BIOPOLYMER SEQUENCING BY HYBRIDIZATION OF PROBES TO FORM TERNARY COMPLEXES AND VARIABLE RANGE ALIGNMENT

• US 20090099786A1
• Filed: 10/01/2008
• Published: 04/16/2009
• Est. Priority Date: 10/01/2007
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John, Goldstein, Peter, Bready, Barrett, Preparata, Franco
• Granted Patent: US 8,278,047 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• 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;

detecting 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; and

determining said whole or partial sequence of said target biopolymer using at least said first probe map.

2. The method of claim 1, 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 one or more locations of said one or more local ternary complexes along a length of said double-stranded biopolymer target molecule, thereby determining said first probe map.

3. 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.

4. The method of claim 1, further comprising the step of 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, wherein said detecting step further comprises detecting 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 wherein said determining step comprises determining said whole or partial sequence of said target biopolymer using at least said first probe map and said second probe map.

5. The method of claim 4, 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.

6. The method of claim 4, wherein said determining step comprises determining a spectrum map from at least said first probe map and said second probe map.

7. The method of claim 6, wherein determining said spectrum map comprises using a combinatorial sequence reconstruction algorithm.

8. The method of claim 6, 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.

9. The method of claim 6, 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.

10. The method of claim 1, wherein the first probe is an n-mer probe, wherein n is an integer from 3 to 10.

11. The method of claim 1, wherein the contacting step further includes contacting the target molecule or fragment thereof with the first probe in the presence of at least one of a recombinational protein or a recombinational enzyme, thereby facilitating hybridization of the first probe with the target molecule.

12. The method of claim 1, wherein the first probe comprises one or more moieties selected from the group consisting of a polyamide, a nucleic acid, and a peptide nucleic acid.

13. The method of claim 1, wherein the first probe comprises a gapped polyamide molecule.

14. 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.

15. 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.

16. 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;

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.

17. The method of claim 16, 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.

18. The method of claim 17, wherein said detecting step is repeated for each of said n probes.