Abstract—A readout approach for the Hamiltonian Path Problem (HPP) in DNA computing based on the real-time polymerase chain reaction (PCR) is re-implemented on DNA Engine Opticon 2 System. Several types of fluorescent probes and detection mechanisms are currently employed in real-time PCR, including SYBR Green, molecular beacons, and hybridization probes. Based on the new approach, real-time amplification performed using the TaqMan probes is adopted, as the TaqMan detection mechanism can be exploited for the design and development of the proposed readout approach. In this study, double-stranded DNA molecules of length 140 base- pairs are selected as the input molecules, which represent the solving path for an HPP instance. These input molecules are prepared via the self-assembly of 20-mer and 30-mer single- stranded DNAs, by parallel overlap assembly. The proposed readout approach consists of two steps: real-time amplification in vitro using TaqMan-based real-time PCR, followed by information processing in silico to assess the results of real-time amplification, which in turn, enables extraction of the Hamiltonian path. The experimental result is compared with that of previously implementation on Roche LightCycler System. Experimental results establish an easier method to interpret the output of real-time PCR for the subsequent in silico information processing. Keywords: DNA computation, graduated PCR, Hamiltonian path problem, readout method, real-time PCR, TaqMan probes. INTRODUCTION Since the discovery of the polymerase chain reaction (PCR) [1], numerous applications have been explored, primarily in the life sciences and medicine, and importantly, in DNA Manuscript received March 15, 2007. This work mainly supported by eScienceFund Research Funding from the Ministry of Science, Technology, and Innovation (MOSTI), Malaysia (Vot: 79033, 79034), and supported partially by Grant-in-Aid for Scientific Research B (18300100; J. Rose, A. Suyama) from the Japan Society for the Promotion of Science (JSPS), and JST-CREST (A. Suyama, J. Rose). Zuwairie Ibrahim is with the Centre for Artificial Intelligence and Robotics, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor Darul Takzim, Malaysia (e-mail: zuwairie@fke.utm.my) Muhammad Faiz Mohamed Saaid is the Faculty of Electrical Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor Darul Takzim, Malaysia Akira Suyama is with the Department of Life Sciences and the Institute of Physics, University of Tokyo, and J.S.T.-CREST (e-mail: suyama@dna.c.u-tokyo.ac.jp) John A. Rose is with the Institute of Information Communication Technology, Ritsumeikan Asia Pacific University, 1-1 Jumonjibaru, Beppu-shi, Oita 874-8577 Japan, and J.S.T.-CREST (e-mail: jarose@apu.ac.jp) computing as well. The subsequent innovation of real-time PCR has rapidly gained popularity and plays a crucial role in molecular medicine and clinical diagnostics [2]. All real-time amplification instruments require a fluorescence reporter molecule for detection and quantitation, whose signal increase is proportional to the amount of amplified product. Although a number of reporter molecules currently exist, it has been found that the mechanism of the TaqMan hydrolysis probe is very suitable for the design and development of a readout method for DNA computing, and is thus selected for the current study.A TaqMan DNA probe is a modified, non-extendable dual-labeled oligonucleotides. The 5’ and 3’ ends of the oligonucleotide are terminated with an attached reporter, such as FAM, and quencher fluorophores dyes, such as TAMRA, respectively, as shown in Fig. 1 [3]. Upon laser excitation at 488 nm, the FAM fluorophore, in isolation emits fluorescence having the maximum spectrum at 518 nm. Given proximity of the TAMRA quencher, however, based on the principle of fluorescence resonance energy transfer (FRET), the excitation energy is not emitted by the FAM fluorophore, but rather is transferred to TAMRA via the dipole-dipole interaction between FAM and TAMRA. As TAMRA emits this absorbed energy at the longer wavelengths (Max. 580 nm), the resulting fluorescence is not observable with a detector adjusted to the FAM emission [4]. The combination of dual-labeled TaqMan DNA probes with forward and reverse primers is a must for a successful real-time PCR. As PCR is a repeated cycle of three steps (denaturation, annealing, and polymerization), a TaqMan DNA probe will anneal to a site within the DNA template in between the forward and reverse primers during the annealing step, if a subsequence of the DNA template is complementary to the sequence of the DNA probe. During polymerization, Thermus aquaticus (Taq) DNA polymerase will extend the primers in a 5’ to 3’ direction. At the same time, the Taq polymerase also acts as a “scissor” to degrade the probe via cleavage, thus separating the reporter from the quencher, as shown in Fig. 2 [5], where R and Q denote the reporter dye and quencher dye, respectively. This separation subsequently allows the reporter to emit its fluorescence [6]. This process occurs in every PCR cycle and does not interfere with the exponential accumulation of PCR product. As a result of PCR, the amount of DNA template increases exponentially, which is accompanied by a proportionate increase in the overall fluorescence intensity emitted by the reporter group of the excised TaqMan probes. Hence, the intensity of the fluorescence at the end of each PCR An Improved Readout Method of Molecular Computation based on Real-Time PCR Implemented on DNA Engine Opticon 2 System Zuwairie Ibrahim, Muhammad Faiz Mohamed Saaid, Adi S. Paramita, Akira Suyama, and John A. Rose 1829 1-4244-1340-0/07$25.00 c 2007 IEEE