Method of attaching adaptors to single-stranded regions of double-stranded polynucleotides
AssigneeOxford Nanopore Technologies PLC
InventorsDaniel George Fordham, Phillip Laurence James
Life of this patent
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Abstract
A method of attaching an adapter to a polynucleotide comprising: providing a double stranded polynucleotide comprising a single stranded break point within its polynucleotide sequence; contacting said double stranded polynucleotide with an exonuclease to form a single stranded region initiated at said break point; attaching an adapter to said single stranded region.
Claims28
A method of attaching an adapter to a polynucleotide comprising:
- (a) providing a double stranded polynucleotide comprising a single stranded break point within its polynucleotide sequence;
- (b) contacting said double stranded polynucleotide with an enzyme having exonuclease activity to form a single stranded region initiated at said break point;
- (c) hybridising an adapter to said single stranded region;
- (d) covalently attaching only the 3′ end of the adapter to the double stranded region of the polynucleotide adjacent to the single stranded region, wherein the 3′ end is covalently attached to the free 5′ end of the double stranded region.
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A method of characterising a polynucleotide, comprising:
- attaching an adapter to a polynucleotide by providing a double stranded polynucleotide comprising a single stranded break point within its polynucleotide sequence;
- contacting said double stranded polynucleotide with an exonuclease to form a single stranded region initiated at said break point;
- attaching an adapter to said single stranded region;
- and attaching a sequencing adapter to the adapter attached to the polynucleotide;
- contacting the adapted polynucleotide with a nanopore such that the polynucleotide translocates through the nanopore;
- and taking one or more measurements as the polynucleotide moves with respect to the nanopore, wherein the measurements are indicative of one or more characteristics of the polynucleotide and thereby characterising the polynucleotide.
A method of amplifying a polynucleotide, comprising attaching an adapter to a polynucleotide by a method disclosed herein;
- hybridising a primer to the adapter attached to the polynucleotide;
- and carrying out an amplification reaction.
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Dependencies are inferred from the claim text.