Vol.3 No.2 2010

61/86

Research paper : Development of an accurate and cost-effective quantitative detection method for specific gene sequences (N. Noda)−164−Synthesiology - English edition Vol.3 No.2 (2010) for multiple samples on site, it is necessary to concentrate on robustness, convenience, and cost performance, while maintaining an equivalent level as the current RT-PCR technology.In this paper, the two quantitative PCR methods developed as new technologies to solve the problem inherent in the current RT-PCR are described, and the cooperation with companies for the practical application of the developed technology will be presented.2 Scenario for the development of gene quantification technology with excellent accuracy and cost performance2.1 Core technology for technology development: quenching phenomenon by the guanine baseTo solve the problem inherent in the current RT-PCR, we developed a new quantitative PCR to overcome the two issues: 1) quantification can be done using only one type of fluorescent probe for different target genes (cost reduction by general-use fluorescent probe), and 2) accurate quantification is possible even with the presence of PCR inhibitors. The core technology in this technological development is the “quenching phenomenon by the guanine base.” Fluorescence means the emission of light as the fluorescent molecule absorbs light, transforms into an excited-state molecule, and then returns to the original ground-state molecule. The difference in energy between the excited and ground states of the molecules is released as the fluorescence energy. When the molecule transforms from the excited state to the ground state, if there is another molecule with high electron density nearby, this molecule acts as an electron donor and gives away electron to the fluorescent molecule. At this moment, the electron excited by the original fluorescent molecule cannot return to the ground state, becomes unable to emit fluorescence, and the fluorescence disappears. This phenomenon is called the photo-induced electron transfer (PET), and is known to occur within and between molecules[6]. Among the bases that comprise the nucleic acid, guanine has the highest electron density, and therefore, tends to cause quenching through this PET reaction. However, not all fluorescent dyes cause quenching, and it is known that some fluorescent dyes such as BODIPY FL and TAMRA are more likely to cause quenching by the guanine base[7].Since the quenching phenomenon by the guanine base is a reversible reaction, it can be used conveniently as a tool to detect and quantify nucleic acids. Completely complementary DNA is prepared for a fluorescent probe of about 20 bases, in which the cytosine base of the terminal is labeled with BODIPY FL. When the temperature and other conditions are adjusted to induce annealing (hybridization) in the same reaction solution, the fluorescence of the BODIPY FL is quenched. Then when the hybridization is broken by raising the temperature or other conditions, the BODIPY FL begins to emit fluorescence again. By controlling the hybridization and separation, the on/off of the fluorescence can be controlled. By measuring the degree of quenching, it becomes possible to estimate the amount of complementary strand of the fluorescent probe. The quantitative PCR method using this phenomenon was developed and put on the market as the quenching probe (QProbe) PCR method through joint research with Dr. Shinya Kurata et al. of the J-Bio 21 Corporation, an AIST venture that spun off from the Institute for Biological Resources and Functions[8]. The author formed the joint research structure with Dr. Kurata’s group, as well as the group of Dr. Satoshi Tsuneda, School of Advanced Science and Engineering, Waseda University, to develop a new technology that extends the QProbe PCR method.2.2 Development of the universal QProbe method that achieves cost reduction through general-use fluorescent probeWhile it is necessary to label the probe with two fluorescent dyes (reporter and quencher dyes) in the TaqMan probe method, which is a method used most frequently as a RT-PCR using the fluorescent probe, only one fluorescent dye is necessary in the QProbe PCR method, which is also a RT-PCR, because it uses the guanine base as the quencher. Moreover, in the QProbe PCR, the adequacy of the amplified product can be checked by a melting curve analysis where the melting temperature of the fluorescent probe that hybridized to the amplified product is measured by raising the temperature gradually from around 40 ºC after the completion of the reaction. This cannot be done in the TaqMan probe method. Although the QProbe PCR has such advantages, it is the same as other fluorescent probe methods in that the fluorescent probes must be designed and synthesized according to the target gene. In the fluorescent probe method, although the specificity of the detection and quantification increases since a fluorescent probe specific to the amplified product is used, the cost increases since the fluorescent probe must be designed and synthesized Fluorescent DNA probe: Strongly hybridizes with joint DNAJoint DNAGene AGene BGene CGene AGene BGene C5’ side: Complementary to target gene3’ side: Complementary to fluorescent DNA probe3’3’3’3’3’3’3’3’3’3’3’5’5’5’5’5’5’5’5’5’3’3’5’5’5’3’3’5’5’5’Multiple genes (genes A, B, and C) can be quantified using one fluorescent DNA probeQuenched by nearby guanine baseQuenched by nearby guanine baseQuenched by nearby guanine baseFig. 2 Universal QProbe method

※このページを正しく表示するにはFlashPlayer9以上が必要です