Hierarchical DNA Memory based on Nested PCR

Satoshi Kashiwamura, Masahito Yamamoto, Atsushi Kameda, Toshikazu Shiba and Azuma Ohuchi,

8th International Workshop on DNA-based Computers, volume 2568 of LNCS. Springer-Verlag, pp. 112-123 (2003)

Summarized by HaYoung Jang

Outline

Nested PCR

Hierarchical DNA Memory (NPMM)

Design of Sequences

Experimental results

Concluding Remarks

Nested PCR

Structure of DNA Memory

Ａ Ｂ Ｃ Data Re

・・・

A0

An・・・・・・

B0

Bn

# of hierarchy : 3 # of sequences in each hierarchy: n

Nested Primer Molecular Memory (n×n×n NPMM)

address part data part

linker sequences

}2,1,0{,,|{

}2,1,0{,,|,,,{

kjitemplateT

kjiReCBAP

ijk

kji

Nested PCR (addressing)

A Ｂ Ｃ Data Re

B Ｃ Data Re

Data ReC

address is represented in the primer sequences and the order of use of them

Hierarchical DNA Memory

NPMM provides a high level of data securityNPMM has a large capacity with a high reaction specificity

Enlarge the address space by using a small number of primer sequencesReduce the total errors by removing (diluting) the error products in each step.

Ease of extracting the target data from MPNN

Hierarchical DNA Memory

M: memory capacity of NPMM.Data: length of the sequence in the data block.Block: number of address blocks.Primer: number of primers in each address block.One base is equal to 2 bits because one base consists of 4 elements (A,T,G,C).

# of hierarchy

# of sequences in each

hierarchy

address space # of required sequences

4 100 1.00×108 407

5 40 1.024×108 208

6 23 1.480358×108

147

7 14 1.054135×108

108

BlockprimerbpDatabitM )(2)(

Readout Process

A B C Data Re

A0A1A2

B0B1B2

C0C1C2

Data0~data26

・ all 27 NPMM units are synthesized・ resulting pools are analyzed after each PCR

A, B, C, Re: 15bpData: 20bp

A0Re

C2Re

B1Re

27mix 9mix 3mix target

Design of Sequences

GC_content

Hamming distance

3’end_complementary

PnumbermaxGCGCCGvalueGC pdefine /__))max((_ 2

nscombinatioALLnumberH_maxtpHtpHvalueH MM _/_)),(),,(max(_

nscombinatioALLerE_max_numbtpEtpEvalueE nM

nM _/)),(),,(max(_

Design of Sequences

Designed sequences of templates and primers

Sequence of data primers

Laboratory Experiments

Extracting target data sequence using PCR

M: 100 bp ladder.

M': DNA marker of length 65, 50, and 35 bp.

Lane 1: B0 after.

Lane 2: B1 after.

Lane 3: B0C0 after.

Lane 4: B0C1 after.

Lane 5: B1C0 after.

Lane 6: B1C1 after.

Laboratory Experiments

Verification method

CC00 Data0

CC00 Data0 CC00 Data0

CC00 Data0 CC00 Data0

Data0 Data1

amplification Non-amplification

35mer

Laboratory ExperimentsDetection of amplified sequence

M: DNA marker of length 65, 50, and 35 bp.

Lane 1: use data000primer. Lane 2: use data001primer.

Lane 3: use data010primer. Lane 4: use data011primer.

Laboratory ExperimentsAmplification using concatenation primer

M: DNA marker of length 65, 50, and 35 bp.

Lane 1: the solution set aside from the thermal cycler at the 17th cycle.

Lane 2: 19th cycle. Lane 3: 21st cycle. Lane 4: 23rd cycle. Lane 5: 25th cycle.

Concluding Remarks

Hierarchical DNA memory based on nested PCR (NPMM)

DNA memory with high capacity, high data security and high specificity of chemical reaction

The feasibility of NPMM through some experiments