Nucleic acid detection method
Inventors
Lamble, Henry John • Egan, Christopher • Lloyd, David • Dunski, Eryk
Assignees
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Publication Number
US-11390909-B2
Publication Date
2022-07-19
Expiration Date
Abstract
The present invention relates to methods for the detection of nucleic acids of defined sequence, and compositions and kits for use in said methods. The methods employ nicking agent(s) and a sequential series of oligonucleotide probes to produce probe fragments in the presence of a target nucleic acid.
Core Innovation
The invention relates to detecting the presence of a target nucleic acid of defined sequence in a sample by sequential hybridisation and cleavage. The method contacts the sample with a first oligonucleotide probe and a nicking agent, where the first probe has a first complementarity region capable of sequence specific hybridisation to the target nucleic acid and a cleavage site for the nicking agent. The nicking agent recognises double-stranded nucleic acid formed by hybridisation and cleaves the first oligonucleotide probe to produce a first probe fragment.
The produced first probe fragment is then contacted with a second oligonucleotide probe and a nicking agent. The second probe includes a second complementarity region capable of sequence specific hybridisation to the first probe fragment and a cleavage site for the nicking agent, and the nicking agent cleaves the second oligonucleotide probe to produce a second probe fragment. The method can optionally include contacting the second probe fragment with a third oligonucleotide probe and a nicking agent, and repeating further sequential rounds using fourth and subsequent sequential (3+n)th oligonucleotide probes.
The method detects the presence of probe fragments produced at the end of the sequential steps as an indicator that the target nucleic acid is present. In the described approach, one or more detected probe fragments is not capable of sequence specific hybridisation to the complementarity region of any preceding oligonucleotide probes to form a site recognised and cleaved by any of the nicking agents, and/or none of the probe fragments produced is capable of sequence specific hybridisation to the first complementarity region of the first oligonucleotide probe. The presence of the detected fragment(s) indicates the presence of the target nucleic acid in the sample.
The patent document further demonstrates embodiments where the sequential probe-cleavage detection approach operates with target nucleic acids derived from double-stranded DNA, including exposing single-stranded target and generating cleavable targets, and also extension to single-stranded RNA targets including direct nicking and restriction-based approaches. Detection embodiments are described using colorimetric and nucleic acid lateral flow formats, including the use of gold/carbon particles and enzyme-conjugated probes, and examples including multiplex detection for bacterial and viral targets.
Claims Coverage
The document provides two independent claims. Both independently define a sequential, stepwise hybridisation of oligonucleotide probes to generate double-stranded nucleic acid recognition sites for nicking agents, followed by detection of the final probe fragment(s), with one claim also requiring solid material attachments and colorimetric detection.
Sequential oligonucleotide probe hybridisation with nicking-agent cleavage and fragment-based target indication
A method for detecting the presence of a target nucleic acid of defined sequence by contacting the sample with a first oligonucleotide probe and a nicking agent, where hybridisation forms double-stranded nucleic acid specifically recognised by the nicking agent and cleaves the first probe to produce a first probe fragment; contacting the first probe fragment with a second oligonucleotide probe and a nicking agent, where hybridisation forms double-stranded nucleic acid specifically recognised by the nicking agent and cleaves the second probe to produce a second probe fragment; optionally repeating sequential probe rounds; and detecting the presence of the probe fragment produced at the end of the final step(s), wherein detected probe fragment(s) is not capable of sequence specific hybridisation to the complementarity region of preceding oligonucleotide probes to form a site recognised and cleaved by the nicking agents, and/or none of the probe fragments produced is capable of sequence specific hybridisation to the first complementarity region of the first oligonucleotide probe, wherein presence indicates the target nucleic acid in the sample.
Solid-material sequential probe format with colorimetric output from cleaved final probe fragment
A method for detecting the presence of a target nucleic acid of defined sequence by contacting the sample with a first oligonucleotide probe attached to a solid material and a nicking agent, wherein hybridisation forms double-stranded nucleic acid specifically recognised by the nicking agent and cleaves the first probe to release a first probe fragment from the solid material; contacting the released first probe fragment with a second oligonucleotide probe attached to a solid material and a nicking agent, wherein hybridisation forms double-stranded nucleic acid specifically recognised by the nicking agent and cleaves the second probe to release a second probe fragment from the solid material; contacting the released second probe fragment with a third oligonucleotide probe attached to a solid material and attached to a colorimetric dye or a moiety capable of attachment to a colorimetric dye, and a nicking agent, wherein hybridisation forms double-stranded nucleic acid specifically recognised by the nicking agent and cleaves the third probe to release a third probe fragment attached to the colorimetric dye or moiety capable of attachment to a colorimetric dye from the solid material, wherein the third probe fragment is not capable of sequence specific hybridisation to the complementarity region of any one of the preceding oligonucleotide probes to form a site specifically recognised and cleaved by any of the nicking agents; and detecting the presence of the third probe fragment at the end of the step by colorimetric detection where the presence of a colorimetric signal indicates the presence of the target nucleic acid.
Across the independent claims, the core claimed coverage is a sequential chain of probe hybridisation to form double-stranded nucleic acid sites recognised by nicking agents, cleaving each bound probe to generate the next probe fragment, and then detecting the presence of the terminal fragment(s) under constraints that prevent further sequence specific hybridisation to preceding probe complementarity regions.
Stated Advantages
Intrinsic exponential signal amplification is described as controllable by probe number.
Increased specificity is described.
Universal or multiplexing capability is described via fixed probe sets with variation of the first probe.
Compatibility with multiple readouts is described, including gel/PAGE, lateral flow, colorimetric (including HRP and β-galactanase), fluorescence, and electrical detection.
Documented Applications
Detection of a target nucleic acid of defined sequence in a sample, with detection performed by gel/PAGE, nucleic acid lateral flow, and colorimetric detection including colorimetric nanoparticle/enzymes (e.g., HRP and β-galactanase).
Integrated detection use cases in which the presence of the target nucleic acid is indicated by a colorimetric signal and also by nucleic acid lateral flow.
Colorimetric detection embodiments, including a colorimetric signal indicating the presence of the target nucleic acid.
Nucleic acid lateral flow detection embodiments using probe fragment(s).
Multiplex detection examples including bacterial and viral targets using lateral flow strips.
Detection of target nucleic acids derived from double-stranded DNA (including restriction digestion exposing single-stranded target, and detection based on probe cleavage).
Extension to single-stranded RNA targets, including direct nicking with Nt.CviPII and restriction-based processing with Avail.
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