Allele-specific capture of nucleic acids

Patent metadata

Field	Value
Publication number	US-10415029-B2
Application number	US-201715701358-A
Country	US
Kind code	B2
Filing date	Sep 11, 2017
Priority date	Jan 20, 2017
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

Abstract

A method for separating a target allele from a mixture of nucleic acids by (a) providing a mixture of nucleic acids in fluidic contact with a stabilized ternary complex that is attached to a solid support, wherein the stabilized ternary complex includes a polymerase, primed nucleic acid template, and next correct nucleotide, wherein the template has a target allele, wherein the next correct nucleotide is a cognate nucleotide for the target allele, and wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and (b) separating the solid support from the mixture of nucleic acids, thereby separating the target allele from the mixture of nucleic acids.

First claim

What is claimed is:

1. A method for separating a target allele from a mixture of nucleic acids, comprising
(a) providing a mixture of nucleic acids in fluidic contact with a stabilized ternary complex that is attached to a solid support,
wherein the stabilized ternary complex comprises a polymerase, primed nucleic acid template, and next correct nucleotide,
wherein the template comprises a target allele,
wherein the next correct nucleotide is a cognate nucleotide for the target allele, and
wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and
(b) separating the solid support from the mixture of nucleic acids, thereby separating the target allele from the mixture of nucleic acids.

2. The method of claim 1 , wherein the target allele occurs at a locus and the mixture of nucleic acids further comprises another allele at the locus.

3. The method of claim 2 , wherein the separating of the solid support from the mixture of nucleic acids thereby separates the target allele from the other allele at the locus.

4. The method of claim 2 , wherein the target allele is a minor allele at the locus, and wherein the minor allele frequency is less than 5% at the locus.

5. The method of claim 1 , further comprising
(c) dissociating the template comprising the target allele from the separated solid support.

6. The method of claim 1 , wherein step (b) further comprises washing the separated solid support to further remove components of the mixture.

7. The method of claim 6 , further comprising
(d) forming a solution comprising second stabilized ternary complex that comprises the formerly dissociated template, a polymerase, and a next correct nucleotide that is a cognate nucleotide for the target allele, wherein the polymerase or the next correct nucleotide is attached to a solid support.

8. The method of claim 7 , further comprising
(e) separating the solid support of step (d) from the solution, thereby separating the formerly dissociated template comprising the target allele from the solution.

9. The method of claim 8 , further comprising
(f) dissociating the formerly dissociated template from the solid support separated in step (e).

10. The method of claim 7 , wherein the solid support of step (a) is the same as the solid support of step (d).

11. The method of claim 7 , wherein the polymerase of step (a) is the same as the polymerase of step (d).

12. The method of claim 1 , wherein step (a) further comprises providing a mixture of nucleic acids comprising the template and contacting the mixture with the polymerase and the next correct nucleotide, thereby providing the mixture of nucleic acids and the stabilized ternary complex.

13. The method of claim 12 , wherein the polymerase or the next correct nucleotide is attached to the solid support prior to forming the stabilized ternary complex on the solid support.

14. The method of claim 12 , wherein the stabilized ternary complex is formed in solution prior to attaching the ternary complex to the solid support.

15. The method of claim 1 , wherein the linkage comprises a receptor-ligand association.

16. The method of claim 1 , wherein the ternary complex lacks exogenous labels.

17. The method of claim 1 , wherein the next correct nucleotide lacks exogenous labels.

18. A method for separating a plurality of target alleles from a mixture of nucleic acids, comprising
(a) providing a mixture of nucleic acids in fluidic contact with a plurality of stabilized ternary complexes that are solid support-attached,
wherein the stabilized ternary complexes each comprises a polymerase, primed nucleic acid template, and next correct nucleotide,
wherein the template comprises a target allele,
wherein the next correct nucleotide is a cognate nucleotide for the target allele, and
wherein each of the stabilized ternary complexes is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and
(b) separating the solid support from the mixture of nucleic acids, thereby separating the target alleles from the mixture of nucleic acids.

19. A method for separating a first allele of a locus from a second allele at the locus, comprising
(a) providing a mixture comprising the second allele in fluidic contact with a stabilized ternary complex that is attached to a solid support,
wherein the stabilized ternary complex comprises a polymerase, primer hybridized to a nucleic acid template, and next correct nucleotide,
wherein the template comprises the first allele,
wherein the next correct nucleotide is a cognate nucleotide for the first allele or the 3′ end of the primer comprises a cognate nucleotide for the first allele, and
wherein the stabilized ternary complex is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and
(b) separating the solid support from the mixture of nucleic acids, thereby separating the first allele from the second allele.

20. A method for separating first alleles at a plurality of loci from second alleles at the plurality of loci, respectively, comprising
(a) providing a mixture of the second alleles at the plurality of loci, respectively, in fluidic contact with a plurality of stabilized ternary complexes that are solid support-attached,
wherein the stabilized ternary complexes each comprises a polymerase, primed nucleic acid template, and next correct nucleotide,
wherein the template comprises a first allele,
wherein the next correct nucleotide is a cognate nucleotide for the first allele or the 3′ end of the primer comprises a cognate nucleotide for the first allele, and
wherein each of the stabilized ternary complexes is attached to the solid support via a linkage between the polymerase and the solid support or via a linkage between the next correct nucleotide and the solid support; and
(b) separating the solid support from the mixture of nucleic acids, thereby separating the first alleles from the second alleles at the plurality of loci.

Assignees

• Omniome Inc

Inventors

• Dambacher Corey M
• Tu Eugene

Classifications

• C12N15/1024

  In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair · CPC title

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2563/137

  Metal/ion, e.g. metal label · CPC title