Allele-specific capture of nucleic acids

Patent metadata

Field	Value
Publication number	US-11180749-B2
Application number	US-201916535009-A
Country	US
Kind code	B2
Filing date	Aug 7, 2019
Priority date	Jan 20, 2017
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

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 plurality of target alleles from a mixture of nucleic acids, comprising
(a) providing a mixture of nucleic acids in fluidic contact with an array of stabilized ternary complexes, wherein each of the stabilized ternary complexes is attached to a feature of the array,
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 array via a linkage between the polymerase and a feature of the array or via a linkage between the next correct nucleotide and a feature of the array; and
(b) separating the array from the mixture of nucleic acids, thereby separating the target alleles from the mixture of nucleic acids.

2. The method of claim 1 , wherein each of the stabilized ternary complexes is attached to the array via a linkage between the polymerase and a feature of the array.

3. The method of claim 1 , wherein one or more of the target alleles occurs at a locus and the mixture of nucleic acids further comprises another allele at the locus.

4. The method of claim 3 , wherein the separating of the array from the mixture of nucleic acids thereby separates the target alleles from the other alleles.

5. The method of claim 3 , wherein at least one of the target alleles is a minor allele.

6. The method of claim 5 , wherein the minor allele frequency is less than 5%.

7. The method of claim 1 , further comprising
(c) dissociating the templates comprising the target alleles from the separated array.

8. The method of claim 7 , further comprising
(d) forming a mixture comprising second stabilized ternary complexes that comprise the formerly dissociated templates, polymerases, and next correct nucleotides that are cognate nucleotides for the target alleles, wherein each of the second stabilized ternary complexes is attached to an array via a linkage between the polymerase and a feature of the array or via a linkage between the next correct nucleotide and a feature of the array.

9. The method of claim 8 , further comprising
(e) separating the array of step (d) from the mixture, thereby separating the formerly dissociated templates comprising the target alleles from the mixture.

10. The method of claim 9 , further comprising
(f) dissociating the formerly dissociated templates from the array separated in step (e).

11. The method of claim 8 , wherein the solid support of step (a) is the same as the solid support of step (d).

12. The method of claim 10 , wherein the polymerase or the next correct nucleotide of step (a) is the same as the polymerase or the next correct nucleotide of step (d).

13. The method of claim 1 , wherein step (a) further comprises providing a mixture of nucleic acids comprising the templates and contacting the mixture with the polymerases and the next correct nucleotides, thereby providing the mixture of nucleic acids in fluidic contact with the array of stabilized ternary complexes.

14. The method of claim 13 , wherein the polymerases or next correct nucleotides are attached to a feature of the array prior to forming the stabilized ternary complexes.

15. The method of claim 13 , wherein the stabilized ternary complexes are formed in solution prior to attaching the ternary complexes to the array.

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

17. The method of claim 16 , wherein step (a) further comprises forming the stabilized ternary complexes in solution and then binding the receptor to the ligand, thereby attaching the ternary complexes to the array.

18. The method of claim 16 , wherein step (a) further comprises providing the polymerase attached to the array and then forming the stabilized ternary complexes on the array.

19. The method of claim 16 , wherein step (a) further comprises providing the next correct nucleotides attached to the array and then forming the stabilized ternary complexes on the array.

20. The method of claim 1 , wherein the ternary complexes lack exogenous labels.

Assignees

• Omniome Inc

Inventors

• Dambacher Corey M
• Tu Eugene

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · 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

• 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

• C12Q2563/137

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