Method and system for sequencing nucleic acids
Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents.
1. A method of determining the identity of the next correct nucleotide for a primed template nucleic acid molecule, the method comprising:
(a) providing a template nucleic acid molecule primed with a primer;
(b) serially contacting the primed template nucleic acid molecule with a plurality of reaction mixtures, each of the reaction mixtures comprising a polymerase and a different combination of two and only two or three and only three different test nucleotides, under conditions that stabilize ternary complexes comprising the primed template nucleic acid molecule, the polymerase and a next correct nucleotide, while precluding incorporation of any nucleotide into the primer;
(c) detecting interaction of the polymerase with the primed template nucleic acid molecule without chemical incorporation of any nucleotide into the primer of the primed template nucleic acid molecule, to determine whether ternary complexes form; and
(d) determining whether a test nucleotide that is common to at least two of the reaction mixtures is the next correct nucleotide for the primed template nucleic acid molecule using the detected interaction.
2. The method of claim 1 , wherein step (d) comprises determining whether a test nucleotide that is common to two of the reaction mixtures is the next correct nucleotide for the primed template nucleic acid molecule using the detected interaction.
3. The method of claim 2 , wherein four different reaction mixtures are serially contacted with the primed template nucleic acid molecule in step (b), wherein in aggregate each different nucleotide is present in two reaction mixtures.
4. The method of claim 1 , wherein step (d) comprises determining whether a test nucleotide that is common to three of the reaction mixtures is the next correct nucleotide for the primed template nucleic acid molecule using the detected interaction.
5. The method of claim 4 , wherein six different reaction mixtures are serially contacted with the primed template nucleic acid molecule in step (b), wherein in aggregate each different nucleotide is present in three reaction mixtures.
6. The method of claim 1 , wherein the conditions also destabilize binary complexes comprising the primed template nucleic acid molecule and the polymerase but not the next correct nucleotide.
7. The method of claim 1 , wherein the ternary complex is stabilized by the presence of a reversible terminator moiety on the 3′ terminal nucleotide of the primer.
8. The method of claim 7 , further comprising, after step (c), removing the reversible terminator moiety on the 3′ terminal nucleotide of the primer.
9. The method of claim 1 , wherein each of the test nucleotides is an unlabeled nucleotide.
10. The method of claim 9 , wherein the polymerase comprises an exogenous label that is detected in step (c).
11. The method of claim 1 , wherein the next correct nucleotide comprises an exogenous label that is detected in step (c).
12. The method of claim 1 , further comprising (e) incorporating a nucleotide at the 3′ end of the primer after step (c).
13. The method of claim 12 , wherein the nucleotide that is incorporated is an unlabeled nucleotide.
14. The method of claim 12 , wherein the nucleotide that is incorporated is an unlabeled reversible terminator nucleotide.
15. The method of claim 12 , further comprising repeating steps (b) through (e) to sequence the primed template nucleic acid molecule.
16. A method of sequencing a primed template nucleic acid, comprising:
(a) contacting a primed template nucleic acid with a polymerase and a first combination of two and only two or three and only three types of test nucleotides under conditions that form a stabilized ternary complex between the polymerase, primed template nucleic acid and a test nucleotide that is complementary to the next base of the primed template nucleic acid;
(b) detecting the ternary complex while precluding incorporation of test nucleotides into the primer;
(c) repeating steps (a) and (b) using the primed template nucleic acid, a polymerase and a second combination of two or three types of test nucleotides, wherein the second combination is different from the first combination;
(d) incorporating into the primer, after step (c), a nucleotide that is complementary to the next base; and
(e) repeating steps (a) through (d) to identify a sequence of the primed template nucleic acid.
17. The method of claim 16 , wherein the first combination comprises two types of test nucleotides.
18. The method of claim 17 , wherein the second combination comprises two types of test nucleotides.
19. The method of claim 18 , wherein steps (a) and (b) are carried out serially for four different combinations of two types of test nucleotides, wherein each different nucleotide type is contacted with the primed template nucleic acid two times in aggregate.
20. The method of claim 18 , wherein steps (a) and (b) are carried out serially for six different combinations of two types of test nucleotides, wherein each different nucleotide type is present three times in aggregate.