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)−163−Synthesiology - English edition Vol.3 No.2 (2010) is necessary to amplify only the gene to be quantified among the various nucleic acid mixtures. There have been various methods developed for the amplification of the target gene, and the one most frequently used is the polymerase chain reaction (PCR) method. The PCR method was developed in 1984 by Kary Mullis, an American researcher who won the Nobel Prize in Chemistry. In this method, the target gene can be amplified exponentially using a simple method where the temperature is changed cyclically using reagents such as heat-resistant polymerase or short DNA fragments (primers) that act as the originators of the reaction. However, since the amount of the final amplified product by PCR does not necessarily reflect the amount of the target gene in the initial reaction solution, the amount of the initial target gene cannot be directly quantified from the amount of the final amplified product. Therefore, to quantify the target gene using PCR (quantitative PCR), it is necessary to find a way to measure the amount of the target gene in the initial reaction solution.In the quantitative PCR, there are several methods with different measurement principles such as the real time[1], the competitive[2], and the most probable number (MPN)[3] methods. The real time PCR (RT-PCR) method is most commonly used. In the RT-PCR, the amount of amplified product is measured at each cycle of PCR, and the number of cycles required for the reaction product to reach a certain amount (or cycle of threshold: Ct) is calculated in the region where exponential amplification reaction is occurring. The relationship between the Ct and the amount of genes in the initial reaction solution is plotted to obtain the standard curve, and the amount of the target gene in the initial reaction solution can be calculated from this standard curve based on the Ct for the unknown sample.In the RT-PCR, it is necessary to measure the amount of the amplified product at each cycle. The method used is to label and quantify the amount of amplified product with fluorescence. The major methods are the method using intercalator such as the SYBR Green[4] or the one using fluorescent probe such as the TaqMan probe[5]. SYBR Green is a special fluorescent dye (intercalator) that emits fluorescence when incorporated into the double-stranded DNA. When the SYBR Green is added to the PCR solution, the SYBR Green intercalates into the double-stranded DNA amplified by PCR and the fluorescence increases. The amount of PCR product can be measured by measuring the intensity of the fluorescence. This method allows using the same reagent for the target genes of any sequence, and it is used widely because of its low cost and convenience. On the other hand, since fluorescence increases with nonspecific amplified product such as primer dimer, there is a disadvantage that the fluorescence intensity and the amount of PCR product may not necessarily correspond. As shown in Fig. 1, the TaqMan probe method is a method using the TaqMan probe, in which one terminal of the oligonucleotide corresponding to the base sequence of the segment of the amplified region of the target gene is labeled with a reporter (fluorescent dye), and the other end is labeled with a quencher to turn off the fluorescence of the reporter. When the TaqMan probe is added to the PCR solution, the TaqMan probe that bonded to the amplified product is broken down by the elongation reaction by the 5’3’ exonuclease activity of DNA polymerase. When the probe is broken down, the reporter fluorescent dye begins to emit its original fluorescence by separating from the quencher. The amount of PCR product can be measured by measuring the fluorescence intensity. Since the TaqMan probe bonds specifically only to the amplified product, it is not affected by any nonspecific amplified product such as the primer dimer, and allows highly specific quantification. While this method is widely used, it requires labeling by two fluorescent dyes.RT-PCR has advantages that it can measure the amount of target gene in a short period (30 min to 2 h), and there is very little contamination of the laboratory with the PCR product since gel electrophoresis is unnecessary. It also has excellent trueness and precision. Also, the detection limit is low due to gene amplification, and the measurement range reaches 105~108 copies. However, it has the following disadvantages: 1) since it is necessary to measure the fluorescence per cycle of PCR to measure the amount of the amplified product, it is necessary to install an expensive RT-PCR device that embodies the fluorescence measurement device and the PCR thermal cycler (problem of initial cost); 2) while the specificity may increase in the fluorescent probe method, it is necessary to design and synthesize the fluorescent probe for each target gene to measure the amount of amplified product (problem of running cost performance); and 3) the amount of the target gene may be undervalued or may produce pseudo-negativeness in the case the measured sample contains a substance that inhibits PCR (problem of robustness). Considering the use of the gene quantification technology Fig. 1 TaqMan probe methodEmissionDNA polymerasePrimerQuencherReporter③ Elongation reaction② Annealing of primer and probe① Heat denaturation3’3’3’5’5’5’TaqMan probe

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