1. 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

2. Outline Nested PCR Hierarchical DNA Memory (NPMM) Design of Sequences Experimental results Concluding Remarks

3. Nested PCR

4. Structure of DNA Memory ＡＢＣ Data Re A0 An ・・・ B0 Bn # of hierarchy : 3 # of sequences in each hierarchy: n Nested Primer Molecular Memory (n×n×n NPMM) address partdata part linker sequences

5. Nested PCR (addressing) A ＢＣ Data Re B Ｃ Data Re Data Re C address is represented in the primer sequences and the order of use of them

6. Hierarchical DNA Memory NPMM provides a high level of data security NPMM has a large capacity with a high reaction specificity Enlarge the address space by using a small number of primer sequences Reduce the total errors by removing (diluting) the error products in each step. Ease of extracting the target data from MPNN

7. 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 e ach hierarchy address space# of required seq uences 41001.00×10 8 407 5401.024×10 8 208 6231.480358×10 8 147 7141.054135×10 8 108

8. Readout Process ABC Data Re A0 A1 A2 B0 B1 B2 C0 C1 C2 Data0~data26 ・ all 27 NPMM units are synthesized ・ resulting pools are analyzed after each PCR A, B, C, Re: 15bp Data: 20bp A0 Re C2 Re B1 Re 27mix 9mix3mix target

9. Design of Sequences GC_content Hamming distance 3’end_complementary

10. Design of Sequences Designed sequences of templates and primers Sequence of data primers

11. 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.

12. Laboratory Experiments Verification method C0C0C0C0Data0 C0C0C0C0 C0C0C0C0 C0C0C0C0 C0C0C0C0 Data1 amplification Non-amplification 35mer

13. Laboratory Experiments Detection 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.

14. Laboratory Experiments Amplification 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: 19 th cycle. Lane 3: 21st cycle. Lane 4: 23rd cycle. Lane 5: 25th cycle.

15. 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