Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

Patent metadata

Field	Value
Publication number	US-11261487-B2
Application number	US-202016782350-A
Country	US
Kind code	B2
Filing date	Feb 5, 2020
Priority date	Jul 25, 2011
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

The claimed invention relates to a method of processing a polynucleotide, by obtaining a sense polynucleotide strand comprising a homopolymeric region that is longer that the reading section of a nanopore; synthesizing an antisense polynucleotide strand under conditions in which a nucleotide analog is incorporated at random in a reverse complement of the homopolymer region, such that the length of the homopolymer region in the antisense polynucleotide strand is shorter than the reading section of the nanopore; and moving the antisense polynucleotide strand through the nanopore such that a proportion of the antisense polynucleotide strand interacts with the nanopore.

First claim

The invention claimed is:

1. A method of processing a polynucleotide, the method comprising:
a) obtaining a sense polynucleotide strand comprising a homopolymeric region than is longer that the reading section of a nanopore;
b) synthesizing an antisense polynucleotide strand under conditions in which a nucleotide analog is incorporated at random in a reverse complement of the homopolymer region, such that the length of the homopolymer region in the antisense polynucleotide strand is shorter than the reading section of the nanopore; and
(c) moving the antisense polynucleotide strand through the nanopore such that a portion of the antisense polynucleotide strand interacts with the nanopore.

2. The method of claim 1 , wherein the sense polynucleotide strand comprises
i) a sequence of nucleotides, wherein each nucleotide in the sequence is selected from a set of nucleotides; and
ii) the homopolymeric region, wherein the homopolymeric region comprising repeated instances of a member of the set of nucleotides, and
wherein step b) comprises combining a polymerase with the same set of nucleotides of step a) and a nucleotide analog, and maintaining the combination under conditions in which the nucleotide analog is incorporated at random in the reverse complement of the homopolymer region.

3. The method of claim 2 further comprising controlling the insertion rate of the nucleotide analog by varying the concentration of the nucleotide analog relative to the set of nucleotides.

4. The method of claim 1 , wherein the sense polynucleotide strand is connected to a single stranded leader.

5. The method of claim 4 , wherein the sense polynucleotide is connected to the single stranded leader via a stretch of abasic nucleotides.

6. The method of claim 1 , wherein the sense polynucleotide strand, in step a), comprises a hairpin loop having a free 3′ end.

7. The method of claim 6 , wherein the antisense polynucleotide strand is synthesized using a polymerase reaction that is nucleated from the free 3′ end of the hairpin loop.

8. The method of claim 1 , further comprising:
d) measuring the current passing through the nanopore during each interaction in step c) and thereby determining the sequence of the antisense polynucleotide strand.

9. The method of claim 8 , further comprising:
e) moving the sense polynucleotide strand through the nanopore such that a proportion of the sense polynucleotide strand interacts with the pore; and
f) measuring the current passing through the nanopore during each interaction in step e) thereby determining the sequence of the antisense polynucleotide strand.

10. The method of claim 9 , further comprising:
g) combining information from steps d) and f) to determine the length of the homopolymeric region.

Assignees

• Oxford Nanopore Tech Ltd
• Oxford Nanopore Tech Plc

Inventors

• Brown Clive Gavin
• Clarke James Anthony
• Hall Graham
• Harper Gavin
• Heron Andrew John
• White James

Classifications

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N27/44717

  Arrangements for investigating the separated zones, e.g. localising zones · 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

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title