US 12,215,384 B2
Methods of characterising target polynucleotides
Richard Alexander Gutierrez, Oxford (GB); Andrew John Heron, Oxford (GB); and James White, Oxford (GB)
Assigned to Oxford Nanopore Technologies PLC, Oxford (GB)
Appl. No. 16/304,077
Filed by Oxford Nanopore Technologies PLC
PCT Filed May 25, 2017, PCT No. PCT/GB2017/051491
§ 371(c)(1), (2) Date Nov. 21, 2018,
PCT Pub. No. WO2017/203268, PCT Pub. Date Nov. 30, 2017.
Claims priority of application No. 1609241 (GB), filed on May 25, 2016; and application No. 1609436 (GB), filed on May 27, 2016.
Prior Publication US 2019/0203288 A1, Jul. 4, 2019
Int. Cl. C12Q 1/6869 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 2565/631 (2013.01)] 19 Claims
 
1. A method of characterising a target polynucleotide, comprising:
(a) providing a transmembrane pore in a membrane having a cis side and a trans side and carrying out steps (b)-(d) under one applied potential;
(b) contacting, on the cis side of the membrane, the target polynucleotide with the transmembrane pore and a molecular brake which controls the movement of a strand of the target polynucleotide through the transmembrane pore;
(c) taking one or more measurements as the molecular brake controls the movement of the strand through the transmembrane pore, wherein the one or more measurements are indicative of one or more characteristics of the target polynucleotide as the target polynucleotide moves through the transmembrane pore; and
(d) decreasing formation of secondary structure by the target polynucleotide by using an endonuclease on the trans side of the membrane to cleave a portion of the strand of the target polynucleotide that has translocated to the trans side of the membrane as the molecular brake controls movement of the strand of the target polynucleotide through the transmembrane pore.