The design, synthesis and characterization of a novel acceptor for real time polymerase chain reaction using both computational and experimental approaches

The design of a novel, fluorescence acceptor for use in real time polymerase chain reaction analysis was carried out with the help of in-depth computational analysis of structure/property relationships. A functionalized indocyanine dye was synthesized in order to quench a fluorescent cyanine dye. The pentamethine indocyanine dye was functionalized with nitro groups so as to enhance its quenching abilities; ad hoc substituents were used to enable conjugation with DNA strands. The visible absorption and photoemission of the indocyanine dye as well as that of an unsubstituted indodicarbocyanine dye, employed as a reference model chromogenic system, were addressed both theoretically and experimentally. A Forster resonance energy transfer analysis was simulated experimentally to evaluate the quenching efficiency of the donor/acceptor couple.