Sequencing kit

Patent metadata

Field	Value
Publication number	US-12553085-B2
Application number	US-202318352701-A
Country	US
Kind code	B2
Filing date	Jul 14, 2023
Priority date	Sep 29, 2016
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

The invention provides a method of detecting a target polynucleotide in a sample comprising: (a) contacting the sample with a guide polynucleotide that binds to a sequence in the target polynucleotide and a polynucleotide-guided effector protein, wherein the guide polynucleotide and polynucleotide-guided effector protein form a complex with any target polynucleotide present in the sample; (b) contacting the sample with a membrane comprising a transmembrane pore; (c) applying a potential to the membrane; and (d) monitoring for the presence or absence of an effect resulting from the interaction of the complex with the transmembrane pore to determine the presence or absence of the complex, thereby detecting the target polynucleotide in the sample.

First claim

The invention claimed is:

1 . A kit comprising:
(a) a guide polynucleotide comprising:
(i) a nucleotide sequence that binds to a sequence in a target polynucleotide,
(ii) a nucleotide sequence that binds to a polynucleotide guided effector protein, and
(iii) an adaptor comprising a leader sequence and/or an anchor capable of coupling to a membrane, and
(b) a polynucleotide-guided effector protein.

2 . The kit of claim 1 , further comprising a polynucleotide binding protein, a microparticle, a membrane, and/or a transmembrane pore.

3 . The kit of claim 1 , wherein the guide polynucleotide is a guide RNA.

4 . The kit of claim 3 , wherein the guide RNA comprises a crRNA and a tracrRNA, and
wherein the anchor or the adaptor is present at the 5′ end of the tracrRNA, the 3′ end of the tracrRNA, or the 3′ end of the crRNA.

5 . The kit of claim 1 , wherein the anchor is a polypeptide anchor or a hydrophobic anchor.

6 . The kit of claim 5 , wherein the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, cholesterol, palmitate, or tocopherol.

7 . The kit of claim 1 , wherein the adaptor comprises a barcode.

8 . The kit of claim 1 , wherein the polynucleotide-guided effector protein is a RNA-guided effector protein.

9 . The kit of claim 8 , wherein the RNA-guided effector protein is a Cas, Cpf1, or C2c2 protein.

10 . The kit of claim 9 , wherein the Cas protein is a Cas9 protein.

11 . A kit comprising:
(a) a first guide polynucleotide comprising:
(i) a nucleotide sequence that binds to a first target sequence,
(ii) a nucleotide sequence that binds to a polynucleotide guided effector protein, and
(iii) an adaptor comprising a leader sequence;
(b) a second guide polynucleotide comprising:
(i) a nucleotide sequence that binds to a second target sequence,
(ii) a nucleotide sequence that binds to a polynucleotide guided effector protein, and
(iii) an anchor capable of coupling to a membrane; and
(c) a polynucleotide-guided effector protein.

12 . The kit of claim 11 , wherein the first target sequence and the second target sequence are nucleic acid sequences belonging to the same target polynucleotide.

13 . The kit of claim 11 , wherein the first target sequence and the second target sequence are nucleic acid sequences belonging to different target polynucleotides.

14 . The kit of claim 11 , further comprising a polynucleotide binding protein, a microparticle, a membrane, and/or a transmembrane pore.

15 . The kit of claim 11 , wherein the first guide polynucleotide and/or the second guide polynucleotide is a guide RNA.

16 . The kit of claim 15 , wherein the guide RNA comprises a crRNA and a tracrRNA, and
wherein the anchor or the adaptor is present at the 5′ end of the tracrRNA, the 3′ end of the tracrRNA, or the 3′ end of the crRNA.

17 . The kit of claim 11 , wherein the anchor is a polypeptide anchor or a hydrophobic anchor.

18 . The kit of claim 11 , wherein the adaptor comprises a barcode.

19 . The kit of claim 11 , wherein the polynucleotide-guided effector protein is a RNA-guided effector protein.

20 . The kit of claim 19 , wherein the RNA-guided effector protein is a Cas, Cpf1, or C2c2 protein.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Heron Andrew John
• Graham James Edward
• Gutierrez Richard Alexander
• Bowen Rebecca Victoria
• White James
• Brown Clive Gavin
• Fordham Daniel George

Classifications

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title