Immobilized nucleic acid complexes for sequence analysis
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.
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.
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.
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.
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.
12. The method of claim 1 , wherein the template localizing moiety comprises a synthetic polymer.
13. The method of claim 4 , 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.
15. The method of claim 4 , wherein the template localizing moiety is a surface-immobilized template localizing moiety.