Nucleic acid detection method
Inventors
Lamble, Henry John • Lloyd, David • Egan, Christopher
Assignees
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Publication Number
US-11591644-B2
Publication Date
2023-02-28
Expiration Date
Abstract
The present invention relates to methods for the detection of nucleic acids of defined sequence and kits for use in said methods. The methods employ nicking agent(s), polymerase and oligonucleotide probes to produce probe fragments in the presence of a target nucleic acid.
Core Innovation
A method is provided for detecting the presence of a target nucleic acid of defined sequence in a sample by producing a double-stranded nucleic acid amplifier. The method uses a first oligonucleotide probe (P1), a polymerase, a first nicking agent, and one or more modified nucleotides, and the double-stranded nucleic acid amplifier comprises a target derived strand containing at least one cleavage site for the first nicking agent and a probe (P1) derived strand.
Following production of the double-stranded nucleic acid amplifier, the first nicking agent specifically recognises the double-stranded nucleic acid amplifier and cleaves the target derived strand at the cleavage site to produce a primer that remains hybridised to the probe (P1) derived strand. The polymerase extends the primer to reproduce the double-stranded nucleic acid amplifier and displaces a target derived strand fragment (F1) that is 3′ of the cleavage site.
The displaced fragment (F1) is then contacted with a second oligonucleotide probe (P2) and a second nicking agent. The second oligonucleotide probe (P2) hybridises to fragment (F1) to produce a cleavage site for the second nicking agent, which specifically recognises the double-stranded nucleic acid formed when P2 hybridises to F1, cleaves P2 to produce a probe fragment (F2), and detection of F2 indicates the presence of the target nucleic acid in the sample.
The first and/or second nicking agent(s) may be a double-strand cleaving agent which functions as a nicking agent due to the double-stranded nucleic acid containing one or more modifications that render one of its strands resistant to cleavage. These modifications are integrated into the probe (P1) derived strand and/or into fragment (F1) by the polymerase using the one or more modified nucleotides.
Claims Coverage
The consolidated independent claim coverage describes a two-stage probe-based nucleic-acid detection workflow using sequential nicking and polymerase extension/displacement to produce a detectable probe fragment (F2). The claim includes four inventive features spanning amplifier generation, cleavage-site design, two-probe signal generation, and strand-resistance via modified nucleotides.
Double-stranded nucleic acid amplifier generation with P1, polymerase, and first nicking agent using modified nucleotides
Producing, in the presence of a target nucleic acid, a double-stranded nucleic acid amplifier comprising a target derived strand with at least one cleavage site for the first nicking agent and a probe (P1) derived strand, wherein P1 comprises a complementarity region capable of sequence specific hybridisation to the target nucleic acid; the method uses a polymerase, a first nicking agent, and one or more modified nucleotides.
Nicking and polymerase extension to create primer and displace target-derived fragment F1
Following production of the double-stranded nucleic acid amplifier, the first nicking agent specifically recognises the double-stranded nucleic acid amplifier and cleaves the target derived strand at the cleavage site to produce a primer that remains hybridised to the probe (P1) derived strand, and the polymerase extends the primer to reproduce the double-stranded nucleic acid amplifier and displaces a target derived strand fragment (F1) that is 3′ of the cleavage site.
P2 hybridisation to F1 and second nicking agent cleavage to produce detectable probe fragment F2
Contacting the fragment (F1) produced in the first stage with a second oligonucleotide probe (P2) and a second nicking agent, wherein P2 comprises a complementarity region capable of sequence specific hybridisation to fragment (F1) which produces a cleavage site for the second nicking agent upon hybridisation; the second nicking agent specifically recognises the double-stranded nucleic acid formed when P2 hybridises to F1 and cleaves P2 to produce a probe fragment (F2), and detecting the presence of probe fragment (F2) indicates the presence of the target nucleic acid.
Double-strand cleaving agent functioning as a nicking agent via modified nucleotides rendering one strand resistant to cleavage
Wherein the first and/or second nicking agent(s) is a double-strand cleaving agent which functions as a nicking agent due to the double-stranded nucleic acid containing one or more modifications that render one of its strands resistant to cleavage by said double-strand cleaving agent, with said modifications integrated into the probe (P1) derived strand and/or into fragment (F1) by the polymerase using the one or more modified nucleotides.
The claim coverage centers on the sequential combination of a first probe (P1) with polymerase and a first nicking agent to generate a cleavage-site-containing double-stranded nucleic acid amplifier, followed by nicking-driven primer extension and displacement to produce F1, then a second probe (P2) and second nicking agent cleavage to release a detectable probe fragment (F2) whose detection indicates the presence of the target nucleic acid. The claim also covers the use of modified nucleotides that render one strand resistant so that a double-strand cleaving agent can function as a nicking agent.
Stated Advantages
Provides a method in which the presence of a detected probe fragment (F2) indicates the presence of the target nucleic acid in the sample.
Uses sequence specific hybridisation of probes (P1 and P2) to the target nucleic acid and fragment (F1) to enable targeted cleavage and probe fragment production.
Employs modified nucleotides integrated by the polymerase so that a double-strand cleaving agent functions as a nicking agent through cleavage resistance of one strand.
Documented Applications
Detection of probe fragment (F2) using colorimetric formats including gold nanoparticles on magnetic beads, lateral flow with gold nanoparticles, lateral flow with carbon nanoparticles, and multiplex readouts (gel and lateral flow) for two or more targets, including 5-plex lateral flow.
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