ABSTRACT
BODIPY FL, developed by Molecular Probes (USA), is a 4,4-diuoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionic acid. BODIPY FL can be attached to a nucleotide by a linker. Horn
TABLE 5.1 Comparison of qPCR Signal Detection Systems
and coworkers9 reported that the uorescence from a probe modied with BODIPY FL was diminished after hybridization, and it was later found10 that the quenching was caused by the interaction between BODIPY FL and a guanine base. The quenching was more dramatic when guanine is positioned opposite BODIPY FL. Compared with a few commercially available uorescent dyes, BODIPY FL linked to an oligonucleotide was found to have the highest quenching rate (95%) by hybridization.11 Based on the effective uorescence quenching of BODIPY FL-modied primer, Kurata and coworkers10 further developed a real-time quantitative PCR (qPCR) system using a PCR primer with BODIPY FL linked to a cytosine at its 5′ end. As the PCR primer is integrated into the product, the uorescence of BODIPY FL was quenched. The quenching rate was proportional to the amount of target DNA. When compared to hybridization probe-based approaches, the above system needs only two primers in the reaction because one PCR primer has the additional function of being the probe as well. This simple design reduces the complexity of the PCR system. Using a guanine base instead of a special dye as the quencher also makes it a cost-effective system. This approach, however, relies on the decrease in total uorescence for monitoring PCR progress.
