Immobilized nucleic acid complexes for sequence analysis

• US 8,481,264 B2
• Filed: 09/18/2009
• Issued: 07/09/2013
• Est. Priority Date: 09/19/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Bibillo, Arek, Christians, Fred, Travers, Kevin, Emig, Robin
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/562,690
• Publication Number:
  US 20100075328A1
• Time in Patent Office: 1,390 Days
• Field of Search: 435/6, 435/91.2, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

Provided are methods for sequencing a nucleic acid that include fixing a template to a surface through a template localizing moiety and sequencing the nucleic acid with a sequencing enzyme, e.g. a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid fixed to a surface via a template localizing moiety, and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Compositions for sequencing reactions are provided. Also provided are sequencing systems comprising reaction regions in which or near which template localizing moieties are immobilized.

Claims

1. A method of sequencing a nucleic acid, the method comprising:

fixing a template nucleic acid to a solid surface through a template localizing moiety, wherein the template localizing moiety topologically encircles the template such that the template can move through the template localizing moiety;

sequencing a portion of at least one strand of the template nucleic acid with a first sequencing enzyme;

exchanging the first sequencing enzyme with a second sequencing enzyme; and

,

continuing sequencing of the strand with the second sequencing enzyme.

2. The method of claim 1, wherein the first sequencing enzyme is a first polymerase, the second sequencing enzyme is a second polymerase, and the template nucleic acid is, a circular template nucleic acid.

3. The method of claim 2, further comprising sequencing the template nucleic acid multiple times with a plurality of polymerases to generate a single nucleic acid strand comprising multiple copies of a polynucleotide complementary to the template nucleic acid.

7. The method of claim 1, wherein exchanging the first sequencing enzyme with the second sequencing enzyme comprises exchanging a photodamaged sequencing enzyme with a non-photodamaged sequencing enzyme.

8. The method of claim 1, wherein sequencing is continued with the second sequencing enzyme, wherein the second sequencing enzyme is non-photodamaged.

12. The method of claim 1, wherein the template localizing moiety comprises a synthetic polymer.

14. The method of claim 1, wherein the template localizing moiety is a surface-immobilized template localizing moiety.

4. A method of sequencing a template nucleic acid, the method comprising:

fixing a circular template nucleic acid to a solid surface through a template localizing moiety, wherein the template localizing moiety topologically encircles the template such that the template can move through the template localizing moiety;

annealing an oligonucleotide primer to the template nucleic acid;

initiating template-directed nascent strand synthesis by a polymerase that is not immobilized to the solid surface;

synthesizing a nascent strand complementary to the template nucleic acid with the polymerase

detecting incorporations of nucleotides into the nascent strand, wherein a temporal sequence of the incorporations is indicative of the sequence of the nucleic acid.

5. The method of claim 4, further comprising sequencing the template nucleic acid multiple times to generate a single nascent stand comprising multiple copies of a polynucleotide complementary to the template nucleic acid.

6. The of claim 4, wherein the polymerase is a plurality of polymerase enzymes, and further wherein only a single of the plurality is engaged in the template-directed nascent strand synthesis on the template nucleic acid at a given time.

9. The method of claim 4, the nucleotides comprise detectable labels that identify the base composition of the nucleotides.

10. The method of claim 4, wherein the incorporations are detected using a luciferase-mediated detection system.

11. The method of claim 4, wherein template nucleic acid is a single-stranded template nucleic acid.

13. The method of claim 4, wherein the template localizing moiety comprises a synthetic polymer.

15. The method of claim 4, wherein the template localizing moiety is a surface-immobilized template localizing moiety.