Coupling method
The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.
1. A method of preparing a double-stranded target polynucleotide for nanopore sequencing, the method comprising:
(a) obtaining a target polynucleotide;
(b) hybridizing a primer with a single stranded leader to the target polynucleotide, extending the primer using a polymerase to copy the target polynucleotide;
(c) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(d) applying a potential difference across the membrane, wherein the polynucleotide is captured by the nanopore via the leader; and
(e) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the nanopore.
2. The method of claim 1 , wherein (b) is repeated by polymerase chain reaction (PCR).
3. The method of claim 1 , wherein the primer comprises a complementary section to the target polynucleotide and a homopolymer section.
4. The method of claim 3 , wherein the primer prevents the polymerase from extending beyond the homopolymer section.
5. The method of claim 4 , wherein the leader comprises a single-stranded polyT section.
6. The method of claim 1 , wherein the membrane is an amphiphilic layer, a lipid bilayer, or a solid state layer.
7. The method of claim 1 , further comprises contacting the membrane with the target polynucleotide, wherein the target polynucleotide is tethered to the membrane via an anchor.
8. The method of claim 7 , wherein the anchor is a hydrophobic anchor selected from a group consisting of a lipid, a fatty acid, a sterol, a carbon nanotube, and an amino acid.
9. The method of claim 7 , wherein the hydrophobic anchor is capable of embedding in the membrane.
10. The method of claim 7 , wherein the polynucleotide is tethered transiently to the membrane.
11. The method of claim 1 , wherein the target polynucleotide is detected based on ion flow through the nanopore that is measured via an electrical means.
12. The method of claim 1 , wherein the polynucleotide is captured by the nanopore via the leader by a DNA handling protein.
13. The method of claim 12 , wherein the DNA handling protein is an exonuclease or a polymerase.
14. The method of claim 1 , wherein the polynucleotide is detected based on ion flow through the nanopore by measuring a current passing through the nanopore.
15. The method of claim 1 , wherein the target polynucleotide is a fragment of genomic DNA.
16. A method for strand sequencing of a double-stranded target polynucleotide, the method comprising:
(a) obtaining a target polynucleotide;
(b) hybridizing a primer with a single stranded leader to the target polynucleotide, extending the primer using a polymerase to copy the target polynucleotide;
(c) providing a membrane in which is present a nanopore that provides a channel through the membrane;
(d) applying a potential difference across the membrane, wherein the polynucleotide is captured by the nanopore via the leader; and
(e) measuring one or more characteristics of the target polynucleotide as the target polynucleotide moves through the nanopore.
17. The method of claim 16 , further comprises contacting the membrane with the target polynucleotide, wherein the target polynucleotide is tethered to the membrane via an anchor.