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
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(_
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.