As a solution, Wyss Institute researchers have developed “ Toehold Probes, ” a deoxyribonucleic acid and RNA detection engineering that is based on specifically engineered nucleic acid sequences. By programming DNA and RNA probes with highly predictable thermodynamic hybridization behaviors, the approach enables the detection of sequences of concern with single-nucleotide preciseness. Toehold Probes besides display high temperature robustness, which allows them to be multiplexed in a individual reaction .
This animation explains how toehold probes consisting of a “probe strand” and a “protector strand” are assembled and how they leverage thermodynamic principles to allow the specific detection of a correct target sequence, or to prevent them from detecting a spurious target sequence that can differ from the correct target sequence by only a single non-matching nucleotide. Credit: Wyss Institute at Harvard University
Their alone advantages are based on the Toehold principle : Toehold Probes contain two strands of deoxyribonucleic acid that are hybridized to each other due to complementary of their nucleotide sequences. One, the “ probe strand ” is besides complementary to a aim sequence, for exercise, in the human genome, while the second “ defender chain ” copies part of the target DNA. Toeholds—short sequences at the ends of the probe strand that are either complementary to the target sequence or the defender strand—initiate two exchange reactions. These either result in the probe maroon being specifically bound to its target DNA/RNA ( to allow its signal detection ) and the defender fibril being released ; or, in reversion, in the probe strand re-engaging with the defender ground and leaving the target DNA/RNA behind. The two competing substitute reactions lead to an balance that is highly predictable and highly sensitive to perturbations such that the presence of a unmarried non-matching nucleotide ( a random variable ) in the target sequence prevents its detection. In addition, Toehold Probes can robustly operate in much broader temperature windows than ceremonious probes, which allows them to be multiplexed.
Toehold probes with their robust single-nucleotide specificities can be leveraged in versatile diagnostic and research applications, including polymerase range reaction ( PCR ), DNA/RNA sequencing-based methods and one cell fluorescence in situ hybridization ( FISH ) techniques for the detection of rare diseased and other sequence variants. The wyoming Institute-launched start-up companies NuProbe and Torus Biosystems are developing toehold probe technology as new preciseness diagnostics for, respectively, cancer and infectious disease medicine .
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