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)−165−Synthesiology - English edition Vol.3 No.2 (2010) in addition to the PCR primer. While the synthetic oligonucleotide DNA without the fluorescent label can be prepared at about 2,000 yen per target gene, the probe labeled with fluorescence costs over 20,000 yen. In case there are several target genes, the fluorescent probe much be designed and synthesized for each target gene, and the cost becomes high. If it is possible to quantify any target gene with one fluorescent probe regardless of the sequence, a new gene quantification method with excellent cost performance may be established, having the advantage of mass-synthesized fluorescent probe.The universal QProbe method was developed based on this thinking (Fig. 2)[9]. While maximizing the principle of the QProbe method that uses the quenching phenomenon by guanine base and realizing the concept of quantifying all types of target genes with one fluorescent probe (the universal QProbe) regardless of the sequence, in the universal QProbe method, we added the idea of the joint DNA that binds both the target gene and the fluorescent probe. The joint DNA is a single-stranded oligo DNA that has a complementary sequence of target gene on the 5’ side and a complementary sequence of fluorescent DNA probe on the 3’ side, and the two sequences are joined with cytosine and thymine. The fluorescent probe is labeled with a dye whose fluorescence is quenched by the guanine base nearby. The joint DNA bonds to both the target gene and the fluorescent probe, and the fluorescence of the fluorescent probe is quenched when the fluorescent probe approaches the guanine base in the target gene. Therefore, it is possible to measure the amount of the target gene by measuring the degree of quenching as in the QProbe method. The fluorescent probe in this method has the adenine base on the 3’ terminal labeled with the fluorescent dye. It is designed so the quenching occurs when the guanine base approaches the fluorescent dye, because this adenine base is positioned across the thymine base of the cytosine-thymine sequence within the joint DNA strand, and the guanine base of the target DNA is positioned across the neighboring cytosine base.The joint DNA must be designed and synthesized for each target gene, but the cost and time of synthesis can be reduced greatly since it is not labeled with the fluorescent dye. This method will enable the quantification using only one type of fluorescent DNA probe, even if the genes under investigation have different sequences.2.3 Development of the alternately binding probe competitive (ABC) PCR that is resistant to PCR inhibitorsIn the RT-PCR method, it is known that the quantification result may be undervalued or show pseudo-negativeness if the sample to be measured contains a substance that inhibits PCR. While such issues are negligible in samples that contain very little inhibitors or samples that are highly purified, amplification inhibitors are thought to be present in blood samples and soil samples that contain decomposed materials, and inhibition of amplification may be an issue. Although the competitive PCR method is a classical method, it solved the issue of such amplification inhibitors. In the competitive PCR, amplification is done using the same primer as the target gene, but uses an internal standard gene with different amplified base length than the target gene. Specifically, the internal standard gene that is shorter or longer than the target gene is created by removing part of the internal sequence of the target gene or by adding extra base. The internal standard gene of known concentration is added to the sample, and PCR is conducted competitively with the target gene. Since the lengths of the strands differ between the target gene and the internal standard gene, the target gene and the internal standard gene are separated by electrophoresis after PCR, the gradation of the bands of the target gene and the internal standard gene are compared quantitatively, and then the amount of the target gene can be measured from the known amount of the internal standard gene. Using this method, even if the PCR inhibitor is present in the sample, accurate quantification is possible since the inhibition equally affects both the target gene and the internal standard gene. While this method allows accurate quantification even in the presence of the PCR inhibitor, it is no longer used recently since it requires labor-intensive and time-consuming post-PCR procedures for the separation of the PCR products by gel electrophoresis.By utilizing the advantage of the competitive PCR that can avoid the issue of PCR inhibitors, and by using the quenching phenomenon by the guanine base, we developed the alternately binding probe competitive (ABC) PCR method as a convenient gene quantification method that eliminates the electrophoresis that was a problem in competitive PCR (Fig. 3)[10]. In the ABC-PCR, internal standard gene ① Competitive amplification of genes② Fluorescence measured after amplification③ Fluorescence value indicates the amount of target geneTarget gene (T)Internal standard gene (C)Target geneInternal standardgeneT:C ratio does notchange before andafter amplificationHybridization with similar affinityFluorescencevalueAmount of target geneHighLargeLowSmallAB-QProbeFluorescence is quenched by the guanine (G)TAMRABODIPY-FLAACCTTCTGAGGGTCGFig. 3 ABC-PCR method

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