Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction

Abstract

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations.

A powerful qPCR-high resolution melting assay with taqman probe in Plasmodium species differentiation

“…But the probe cost is distribution across thousands of PCR reactions making low impact on the cost of sample analysis. Despite using a Taqman probe, the developed assay remains cost-efficient compared to a fluorescence resonance energy transfer (FRET) more expensive than the Taqman probes [ 37 – 39 ]. The assay without the Taqman probe will indeed identify any Plasmodium infection through the specific melting temperature.…”

Absolute quantification of real-time PCR data with stage signal difference analysis

“…With the mechanism of sequence complementarity of DNA molecules, the signal can be amplified with high specificity. These features give qPCR the ability to accurately detect low concentration sample with great specificity when combining with fluorescence technique, such as passive fluorescence dyes, or fluorescent reporter probe or short Förster resonance energy transfer probes [4]. As a direct result, the step-wise fluorescence signal is expected to be linearly proportion to the amount of the double-strain DNA (dsDNA) presented under the same thermodynamic conditions.…”