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)−167−Synthesiology - English edition Vol.3 No.2 (2010) the relationship of the number of cycles and fluorescent quenching rate when the -actin gene was quantified. The fluorescent quenching rate was about 30~40 %, or about the same as the QProbe PCR. Figure 5 shows the standard curve created by calculating Ct from Fig. 4. The lower limit of quantification was 10 copies, and the correlation coefficient R2 of the standard curve was 0.9967. Both the lower limit of quantification and the correlation coefficient were about the same as the QProbe PCR. The amplified product can be checked by conducting the melting curve analysis where the temperature at which the complex of the fluorescent probe and joint DNA dissociates from the amplified product is measured by gradually raising the temperature from around 40 ºC after PCR. Results with equivalent quantification precision were obtained for the albumin and -globin genes as well as the -actin gene. Our initial objective of developing the quantification of multiple target gene sequences using one fluorescent DNA probe while maintaining the same quantitative quality as the QProbe PCR was achieved with this universal QProbe PCR [9].We applied the universal QProbe PCR method to the genetic analysis of single nucleotide polymorphisms (SNP) in the human gene. SNP is a single base DNA variation occurring in the genome, and is defined as a mutation seen at 1 % or higher frequency in a certain group. Recently, through the advancement in human genome and genetic analysis research, SNP is drawing attention as one of the causes of the individual differences such as susceptibility to disease or reactivity to drugs. SNP is said to occur on average in one place among 1000 bases, and there are over 3 million SNPs in the 3 billion base pairs on a human genome. We differentiated these SNP gene types through the melting curve analysis using the universal QProbe PCR. The joint DNA was designed to be completely complementary to one allele, and had one base mismatch on the other allele. After annealing the fluorescent probe and joint DNA complex with the PCR amplified product by decreasing the temperature, the SNP was analyzed by obtaining the melting curve from the fluorescence emitted by the quenched probe as the temperature was raised. The fluorescence is emitted as the dissociation occurs at low temperature if there is a mismatch, while the fluorescence is emitted at higher temperature if there is a perfect match (Fig. 6). Figure 7 shows the result of the analysis of the three gene types for SNP: wild homo, mutant homo, and hetero types. Separation was easy since the positions of the peak of emission differ for the wild and mutant types. Both peaks were observed in the hetero type where the wild and mutant types were mixed. Since the universal QProbe PCR allows quantifying multiple target genes with one fluorescent probe, it is expected to be an effective tool in genotyping the SNP, since it is said that 3 million SNPs exist in the human genome. 3.2 Alternately binding probe competitive (ABC) PCR methodWe investigated the quantitative property in the ABC-PCR method using the gfp gene, which is the famous green fluorescent protein, as the target gene. The internal standard gene was created based on the sequence of the gfp gene, and the verification of ABC-PCR was conducted. The quenching rate calculated from the fluorescence values after the PCR that was corrected by several background fluorescence values was set as the relative fluorescence intensity. Figure 8 shows the graph of the relationship between this relative fluorescence intensity and the amount of the target gene in the initial template. Using this method, the standard curve could be regressed to a sigmoid curve just as in the standard curve obtained by other general competitive measurement methods such as the competitive ELISA. According to Fig. 8, the correlation coefficient of the standard curve was 0.9997. The lower limit of quantification was 103 copies. Since this method is competitive, the quantifiable range using one standard curve is in the order of 2~3, but the quantifiable range can be adjusted by changing the concentration of the internal standard gene. It is also possible to calculate Fig. 6 Principle of SNP typing by the universal QProbe methodACCT5’3’GG5’3’GTACCT5’3’3’5’5’3’ACCGTT3’3’5’5’5’3’ACCGGTTemperatureFluorescence value ismeasured asthe temperature is raisedSNP genotyping is possibleHeterotypeMutanttypeWildtypeWild typeFluorescence intensityMutant typeIncreasedtemperatureSNP siteDissociation occurs at high temperature since mismatch does not existDissociation occurs at low temperature since mismatch exists0.040.02Wild typeMutant typeHetero typeTemperature (ºC)-d (fluorescence intensity) /dT0-0.02-0.06-0.08-0.1-0.12-0.14-0.04405060708090Fig. 7 Result of SNP genotyping by the universal QProbe methodSNP genotyping is conducted from the analysis of dissociation curve where the fluorescence values are measured as the temperature is gradually raised from 40 ºC to 90 ºC. The vertical axis shows the value obtained by the primary derivation of fluorescent value by time.

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