Method of characterizing a polynucleotide

Patent metadata

Field	Value
Publication number	US-11920193-B2
Application number	US-201916972039-A
Country	US
Kind code	B2
Filing date	Jun 6, 2019
Priority date	Jun 6, 2018
Publication date	Mar 5, 2024
Grant date	Mar 5, 2024

Abstract

Methods of characterizing an analyte using a detector such as a nanopore and an enzyme are provided. One aspect features methods for characterizing a double-stranded polynucleotide using a detector, e.g., without using a hairpin connecting a template and a complement of the double-stranded polynucleotide. Another aspect features methods for characterizing an analyte using a tag-modified nanopore with increased sensitivity and/or higher throughput. Compositions and systems including, e.g., adaptors for attachment to double-stranded polynucleotides and tag-modified nanopores, which can be used in the methods are also provided.

First claim

The invention claimed is:

1. A method of characterizing a polynucleotide, the method comprising:
(i) combining in a solution:
a) a construct comprising a double-stranded polynucleotide, having a template strand and a complement strand, wherein the template strand and the complement strand are not covalently linked, with
b) a detector disposed in a membrane, wherein at least one tag that binds to the complement strand is conjugated to the detector; and
c) a polymerase and nucleotides;

(ii) processing the template strand using the polymerase to produce a product and by-products, the product comprising a third polynucleotide strand complementary to the template strand, wherein as the template strand of the double-stranded polynucleotide is processed, the complement strand becomes bound to the detector via the at least one tag conjugated to the detector; and
(iii) detecting the by-products through addition of each nucleotide to the third polynucleotide strand by the polymerase.

2. A method according to claim 1 , wherein after processing of the template strand by the polymerase, the complement strand of the construct is processed by a polymerase and the by-products of the processing of the complement strand are detected by the detector.

3. A method according to claim 1 , further comprising identifying a nucleotide sequence of the template strand based on the detecting in (iii).

4. A method according to claim 1 , wherein each type of nucleotide in the solution is distinguishably labelled according to the type of nucleotide.

5. A method according to claim 4 , wherein each type of nucleotide in the solution is distinguishably labelled with optical labels and/or polymer tags.

6. A method according to claim 1 , wherein the by-products of the processing in (ii) are sequentially released as nucleotides are sequentially added by the polymerase to the polynucleotide strand.

7. A method according to claim 6 , wherein the by-products of the processing step are labelled phosphate species.

8. A method according claim 1 , wherein the detector is selected from (i) a zero-mode waveguide, (ii) a field-effect transistor; (iii) an AFM tip; (iv) a nanotube; and (v) a nanopore.

9. A method according to claim 1 , wherein an adapter is attached to one or both of the two ends of the double-stranded polynucleotide prior to processing of the construct by the polymerase.

10. A method according to claim 1 , wherein the detector is a nanopore and the polymerase is provided within the lumen of the nanopore.

11. A method according to claim 1 , wherein the detector is a nanopore and the at least one tag that binds to the complement strand is conjugated to an outer rim of the nanopore.

12. A method according to claim 1 , wherein processing of the template strand by the polymerase reveals a portion of the complement strand for hybridization with the tag.

13. A method according to claim 1 , wherein an adapter comprising a duplex stem and a first single strand extending from the duplex stem is attached to at least one end of the double-stranded polynucleotide such that the first single strand of the adaptor is contiguous with the complement strand.

14. A method according to claim 1 , wherein the detector is a nanopore, wherein step (ii) comprises applying a potential difference across the nanopore so as to permit the by-products of the processing reaction to enter the nanopore; and the potential difference is maintained across the nanopore for a sufficient period of time so as to permit translocation of at least a portion of the by-products of the processing reaction through the nanopore.

15. The method according to claim 1 , further comprising
determining a sequence of the template strand based on changes measured in a property indicative of the by-products of the processing of the template strand by the polymerase,
determining a sequence of the complement strand based on changes measured in a property indicative of the by-products of the processing of the complement strand by a polymerase, and
comparing the sequence of the template strand with the sequence of the complement strand to establish a sequence of the polynucleotide.

16. A method according to claim 1 , wherein detecting the by-products of the processing involves measuring a property indicative of the by-products of the processing; and
wherein the polynucleotide is characterized based on a measured property of the by-products of the processing.

17. A method according to claim 16 , wherein the detector is a nanopore and the solution is ionic and the measured property is ion current flow through the nanopore and the polynucleotide is characterized based on the change in ionic current flow through the nanopore measured as the by-products of the processing translocate through the nanopore.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Clarke James Anthony
• White James
• Muscat Richard
• Johnson Jessica Mary May
• Nathani Ramiz Iqbal
• Heron Andrew John
• Bruce Mark John
• Jayasinghe Lakmal Nishantha
• Caprotti Domenico
• Stoddart David Jackson
• Bowen Rebecca Victoria
• Wright Christopher James
• Moody Paul Richard

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• 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