Analysis of a polynucleotide via a nanopore system
A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.
1. A method of expanding a target polynucleotide that comprises at each nucleotide position a member of a set of different nucleotides, the method comprising:
expanding, by a technique employing a restriction enzyme, each member of the set of different nucleotides in the target polynucleotide in order of its nucleotide position into an ordinally corresponding expanded unit to form an expanded polynucleotide, wherein each expanded unit comprises:
i) a clock nucleotide sequence, the clock nucleotide sequence being common among expanded units of the expanded polynucleotide; and
ii) at least one signal nucleotide, the at least one signal nucleotide being indicative of the member of the set of different nucleotides at the particular nucleotide position;
wherein the clock nucleotide sequence comprises:
1) a plurality of nucleotides that is a restriction site for the restriction enzyme and
2) at least one further nucleotide.
2. A method according to claim 1 , wherein the at least one signal nucleotide consists of a single signal nucleotide.
3. A method according to claim 2 , wherein the single signal nucleotide has the same identity as the member of the set of different nucleotides at the particular nucleotide position.
4. A method according to claim 1 , wherein the at least one signal nucleotide consists of a signal nucleotide sequence of plural signal nucleotides, wherein the signal nucleotide sequence is indicative of the member of the set of different nucleotides at the particular nucleotide position.
5. A method according to claim 4 , wherein one of the plural signal nucleotides has the same identity as the member of the set of different nucleotides at the particular nucleotide position.
6. A method according to claim 4 or 5 , wherein the clock nucleotide sequence and the signal nucleotide sequence are in a predetermined order in which the signal nucleotide sequence is contiguous.