Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

• US 10,597,713 B2
• Filed: 04/03/2018
• Issued: 03/24/2020
• Est. Priority Date: 07/25/2011
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Brown, Clive Gavin, Clarke, James Anthony, Hall, Graham, Harper, Gavin, Heron, Andrew John, White, James
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/944,365
• Publication Number:
  US 20180291441A1
• Time in Patent Office: 721 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 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

Abstract

The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

Claims

1. A method for nanopore sensing, the method comprising:

i) coupling a nucleic acid construct transiently to a membrane via a 3′

cholesterol TEG tether,

wherein the membrane comprises an amphiphilic layer that is a 1,2-diphytanoyl-glycero-3-phosphocholine lipid bilayer, and wherein a MspA transmembrane pore is present in the membrane, wherein the MspA transmembrane pore is a nanopore, and is configured to permit flow of ions through the transmembrane pore from one side of the membrane to the other side of the membrane,

wherein the construct comprises a target polynucleotide having a target portion, a leader, and the 3′

cholesterol TEG tether, wherein the construct is configured such that the leader facilitates engagement of the construct with the transmembrane pore while the construct is transiently coupled to the membrane, promoting passage of the nucleic acid construct through the nanopore, and

wherein the step of transiently coupling the construct to the membrane comprises contacting the membrane with the construct, wherein the construct comprises, prior to the contacting step, the target polynucleotide, the leader, and the 3′

cholesterol TEG tether; and

ii) sensing at least the target portion of the target polynucleotide upon movement of the construct through the nanopore, wherein sensing comprises reading nucleotides of the nucleic acid construct as the nucleotides pass through the nanopore in order to sequence the target portion, wherein the nucleotides are read from single channel currents obtained from electrodes connected to a buffered solution on cis and trans sides of the lipid bilayer comprising the nanopore.