Molecular Beacon DNA Probes with Fluorescein Bifluorophore

Abstract— An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of “molecular beacon”-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or “click reaction” (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔCt = 1–2.

Fluorescence Energy Transfer between Fluorescein Label and DNA Intercalators to Detect Nucleic Acids Hybridization in Homogeneous Media

A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5′), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5′), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.

Fluorescein-Labeled DNA Probes for Homogeneous Hybridization Assays: Application to DNA E. Coli Renaturation

The sensitivity of the emission intensity of fluorescein to environmental changes opens the possibility for developing homogeneous DNA assays. In this research, we were able to quickly and easily detect nucleic acid renaturation by using an E. coli DNA with approximately 4% conjugation of fluorescein to the N4-aminoethylmodifled cytosine residues. We determined how the emission intensity of fluorescein attached to single-stranded DNA decreases when renatured to double-stranded DNA. The decrease in fluorescence efficiency is due to changes in the fluorescein monoanion–dianion ratio because of pH modifications in the surrounding of the fluorescein label. The decrease in fluorescence intensity was used for analysis of DNA E. coli renaturation kinetics.

Homogeneous Fluoroimmunoassay Using Lucifer Yellow VS: Determination of Albumin Plasma

Non-separation fluoroimmunoassays are well suited to automation. The potential sensitivity of such assays has not been realised because the most commonly used label (fluorescein) has absorption and emission spectra which coincide with those of blood constituents and because it has a very small Stokes shift. Lucifer yellow VS has a larger Stokes shift than fluorescein and an emission maximum at longer wavelength. We describe an energy transfer fluoroimmunoassay for plasma albumin in which Lucifer yellow VS is used to label albumin and rhodamine B isothiocyanate is used to label anti-albumin antibodies. The assay shows good correlation with a dye-binding method for albumin and has sensitivity and precision which compare favourably with similar assays using a fluorescein label.