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US 10,221,450 B2Granted
Enzyme stalling method
AssigneeOxford Nanopore Technologies PLC
InventorsAndrew John Heron, David Antoni Alves, James Anthony Clarke, Marion Louise Crawford, Daniel Ryan Garalde, Graham Hall, Daniel John Turner, James White
Life of this patent
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Abstract
The invention relates to new methods of moving helicases past spacers on polynucleotides and controlling the loading of helicases on polynucleotides. The invention also relates to new methods of characterizing target polynucleotides using helicases.
Claims12
A method comprising:
- (a) providing a complex comprising one or more helicases bound, in an active mode, to a polynucleotide, wherein the polynucleotide comprises one or more spacers that are configured such that movement of the one or more helicases relative to the polynucleotide is stalled:
- (b) contacting a transmembrane pore, which is present in a membrane, with the complex, wherein the transmembrane pore comprises an opening that permits passage of the polynucleotide, but not the one or more helicases, across the membrane;
- and (c) applying a potential difference across the membrane such that the polynucleotide moves through the transmembrane pore and such that the one or more helicases pass across the one or more spacers of the polynucleotide.
A method of comprising:
- (a) providing a target polynucleotide comprising one or more spacers that are configured such that movement of a helicase relative to the target polynucleotide is stalled;
- (b) contacting the target polynucleotide with one or more helicases such that the one or more helicases, in an active mode, bind to the target polynucleotide and stall at the one or more spacers;
- (c) contacting a transmembrane pore, which is present in a membrane, with the target polynucleotide and the one or more stalled helicases, wherein the transmembrane pore comprises an opening that permits passage of the target polynucleotide, but not the one or more helicases, across the membrane;
- and
- (d) applying a potential difference across the membrane such that the target polynucleotide moves through the transmembrane pore;
- and such that the one or more helicases pass across the one or more spacers of the target polynucleotide and control the movement of the target polynucleotide through the transmembrane pore.
3 more elements
Dependencies are inferred from the claim text.