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US 12,503,729 B2Granted
Enzyme method
AssigneeOxford Nanopore Technologies PLC
InventorsRuth Moysey, Andrew John Heron
Life of this patent
Estimating the term…
Abstract
The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.
Claims20
A method of characterising a target polynucleotide that is partially translocated through a transmembrane pore having a cis side and a trans side, comprising:
- (a) contacting the target polynucleotide with a first helicase capable of controlling movement of a polynucleotide and located on the cis side of the transmembrane pore, such that the first helicase controls the movement of the target polynucleotide through the transmembrane pore from the trans side to the cis side, wherein the polynucleotide comprises a spacer connected to a label or tag, and wherein a potential difference is applied across the transmembrane pore;
- (b) further contacting the target polynucleotide with a second helicase capable of controlling movement of a polynucleotide and located on the cis side of the transmembrane pore as the first helicase controls the movement of the target polynucleotide through the transmembrane pore, wherein the second helicase binds to an internal polynucleotide of the target polynucleotide at a location on the polynucleotide different than where the first helicase is bound without concurrent binding to a terminal nucleotide of the target polynucleotide, and wherein the second helicase controls movement of the target polynucleotide through the transmembrane pore from the trans side to the cis side after the first helicase disengages from the target polynucleotide;
- and
- (c) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the transmembrane pore;
- thereby characterising the target polynucleotide.
2 more elements
A method comprising:
- (a) applying a potential difference across a transmembrane pore having a cis side and a trans side such that a target polynucleotide moves from the cis side to the trans side, wherein the polynucleotide comprises a spacer connected to a label or tag;
- (b) controlling movement of the target polynucleotide, with a first helicase capable of controlling movement of a polynucleotide, through the transmembrane pore from the trans side to the cis side, wherein the first helicase is bound to the target polynucleotide on the cis side;
- (c) measuring one or more characteristics of a portion of the target polynucleotide as the target polynucleotide moves through the transmembrane pore from the trans side to the cis side, thereby characterising the target polynucleotide;
- (d) further contacting the target polynucleotide, on the cis side of the transmembrane pore, with a second helicase capable of controlling movement of a polynucleotide as the first helicase controls the movement of the target polynucleotide through the transmembrane pore, wherein the second helicase binds to an internal polynucleotide of the target polynucleotide at a location on the polynucleotide different than where the first helicase is bound without concurrent binding to a terminal nucleotide of the target polynucleotide, and wherein the second helicase controls movement of the target polynucleotide through the transmembrane pore from the trans side to the cis side after the first helicase disengages from the target polynucleotide;
- and
- (e) measuring one or more characteristics of the same portion of the target polynucleotide as measured in (c) as the target polynucleotide moves through the transmembrane pore from the trans side to the cis side, thereby re-characterising the target polynucleotide.
3 more elements
Dependencies are inferred from the claim text.