Genotyping by polymerase binding
A method for identifying target alleles, that includes steps of (a) forming a plurality of stabilized ternary complexes at a plurality of features on an array, wherein the stabilized ternary complexes each has a polymerase, a template nucleic acid having a target allele of a locus, a primer hybridized to the locus, and a next correct nucleotide having a cognate in the locus, wherein either (i) the primer is an allele-specific primer having a 3′ nucleotide that is a cognate nucleotide for the target allele, or (ii) the primer is a locus-specific primer and the next correct nucleotide hybridizes to the target allele; and (b) detecting stabilized ternary complexes at the features, thereby identifying the target alleles.
1. A method for identifying target alleles, comprising
(a) forming first ternary complexes on an array of primers, wherein each of the first ternary complexes comprises a polymerase, a first template nucleic acid comprising a target allele of a locus, a primer of the array, and a next correct nucleotide that is a cognate to the target allele of the first template nucleic acid; and
(b) detecting the polymerase, first template nucleic acid or next correct nucleotide in the first ternary complexes, thereby identifying target alleles of the first template nucleic acid;
(c) dissociating the first ternary complexes from the array of primers;
(d) forming second ternary complexes on the array of primers, wherein each of the second ternary complexes comprises a polymerase, a second template nucleic acid comprising a target allele of the locus, a primer of the array, and a next correct nucleotide that is a cognate to the target allele of the second template nucleic acid; and
(e) detecting the polymerase, second template nucleic acid or next correct nucleotide in the second ternary complexes, thereby identifying target alleles of the second template nucleic acid.
2. The method of claim 1 , wherein the primers of the array comprise blocking groups that preclude enzymatic incorporation of the next correct nucleotide into the primer.
3. The method of claim 2 , wherein the blocking groups comprise reversible terminators.
4. The method of claim 2 , wherein the blocking groups comprise irreversible terminators.
5. The method of claim 1 , wherein the next correct nucleotide of the first ternary complex comprises an exogenous label that is detected in step (b).
6. The method of claim 5 , wherein the next correct nucleotide of the second ternary complex comprises an exogenous label that is detected in step (e).
7. The method of claim 1 , wherein the first ternary complexes comprise at least two alleles at the locus and wherein different exogenous labels distinguish at least two alleles at the locus in step (b).
8. The method of claim 1 , wherein the array of primers is in a flow cell.
9. The method of claim 8 , further comprising washing the array after the dissociating of the first ternary complexes from the array of primers, thereby removing the polymerase, the first template nucleic acid and the next correct nucleotide from the flow cell.
10. The method of claim 1 , wherein the polymerase of the first ternary complex comprises an exogenous label that is detected in step (b).
11. The method of claim 10 , wherein the polymerase of the second ternary complex comprises an exogenous label that is detected in step (e).
12. The method of claim 1 , wherein the primers are chemically unchanged by the polymerases of the first ternary complexes.
13. The method of claim 1 , wherein the array of primers comprises allele-specific primers that form first ternary complexes in step (a) and second ternary complexes in step (d).
14. The method of claim 1 , wherein the array of primers comprises locus-specific primers that form first ternary complexes in step (a) and second ternary complexes in step (d).
15. The method of claim 1 , wherein the first ternary complexes are stabilized by a non-catalytic metal ion or a blocking group on the primers.
16. The method of claim 1 , wherein the target alleles comprise single nucleotide polymorphisms.
17. The method of claim 1 , wherein the target alleles comprise insertions or deletions.
18. The method of claim 1 , wherein the target alleles comprise alternative splicing polymorphisms.
19. The method of claim 1 , wherein the first template nucleic acid comprises an exogenous label that is detected in step (b).
20. The method of claim 19 , wherein the first template nucleic acid comprises an exogenous label that is detected in step (e).