# US 2021/0340609 A1 · Genotyping by polymerase binding

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- Number: US 2021/0340609 A1
- Document ID: US20210340609A1
- Status: Published application
- Kind: A1
- Application number: 17/198,932
- Filed: 2021-03-11
- Published: 2021-11-04
- Earliest claimed: 2017-01-20
- Assignees: Omniome, Inc.
- Applicants: Omniome, Inc.
- Inventors: Corey M. DAMBACHER; Michael VAN NGUYEN
- Web page: https://patentgrep.com/patent/US20210340609A1

## CPC

- C12Q 1/6853: using modified primers or templates
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6872: involving mass spectrometry
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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.

## Claims

### Claim at position 1

-20. (canceled)

### Claim 21

A method for identifying target alleles in a mixture of nucleic acids, comprising
(a) providing an array of features, wherein different template nucleic acids are attached at different features of the array;
(b) contacting the array with a plurality of primers, polymerases and nucleotides to form a plurality of stabilized ternary complexes at a plurality of the features, wherein the stabilized ternary complexes at the features each comprises a polymerase, a template nucleic acid attached to a feature of the array and comprising 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 comprising 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
(c) detecting stabilized ternary complexes at the features, thereby identifying the target alleles.

### Claim 22 (depends on 21)

The method of claim 21, wherein the primers of the array comprise blocking groups that preclude enzymatic incorporation of the next correct nucleotide into the primer.

### Claim 23 (depends on 22)

The method of claim 22, wherein the blocking groups comprise reversible terminators.

### Claim 24 (depends on 22)

The method of claim 22, wherein the blocking groups comprise irreversible terminators.

### Claim 25 (depends on 21)

The method of claim 21, wherein the next correct nucleotide comprises an exogenous label that is detected in step (c).

### Claim 26 (depends on 21)

The method of claim 21, wherein the array of features is in a flow cell.

### Claim 27 (depends on 21)

The method of claim 21, wherein the stabilized ternary complexes are stabilized by a non-catalytic metal ion or a blocking group on the primers.

### Claim 28 (depends on 21)

The method of claim 21, wherein the primer is an allele-specific primer comprising a 3′ nucleotide that is a cognate nucleotide for the target allele.

### Claim 29 (depends on 21)

The method of claim 21, wherein the primer is a locus-specific primer and the next correct nucleotide hybridizes to the target allele.

### Claim 30 (depends on 21)

The method of claim 21, wherein the next correct nucleotides are non-covalently bound to the stabilized ternary complexes during step (c).

### Claim 31 (depends on 21)

The method of claim 21, wherein the target allele occurs at a locus and the different template nucleic acids further comprises another allele at the locus.

### Claim 32 (depends on 31)

The method of claim 31, wherein the locus comprises a single nucleotide polymorphism, INDEL polymorphism, or alternative mRNA splice site polymorphism.

### Claim 33 (depends on 25)

The method of claim 25, wherein (c) comprises detecting stabilized ternary complexes at the features by detecting the exogenous labels, thereby identifying the target alleles.

### Claim 34 (depends on 21)

The method of claim 21, wherein at least two types of nucleotides are contacted with the array in step (b), and wherein different exogenous labels distinguish at least two alleles at each locus in step (c).

### Claim 35 (depends on 21)

The method of claim 21, wherein a single type of nucleotide is contacted with the array in step (b).

### Claim 36 (depends on 21)

The method of claim 21, further comprising
(d) dissociating the next correct nucleotide from each feature of the array after the detecting of the stabilized ternary complexes.

### Claim 37 (depends on 21)

The method of claim 21, further comprising washing the array after the dissociating.

### Claim 38 (depends on 21)

The method of claim 21, wherein the polymerases comprise exogenous labels.

### Claim 39 (depends on 38)

The method of claim 38, wherein (c) comprises detecting stabilized ternary complexes at the features by detecting the exogenous labels, thereby identifying the target alleles.

### Claim 40 (depends on 21)

The method of claim 21, wherein the ternary complex that is detected in step (c) lacks exogenous labels.