Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment
Methods for sequencing a biopolymer by forming local ternary complexes along the length of the double-stranded biopolymer target molecule using one or more probes and obtaining information about the location of the probe(s) using a detector. These methods offer particular advantage when implemented with nanopore (including micropore) detection systems.
1. A method for determining a whole or partial sequence of a target biopolymer, the method comprising the steps of:
providing a double-stranded biopolymer target molecule;
contacting said target molecule or fragment thereof with a first probe having specificity for one or more first recognition sites of said target molecule, thereby forming one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof, said one or more first recognition sites each having a first known sequence, wherein the first probe is a member selected from the group consisting of an oligonucleotide, a peptide, and a polyamide;
contacting said target molecule or fragment thereof with a second probe having specificity for one or more second recognition sites of said target molecule, thereby forming one or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof, said one or more second recognition sites each having a second known sequence;
passing said double-stranded biopolymer target molecule or fragment thereof through a nanopore or channel;
detecting an electrical signal indicative of one or more locations of at least a subset of said one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof to determine a first probe map;
detecting an electrical signal indicative of one or more locations of at least a subset of said one or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof to determine a second probe map; and
determining said whole or partial sequence of said target biopolymer using at least said first probe map and said second probe map.
2. The method of claim 1 , wherein said first probe map comprises at least one of the following:
(a) relative positional information of two or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(b) absolute positional information of one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof; or
(c) error of positional information of one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof.
3. The method of claim 1 , wherein at least one of said first probe map or said second probe map comprises one or more of the following:
(a) relative positional information of two or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(b) relative positional information of two or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(c) relative positional information of one or more first local ternary complexes and one or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(d) absolute positional information of one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(e) absolute positional information of one or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof;
(f) error of positional information of one or more first local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof; or
(g) error of positional information of one or more second local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof.
4. The method of claim 1 , wherein said determining step comprises determining a spectrum map from at least said first probe map and said second probe map.
5. The method of claim 4 , wherein determining said spectrum map comprises using a combinatorial sequence reconstruction algorithm.
6. The method of claim 4 , wherein determining said spectrum map comprises determining a candidate sequence by ordering at least two probe sequences using positional information and error of positional information.
7. The method of claim 4 , wherein said first recognition site sequence and said second recognition site sequence overlap, and wherein determining said spectrum map comprises aligning said recognition site sequences.
8. The method of claim 1 , wherein the first probe is an n-mer probe, wherein n is an integer from 3 to 10.
9. The method of claim 1 , wherein the first probe comprises a gapped polyamide molecule.
10. The method of claim 1 , wherein the providing step comprises enzymatically synthesizing at least a portion of a second strand upon a single-stranded template.
11. The method of claim 1 , wherein the contacting step comprises binding the first probe to a minor groove of the target molecule or fragment thereof.
12. The method of claim 1 , wherein the first probe is a polyamide selected from the group consisting of a pyrrole and an imidazole.
13. The method of claim 12 , wherein the first probe is pyrrole or a substituted pyrrole.
14. The method of claim 13 , wherein the first probe is a member selected from the group consisting of pyrrole, hydroxyl-pyrrole, N-methylpyrrole, 3-hydroxy-N-methylpyrrole, N-methylpyrazole, and 3-methylthiophene.
15. The method of claim 12 , wherein the first probe is imidazole or a substituted imidazole.
16. The method of claim 15 , wherein the first probe is a member selected from the group consisting of imidazole, benzimidazole, hydroxybenzimidazole, N-methylimidazole, and imidazopyridine.
17. The method of claim 1 , wherein the first probe is a polyamide comprising a molecule containing a heterocyclic ring structure.
18. The method of claim 1 , further comprising:
fragmenting the double-stranded biopolymer target molecule prior to performing the contacting steps,
wherein determining said whole or partial sequence of said target biopolymer comprises constructing a probe map by matching a pattern between overlapping regions of multiple fragments to arrive at an aggregated hybridization pattern for each probe.
19. The method of claim 1 , wherein a sequence of said first recognition site and a sequence of said second recognition site overlap.
20. The method of claim 1 , wherein said first and second contacting steps are performed separately with different copies of the same target molecule.
21. The method of claim 1 , wherein the second probe is a member selected from the group consisting of an oligonucleotide, a peptide, and a polyamide.
22. A method for determining a whole or partial sequence of a target biopolymer, the method comprising the steps of:
providing a double-stranded biopolymer target molecule;
contacting said target molecule or fragment thereof with a plurality of n probes each having specificity for one or more recognition sites of said target molecule, thereby forming local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof, each of said recognition sites having a known sequence, wherein each of the n probes is individually a member selected from the group consisting of an oligonucleotide, a peptide, and a polyamide;
detecting at least a subset of said local ternary complexes along said double-stranded biopolymer target molecule or fragment thereof to determine one or more probe maps; and
determining said whole or partial sequence of said target biopolymer using at least said one or more probe maps.
23. The method of claim 22 , wherein said detecting step comprises passing said double-stranded biopolymer target molecule or fragment thereof through a nanopore and detecting an electrical signal indicative of locations of said local ternary complexes along a length of said double-stranded biopolymer target molecule, thereby determining said one or more probe maps.
24. The method of claim 23 , wherein said detecting step is repeated for each of said n probes.
25. The method of claim 22 , wherein at least one of the n probes is a polyamide selected from the group consisting of a pyrrole and an imidazole.
26. The method of claim 25 , wherein the at least one n probe is pyrrole or a substituted pyrrole.
27. The method of claim 26 , wherein the at least one n probe is a member selected from the group consisting of pyrrole, hydroxyl-pyrrole, N-methylpyrrole, 3-hydroxy-N-methylpyrrole, N-methylpyrazole, and 3-methylthiophene.
28. The method of claim 25 , wherein the at least one n probe is imidazole or a substituted imidazole.
29. The method of claim 28 , wherein the at least one n probe is a member selected from the group consisting of imidazole, benzimidazole, hydroxybenzimidazole, N-methylimidazole, and imidazopyridine.
30. The method of claim 22 , wherein at least one n probe is a polyamide comprising a molecule containing a heterocyclic ring structure.
31. The method of claim 22 , further comprising:
fragmenting the double-stranded biopolymer target molecule prior to performing the contacting step.
32. The method of claim 31 , wherein determining said whole or partial sequence of said target biopolymer comprises constructing a probe map by matching a pattern between overlapping regions of multiple fragments to arrive at an aggregated hybridization pattern for each probe.
33. The method of claim 22 , wherein said contacting step is performed separately for each of said n probes using different copies of the same target molecule.