Methods for determining sequence profiles

• US 10,253,352 B2
• Filed: 11/17/2016
• Issued: 04/09/2019
• Est. Priority Date: 11/17/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: Nguyen, Michael, Tu, Eugene
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/354,941
• Publication Number:
  US 20170137873A1
• Time in Patent Office: 873 Days
• Field of Search: 435 912
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

Provided herein are methods for analyzing a signature sequence in a nucleic acid sample by rapid sequencing of a target nucleic acid region. The method examines the target nucleic acid directly and minimizes the number of examination steps needed to determine a signature that is characteristic of a genetic feature of the nucleic acid sample.

Claims

1. A method of detecting a preselected signature in a nucleic acid, comprising:

(a) incorporating a subset of nucleic acid base types into a primed nucleic acid to form an extended nucleic acid, wherein the subset of nucleic acid base types is complementary to at least one and no more than three nucleic acid base types in the primed nucleic acid, wherein incorporation of each of the individual nucleic acid base types is not detected among the subset of nucleic acid base types;

(b) contacting the extended nucleic acid with a polymerase and a nucleotide with a preselected base type different from the nucleic acid base types in the subset of step (a);

(c) detecting a ternary complex comprising the extended nucleic acid, the polymerase and the nucleotide with the preselected nucleic acid base type thereby detecting the preselected signature, wherein the nucleotide with the preselected base type is prevented from incorporating into the extended nucleic acid during the detecting;

(d) incorporating, after step (b), the nucleotide with the preselected based type into the extended nucleic acid;

(e) repeating steps (a) and (b) at least one time after step (d); and

(f) determining a number of times the preselected nucleic acid base type is detected to determine a number of times the preselected signature occurs.

2. The method of claim 1, wherein the primed nucleic acid comprises a plurality of repeat units.

3. The method of claim 2, wherein the number of times the ternary complex is detected is the number of repeat units within the primed nucleic acid.

4. The method of claim 1, further comprising a wash step prior to step (b).

5. The method of claim 1, wherein the nucleotide comprising the preselected nucleic acid base type in step (b) is labeled.

6. The method of claim 5, wherein the subset of nucleic acid base types comprises unlabeled nucleic acid base types.

18. The method of claim 1, wherein the subset of nucleic acid base types is complementary to at least two and no more than three nucleic acid base types in the primed nucleic acid.

19. The method of claim 1, wherein the subset of nucleic acid base types is complementary to at least three and no more than three nucleic acid base types in the primed nucleic acid.

20. The method of claim 1, wherein the nucleic acid base types that are incorporated in step (a) do not comprise exogenous labels.

7. A method of distinguishing target sequences, comprising:

(a) providing a primed nucleic acid that comprises a primer hybridized to a template strand, wherein the template strand comprises a candidate sequence selected from at least two known target sequences;

(b) contacting the primed nucleic acid with a polymerase and a subset of nucleic acid base types,

whereby the subset of nucleic acid base types incorporates into the primer to produce an extended primed nucleic acid;

(c) contacting the extended primed nucleic acid with a polymerase and a preselected nucleic acid base type different from the subset of nucleic acid base types,

whereby a ternary complex forms if the preselected nucleic acid base type is the next correct nucleotide for the extended primed nucleic acid product, and

wherein the ternary complex is stabilized to prevent incorporation of the preselected nucleic acid base type into the extended primed nucleic acid;

(d) acquiring a signal produced by the ternary complex; and

(e) repeating (b) through (d) at least one time to obtain a series of signals, wherein the series of signals comprises a signature that identifies the candidate sequence.

8. The method of claim 7, wherein the two known target sequences are different alleles of a single locus, and wherein one of the alleles is identical to or complementary to the preselected nucleic acid base type.

9. The method of claim 7, wherein the candidate sequence comprises an indel comprising at least one nucleic acid base type that is identical to or complementary to the preselected nucleic acid base type.

10. The method of claim 7, further comprising performing the method for a second primed nucleic acid that comprises a primer hybridized to a second template strand, wherein the template strand comprises a second candidate sequence selected from the at least two known target sequences, thereby obtaining a second set of signals.

11. The method of claim 10, further comprising comparing the second series of signals to the series of signals acquired in (d), thereby distinguishing the at least two known target sequences.

12. The method of claim 7, further comprising

(f) repeating (b) and (c) at least one time using a second subset of nucleic acid base types that differs from the subset of nucleic acid base types,

whereby the second subset of nucleic acid base types incorporates into the extended primed nucleic acid to produce a further extended primed nucleic acid, and

whereby a second ternary complex forms for the further extended primed nucleic acid product;

and

(g) acquiring a second signal produced by the second ternary complex, wherein the first and second signals comprises a signature that distinguishes the target sequence from another of the at least two known target sequences.

13. The method of claim 12, wherein the first preselected nucleic acid base type and the second preselected nucleic acid base type are complementary to different nucleic acid base types in the template strand.

14. The method of claim 12, wherein the first preselected nucleic acid base type and the second preselected nucleic acid base type are complementary to the same nucleic acid base type in the template strand.

15. The method of claim 7, wherein the same type of polymerase is used in step (b) and step (c).

16. A method of determining the presence of a repeat unit within a nucleic acid region in a sample, comprising:

(a) providing a primed nucleic acid that comprises a primer hybridized to a template strand with at least one repeat unit downstream of the primer;

(b) contacting the primed nucleic acid with a polymerase and a subset of nucleic acid base types,

whereby the subset of nucleic acid base types incorporates into the primer to produce an extended primed nucleic acid;

(c) contacting the extended primed nucleic acid with a polymerase and a preselected nucleic acid base type different from the subset of nucleic acid base types,

whereby a ternary complex forms if the preselected nucleic acid base type is the next correct nucleotide for the extended primed nucleic acid product, and

wherein the ternary complex is stabilized to prevent incorporation of the preselected nucleic acid based type into the extended primed nucleic acid; and

(d) detecting the ternary complex while preventing incorporation of the preselected nucleic acid base type into the extended primed nucleic acid, thereby determining the presence of the repeat unit within the nucleic acid region.

17. The method of claim 16, further comprising the steps of:

(f) repeating steps (b), (c), (d), and (e) at least one time until the ternary complex is no longer detected; and

(g) determining that the number of repeat units within the nucleic acid region is the same as the number of times the ternary complex is detected.