Systems and methods for sequencing by hybridization

• US 20030064382A1
• Filed: 03/07/2002
• Published: 04/03/2003
• Est. Priority Date: 10/13/1998
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: Brown University
• Inventors: Oliver, John S., Preparata, Franco P., Upfal, Eliezer
• Granted Patent: US 7,071,324 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

The systems and methods described herein relate to nucleic acid probes comprising a pattern of universal and designate nucleotides (or analogs thereof), or ‘gapped’ probes, and the use of sets of gapped probes in sequencing by hybridization to determine the sequence of nucleic acid sequences. The inclusion of universal nucleotides in the probes allows for efficient and rapid sequencing of longer nucleotide sequences than can be sequenced using traditional probes. The systems and methods described herein also relate to apparatus for sequencing nucleic acids which include gapped probes, as well as computer systems capable of analyzing data generated using gapped probes in such apparatus.

Claims

1. A gapped probe, comprising a sequence of universal and designate nucleotides and/or nucleotide analogs ordered in a predetermined pattern, wherein the pattern has a reduced shift-overlap count.

2. The probe of claim 1, having a universal nucleotide selected from the group consisting of 5-nitroindole, 3-nitropyrrole and deoxyinosine.

3. The probe of claim 1, further comprising at least two contiguous designate nucleotides and/or nucleotide analogs bound to an end of the sequence.

4. The probe of claim 1, wherein at least one of the designate nucleotides and/or nucleotide analogs is a nucleotide analog.

5. The probe of claim 4, wherein the nucleotide analog is selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo[3.2.1]DNA, a bicyclo[3.3.0]DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

6. A gapped probe, comprising a sequence of universal and designate nucleotides and/or nucleotide analogs ordered in a pattern, wherein

(a) the pattern comprises a first string of universal nucleotides followed by a first segment, and a second string of universal nucleotides followed by a second segment,

(b) the first string and the second string each comprise two or more consecutive universal nucleotides;

(c) the first segment and the second segment each comprise a designate nucleotide; and

wherein at least one of the designate nucleotides is a nucleotide analog selected from the group consisting of;

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

7. An gapped probe, comprising a sequence of universal and designate nucleotides and/or nucleotide analogs ordered in a pattern, wherein the pattern comprises a root and an iterated unit, and wherein the length of the root is identical to the length of the iterated unit, and wherein the probe comprises at least one nucleotide analog selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a bicyclo[3.3.0]DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

8. A gapped probe of claim 7, wherein each iterated unit comprises a string of universal nucleotides and/or nucleotide analogs followed by one or more designate nucleotide and/or nucleotide analog.

9. A set of gapped probes, comprising a plurality of instances of a sequence of universal and designate nucleotides and/or nucleotide analogs ordered in a predetermined pattern, wherein the pattern has a reduced shift-overlap count.

10. The set of gapped probes of claim 9, comprising a universal nucleotide selected from the group consisting of 5-nitroindole, 3-nitropyrrole and deoxyinosine.

11. The set of gapped probes of claim 9, wherein the probes are displayed in a spatially defined array.

12. The set of gapped probes of claim 9, wherein the probes are displayed on a solid support.

13. The set of gapped probes of claim 9, wherein the probes are displayed on or in a semisolid matrix.

14. The set of gapped probes of claim 9, wherein the probes are displayed in a matrix of fluid-containing chambers.

15. The set of gapped probes of claim 9, wherein at least one of the nucleotides and/or nucleotide analogs is a nucleotide analog.

16. The set of gapped probes of claim 15, wherein the nucleotide analog is selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a bicyclo-DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

17. A probe array, comprising

a substrate, and

a set of gapped probes disposed thereon, wherein each probe comprises an instance of a predetermined pattern of universal and designate nucleotides and/or nucleotide analogs such that the set comprises a plurality of instances of the pattern.

18. The array of claim 17, wherein the predetermined pattern has a reduced shift-overlap count.

19. The array of claim 17, wherein the predetermined pattern is iterative.

20. The array of claim 17, having a universal nucleotide selected from the group consisting of 5-nitroindole, 3-nitropyrrole and inosine.

21. The array of claim 17, wherein each particular instance of a predetermined pattern is associated with a particular location on the array.

22. The array of claim 17, wherein each probe further comprises a sequence of at least two contiguous designate nucleotides bound to an end of the pattern.

23. The array of claim 17, wherein at least one of the nucleotides and/or nucleotide analogs is a nucleotide analog.

24. The array of claim 23, wherein the nucleotide analog is selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a bicyclo-DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

25. A set of gapped probes, comprising a plurality of instances of a sequence of universal and designate nucleotides and/or nucleotide analogs ordered in a predetermined pattern, wherein the each gapped probe of the set comprises greater than five nucleotides and/or nucleotide analogs.

26. The set of gapped probes of claim 25, wherein each gapped probe of the set comprises greater than ten nucleotides and/or nucleotide analogs.

27. A method for analyzing a nucleic acid sequence, comprising

providing a set of probes wherein each probe comprises an instance of a pattern of universal and designate nucleotides and/or nucleotide analogs such that the set comprises a plurality of instances of the pattern,

determining a spectrum of probes representative of the probes in the set of probes which hybridize to a test sequence, and

ordering the spectrum of probes to determine a sequence of a portion of the test sequence.

32. The method of claim 27, wherein the predetermined pattern has a reduced shift-overlap count.

33. The method of claim 27, wherein each probe comprises greater than five nucleotides and/or nucleotide analogs.

34. The method of claim 27, wherein each probe comprises greater than ten nucleotides and/or nucleotide analogs.

35. The method of claim 27, wherein:

(a) the pattern comprises a first string of universal nucleotides and/or nucleotide analogs followed by a first segment, and a second string of universal nucleotides and/or nucleotide analogs followed by a second segment,

(b) the first string and the second string each comprise two or more consecutive universal nucleotides and/or nucleotide analogs;

(c) the first segment and the second segment each comprise a designate nucleotide and/or nucleotide analog.

36. The method of claim 27, wherein at least one of the nucleotides and/or nucleotide analogs is a nucleotide analog.

38. The method of claim 36, wherein the nucleotide analog is selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a bicyclo-DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.

28. A method for ordering a spectrum of probes to determine a sequence of a portion of a test sequence, comprising

i) providing a spectrum of probes that hybridize to a test sequence, wherein each probe in the spectrum is an instance of a pattern of universal and designate nucleotides and/or nucleotide analogs, which pattern requires a designate nucleotide and/or nucleotide analog at an mth position and an nth position,

ii) identifying a first subset of probes from the spectrum whose first m−

1 nucleotides and/or nucleotide analogs correspond to a last m−

1 nucleotides of a growing sequence,

iii) appending the nucleotide at the mth position to the growing sequence if a single nucleotide and/or nucleotide analog occurs at the mth position of all probes in the first subset.

29. The method of claim 28, further comprising

iv) if two or more nucleotides and/or nucleotide analogs occur at the mth position of the probes in the first subset, designating a new growing sequence for each of the nucleotides and/or nucleotide analogs which occur at the mth position of the probes in the first subset, and

v) repeating the steps of identifying and appending for each new growing sequence until the step of identifying identifies zero probes.

30. The method of claim 29, further comprising

vi) if two or more nucleotides and/or nucleotide analogs occur at the mth position of the probes in the first subset, selecting a second subset of probes from the spectrum whose first n−

1 nucleotides and/or nucleotide analogs correspond to a last n−

1 nucleotides of the growing sequence, and

vii) appending a nucleotide to the growing sequence that uniquely occurs at the mth position of the probes in the first subset and at the nth position of the probes in the second subset.

31. The method of claim 30, further comprising

viii) if two or more nucleotides and/or nucleotide analogs occur at the mth position of the probes in the first subset and at the nth position of the probes in the second subset, designating a new growing sequence for each of the nucleotides and/or nucleotide analogs which occur at the mth position of the probes in the first subset and at the nth position of the probes in the second subset, and

ix) repeating steps ii) to vii) for each new growing sequence until the step of identifying identifies zero probes.

37. The method of claim 28, wherein at least one of the nucleotides and/or nucleotide analogs is a nucleotide anaolog.

39. The method of claim 37, wherein the nucleotide analog is selected from the group consisting of:

a peptide nucleic acid, pyranosyl-RNA, a hexitol nucleic acid, a mannitol nucleic acid, an altritol nucleic acid, a 2′

-5′

nucleic acid, a locked nucleic acid, a seco-locked nucleic acid, a bicyclo-DNA, a bicyclo-DNA, a tricyclo-DNA, 3-hydroxy-N-acetylprolinol substituted nucleic acid, a carbocyclic nucleic acid, a carbocyclic/bicyclic nucleic acid, a nucleic acid with a triazole backbone, a nucleic acid with an imidazole backbone, a 1-phenylserinol nucleic acid, a nucleic acid with an alpha anomer backbone, and a metal-linked nucleic acid.