Adaptors for nucleic acid constructs in transmembrane sequencing
AssigneeOxford Nanopore Technolgies PLC
InventorBrian Mckeown
Life of this patent
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Abstract
The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.
Claims23
1-34. (canceled)
A method of sequencing a double stranded nucleic acid template, comprising:
- (a) providing a construct in which the two strands of the double stranded nucleic acid template are covalently linked via an adaptor, wherein the adaptor comprises a region of double stranded nucleic acid which is formed by hybridization between two separate regions of a single stranded nucleic acid and comprises a hairpin loop;
- (b) denaturing the construct to form a single stranded construct;
- (c) contacting the construct with a transmembrane pore and a nucleic acid handling enzyme so that the enzyme provides controlled and stepwise translocation of the construct through the pore and a proportion of the nucleotides in the construct interacts with the pore;
- and
- (d) measuring the current passing through the pore during each interaction and thereby determining the sequence of the double stranded nucleic acid template.
A method of sequencing a double stranded nucleic acid template comprising:
- (a) providing a construct in which the two strands of the double stranded nucleic acid template are covalently linked via an adaptor, wherein the adaptor comprises a region of double stranded nucleic acid which is formed by hybridization between two separate regions of a single stranded nucleic acid and comprises a hairpin loop;
- (b) denaturing the construct to form a single stranded construct;
- (c) contacting the construct with a transmembrane pore having an exonuclease and a molecular adaptor covalently attached thereto so that the exonuclease digests an individual nucleotide from one end of the construct;
- (d) contacting the nucleotide with the pore so that the nucleotide interacts with the molecular adaptor;
- (e) measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide;
- and
- (f) repeating steps (c) to (e) at the same end of the construct and thereby determining the sequence of the double stranded nucleic acid template.
An adaptor for sequencing nucleic acids, which comprises a region of double stranded nucleic acid, wherein the adaptor is differentially selectable from another adaptor on the basis of differential or selective binding to a surface and wherein the region of double stranded nucleic acid is formed by hybridization between two separate regions of a single stranded nucleic acid and the adaptor comprises a hairpin loop.
Dependencies are inferred from the claim text.