Vol.3 No.2 2010

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Research paper : Development of an accurate and cost-effective quantitative detection method for specific gene sequences (N. Noda)−168−Synthesiology - English edition Vol.3 No.2 (2010) the amount of the standard gene from the diluted sample within the quantifiable range by conducting measurement by creating a dilution series of the unknown sample. This method can be also used as genotyping to identify the SNP as in the universal QProbe as well as for gene quantification[10].We evaluated the effect on the quantification value in the ABC-PCR and RT-PCR methods by adding humic acid that is found in the soil and is known as a DNA amplification inhibitor. As a result, in the RT-PCR, the quantification value turned out to be lower than the true value as the concentration of the humic acid increased, while in the ABC-PCR, the quantification value was almost the same as the true value even in the presence of humic acid[10]. In the experiment using urea and Triton X-100 as the DNA amplification inhibitor, it was found that the ABC-PCR was capable of highly accurate quantification compared to the RT-PCR[12].The ABC method not only is capable of accurate quantification in the presence of the DNA amplification inhibitor, but also is capable of quantifying the target gene by measuring the fluorescence after the gene amplification reaction. This means that the target gene can be quantified in a similar manner whether the gene amplification reaction is PCR or some other technique. Recently, methods such as the loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA) have been developed as the isothermal gene amplification, as alternative to the PCR. By combining such isothermal gene amplification methods with the ABC method, similar quantification as the ABC-PCR can be done. The ABC method is not only accurate, but is also highly universal from the perspective of combining with the gene amplification methods.4 Evaluation of the development technology and scenario for realizationThe advantages and the disadvantages of the two newly-developed gene quantification technologies, universal QProbe PCR and ABC-PCR methods, will be compared to the current technologies, and the scenario for their realization to maximize the advantages of each technology will be discussed (Fig. 9). Table 1 shows the comparison of the properties of the current technologies (TaqMan probe, QProbe, intercalator, competitive methods) and the universal QProbe and ABC-PCR methods. Since the technologies have their advantages and disadvantages, it is necessary to consider the ways to realize them by thoroughly understanding the properties of the technologies. The business for the realization of the universal QProbe PCR and the ABC-PCR is currently undertaken by the J-Bio 21 Corporation, the partner of the joint research.Table 2 shows the comparison of the characteristics of the universal QProbe PCR with the conventional RT-PCR (fluorescent probe and intercalator methods). As it can be seen from Table 2, the universal QProbe PCR is a technology that takes the advantages of the fluorescent probe and the intercalator methods. Although this paper does not refer to multicolor detection, four different colors can be used as dyes where the fluorescence is quenched by guanine, and this method can be used in the multicolor detection and quantification. Since the universal QProbe PCR is a RT-PCR, the thermal cycler for RT-PCR is necessary. However, thinking alternatively, this method can be used immediately if there is a thermal cycler for RT-PCR available. Therefore, the prime strength in realizing this technology is that we can recommend it to users who are already using the RT-PCR method. In the conventional RT-PCR, the reagent kits are commercially available for the detection and quantification of specific genes (such as pathogenic bacteria, virus, or certain SNP). However, such pre-marketing method is not compatible for the universal QProbe PCR. One of the advantages of the reagent kit is the cost merit of mass synthesizing the fluorescent probe to detect a specific gene. However, since only one type of fluorescent probe is necessary for various gene sequences in the universal QProbe PCR, there is hardly any cost advantage in providing the reagent kit. Therefore, as business plans to optimize the advantage of this method, the joint DNA and fluorescent probe according to the client’s target gene sequence can be provided, or the genetic analysis service can be subcontracted to detect and quantify the client’s target gene sequence. In these business plans, the low cost of the fluorescent probe and the short time for the preparation of the fluorescent probe synthesis that are the characteristics of this method can be optimized fully, to provide a low-cost, quick-delivery gene analysis service. One of the clients who may benefit from the low cost and quick delivery may be a company in the field of Initial template (copies)Relative fluorescence intensity102103104105106-0.20.100.20.30.40.5-0.1Y = {-8.87×103/(1.58×104 +X)}+0.430R = 0.9997Fig. 8 Standard curve in the ABC-PCR methodThis is the relationship between the amount of target genes in the initial template and the relative fluorescence intensity. The relative fluorescence intensity is the value where the fluorescence value obtained after the completion of PCR is corrected by some background fluorescence values[9]. The plot obtained is regressed by equilateral hyperbola. R is the correlation coefficient.

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