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US 12,215,384 B2Granted
Methods of characterising target polynucleotides
AssigneeOxford Nanopore Technologies PLC
InventorsRichard Alexander Gutierrez, Andrew John Heron, James White
Life of this patent
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Abstract
The invention relates to a new method of characterising a target polynucleotide using a pore. The method involves controlling the formation of secondary structure by the target polynucleotide after the polynucleotide has moved through the pore.
Claims19
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.
2 more elements
A method characterising a target polynucleotide, wherein the target polynucleotide is double stranded and the method comprises:
- (a) providing a construct comprising the target polynucleotide in which the two strands of the target polynucleotide are linked at one end of the target polynucleotide by a hairpin loop;
- (b) providing a transmembrane pore in a membrane having a cis side and a trans side and carrying out steps (c)-(e) under one applied potential;
- (c) contacting, on the cis side of the membrane, the construct with the transmembrane pore and a molecular brake which separates the two strands of the construct and controls the movement of the construct through the transmembrane pore one strand at a time;
- (d) 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 construct moves through the transmembrane pore;
- and
- (e) 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 each strand of the target polynucleotide that has translocated to the trans side of the membrane as the molecular brake controls movement of the construct through the transmembrane pore one strand at a time.
3 more elements
Dependencies are inferred from the claim text.