A Fluorescent Confirmation Method for DNA Amplification in PCR through a Fluorescent Pyrophosphate Sensor

Pyrophosphate (PPi) is involved in several important enzymatic reactions such as the adenylate cyclase catalyzing synthesis of cyclic AMP from ATP, the attachment of a given amino acid to a particular tRNA catalyzed by an aminoacyl-tRNA synthetase in protein synthesis, and the DNA replication catalyzed by a DNA polymerase.1,2 In addition, patients with calcium PPi dihydrate (CPPD) crystals and chondrocalcinosis have been shown to have high synovial fluid PPi levels.3 Therefore, the selective and sensitive detection of PPi is a prerequisite for monitoring the above enzyme reactions and for diagnosing the diseases related to the release of certain levels of PPi. The most common way to confirm the target nucleic acids amplified after PCR (polymerase chain reaction) is by running a gel electrophoresis. Chemical and enzymatic assays have been developed to verify PCR results.4-7 How-ever, these methods may be too slow and laborious for quick confirmation of DNA amplification in PCR. PPi detection methods based on the formation of visible precipitates with transition metal ions are insufficiently precise.8 The colori-metric detection for PPi by 1,10-phenanthroline complex with ferrous ion needs incubation at high temperature.9 Recently, we developed a fluorescent sensor (1·2Zn, Figure 1), which showed a high sensitivity and selectivity for PPi over other anions including nucleotide triphosphates in aqueous solvent.10 We expected that our sensor can be used as a new fluorescent confirmation method for DNA amplification after PCR, because the sensor can detect a small amount of PPi in the presence of a large excess of ATP.10 In this paper, we present a fluorescence method that can determine PPi released during PCR through 1·2Zn sensor (1·2Zn, Figure 1). We expect that the degree of DNA poly-merization can be determined by the concentration of PPi released during PCR. The PPi released from the polymerization reaction bound to the dinuclear zinc complex of the 1·2Zn sensor (see th