A new class of homogeneous nucleic acid probes based on specific displacement hybridization

TLDR

The probes are intended for high‑specificity nucleic acid detection in diverse applications. The probes are duplex oligodeoxyribonucleotides labeled with a fluorophore and quencher that are quenched until target‑induced displacement hybridization restores fluorescence, and they can also function as primers in real‑time amplification. The probes achieve complete discrimination between perfectly matched and single‑nucleotide mismatched targets, enhance specificity compared to linear probes, enable multiplex detection of single‑nucleotide variants in a single solution, and offer low cost, high sensitivity, and broad labeling flexibility.

Abstract

We have developed a new class of probes for homogeneous nucleic acid detection based on the proposed displacement hybridization. Our probes consist of two complementary oligodeoxyribonucleotides of different length labeled with a fluorophore and a quencher in close proximity in the duplex. The probes on their own are quenched, but they become fluorescent upon displacement hybridization with the target. These probes display complete discrimination between a perfectly matched target and single nucleotide mismatch targets. A comparison of double-stranded probes with corresponding linear probes confirms that the presence of the complementary strand significantly enhances their specificity. Using four such probes labeled with different color fluorophores, each designed to recognize a different target, we have demonstrated that multiple targets can be distinguished in the same solution, even if they differ from one another by as little as a single nucleotide. Double-stranded probes were used in real-time nucleic acid amplifications as either probes or as primers. In addition to its extreme specificity and flexibility, the new class of probes is simple to design and synthesize, has low cost and high sensitivity and is accessible to a wide range of labels. This class of probes should find applications in a variety of areas wherever high specificity of nucleic acid hybridization is relevant.