[A novel quantitative PCR with fluorogenic probe].
The polymerase chain reaction(PCR) is a powerful tool to amplify small amounts of DNA or RNA for various molecular analysis. However, in these analyses, PCR only provides qualitative results. The availability of quantitative PCR provides valuable additional information in various applications. It is difficult to establish absolute quantitation, because PCR amplification is a complicated reaction process of exponential growth. To trace the amplification process, the initial amount of template and the efficiency of amplification in each cycle, has to be determined. Conventional methods have not achieved absolute quantitative analysis. The ABI PRISM 7700 Sequence Detection System has solved these problems with real-time monitoring of the PCR process. The real-time detection system provides essential information to quantify the initial target copy number, because it can draw an amplification curve. Using the 5' nuclease assay, a specific fluorescent signal is generated and measured at every cycle during a run. This system can perform a variety of applications including, quantitation, allele discrimination, PCR optimization and viral screening. Using the ABI PRISM 7700 Sequence Detection System, the rice genome has been quantitatively analyzed. To monitor maturation of the chloroplast genome from proplastid during germ development, 5' nuclease assay set up for Cab and rbcL genes which are located in the nuclear genome and chloroplast genome, respectively. Cab was used as an internal standard for normalization of cell numbers. The maturation process of chloroplast was estimated using the ratio of gene dosage, [rbcL]/[Cab]. After development of cotyledon, a significant increase in copy numbers of the chloroplast was observed. These results indicate that a light-induced chloroplast maturation process is coupled with an increase in chloroplast genome copy numbers.
Isono
K
K
Perkin-Elmer Japan Applied Biosystems Division, Urayasu.
D002736:Chloroplasts / Q000235:genetics; D017931:DNA Primers; D018744:DNA, Plant / Q000032:analysis; D004735:Energy Transfer; D005456:Fluorescent Dyes; D018745:Genome, Plant; D012275:Oryza / Q000235:genetics / Q000254:growth & development; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*
D017931:DNA Primers; D018744:DNA, Plant; D005456:Fluorescent Dyes; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I