Biopolymer sequencing by hybridization of probes to form ternary complexes and variable range alignment

• US 8,278,047 B2
• Filed: 10/01/2008
• Issued: 10/02/2012
• Est. Priority Date: 10/01/2007
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John, Bready, Barrett, Goldstein, Peter, Preparata, Franco
• Primary Examiner(s): Clow, Lori A
• Application Number:
  US12/243,451
• Publication Number:
  US 20090099786A1
• Time in Patent Office: 1,462 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

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.

Claims

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.