Kameda, Toshikazu Shiba, Azuma Ohuchi. Introduction. Nested Primer Molecular Memory Data structure of NPMM Addressing of the data Merits of NPMM NPMM design strategy of primer design Experiments - PowerPoint PPT Presentation

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Page 1: Hierarchical DNA Memory based on Nested PCR

Hierarchical DNA Memory based oHierarchical DNA Memory based on Nested PCRn Nested PCR

Satoshi Kashiwamura, Masahito YamamotSatoshi Kashiwamura, Masahito Yamamoto, Atsushi Kameda,o, Atsushi Kameda,

Toshikazu Shiba, Azuma OhuchiToshikazu Shiba, Azuma Ohuchi

Page 2: Hierarchical DNA Memory based on Nested PCR

IntroductionIntroduction

Nested Primer Molecular MemoryNested Primer Molecular Memory Data structure of NPMMData structure of NPMM Addressing of the dataAddressing of the data Merits of NPMMMerits of NPMM NPMM designNPMM design strategy of primer designstrategy of primer design ExperimentsExperiments dissussion dissussion

Page 3: Hierarchical DNA Memory based on Nested PCR

Nested PCRNested PCR

Page 4: Hierarchical DNA Memory based on Nested PCR

Data structure of NPMMData structure of NPMM

3 Address block : Ai / Bj / Ck I,j,k { 0,1,2 }∈ Re : reverse primer (common primer)

Example data 101 = A1 , B0 , C1

Page 5: Hierarchical DNA Memory based on Nested PCR

Addressing of the dataAddressing of the data

1. Select a p ∈ P and then perform PCR to NPMM with p and Re

2. Select another p’∈ P, then perform PCR to the diluted solution after previous PCR.

3. Repeat process 2 for an appropriate number of times

Page 6: Hierarchical DNA Memory based on Nested PCR

Merits of NPMMMerits of NPMMThe level of data securityThe level of data security : :

it is impossible to read data without primers it is impossible to read data without primers each primer works as a keyseach primer works as a keyseach key is independent of other keyseach key is independent of other keys

A large capacity with a high reaction sA large capacity with a high reaction specificity : pecificity :

M (bit) = 2 * Data(bp) * Primer block

M : the memory capacity of NPMMData : the length of the sequence in the data block Block : the number of address blocksPrimer : the number of primers in each address block

Page 7: Hierarchical DNA Memory based on Nested PCR

NPMM designNPMM design Size of NPMM :Size of NPMM : data sequence : 20bp data sequence : 20bp primers : all 15bp primers : all 15bp

template : 80bp ( 15 + 15 + 15 + 20 + 15 )template : 80bp ( 15 + 15 + 15 + 20 + 15 )

Several regards Several regards GC content GC content hamming distance hamming distance

3’ end complementary3’ end complementary

Fitness = GC weight * GC value + Fitness = GC weight * GC value + H weight * H value +H weight * H value + E weight * E valueE weight * E value

Page 8: Hierarchical DNA Memory based on Nested PCR
Page 9: Hierarchical DNA Memory based on Nested PCR

ExperimentsExperiments

expected result

data 000 = [ Bo , Co ]

data 001 = [ Bo, C1 ]

data 010 = [ B1 , Co ]

data 011 = [ B1 , C1 ]

Page 10: Hierarchical DNA Memory based on Nested PCR

Amplify the target sequenceAmplify the target sequence

using Bo or B1 / Re

perform 23 cycle

denature 94C for 10sec

anneal 50C for 30 sec

extension 72C for 5 sec

using Co or C1 / Re

same condition

Page 11: Hierarchical DNA Memory based on Nested PCR

Result 1Result 1

Run 10% poly acrylamide gelRun 10% poly acrylamide gel

Page 12: Hierarchical DNA Memory based on Nested PCR

Detection of amplified sequenceDetection of amplified sequence

50bp is too short to sequencing ---- need another PCR for detection

Bo Co Data ooo Re

5’ end

15 mer

Page 13: Hierarchical DNA Memory based on Nested PCR

Result 2Result 2

using data 000 / data 001 data 010 / data 011 primers

perform 25 cycle using same condition

10% Poly acrylamide gel

Page 14: Hierarchical DNA Memory based on Nested PCR

Amplify using concatenation Amplify using concatenation primerprimer

whether we can extract the target data by once PCR. whether we can extract the target data by once PCR.

Anneal 50 C or 58 C or 74 C for 30 secAnneal 50 C or 58 C or 74 C for 30 sec

PCR to NPMM using B0 C0PCR to NPMM using B0 C0

Detection of the sequence amplified with B0 C0Detection of the sequence amplified with B0 C0

Bo Co Data ooo Re

1st 2nd 1st / 2nd

Page 15: Hierarchical DNA Memory based on Nested PCR

Result 3Result 3

using data 000 / data 001 data 010 / data 011 primers

10% Poly acrylamide gel

Page 16: Hierarchical DNA Memory based on Nested PCR

DiscussionDiscussionProposed a DNA memory with

A high capacuity / high data security / high specificity ..

Future work

Scaling up of NPMM

Design strategy for set of primers without dependence on the data sequence