# US 10,246,744 B2 · Method and system for sequencing nucleic acids

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- Number: US 10,246,744 B2
- Document ID: US10246744B2
- Status: Granted
- Kind: B2
- Application number: 15/724,570
- Filed: 2017-10-04
- Published: 2018-03-22
- Granted: 2019-04-02
- Earliest claimed: 2016-08-15
- Assignees: Omniome, Inc.
- Applicants: Omniome, Inc.
- Inventors: Kandaswamy Vijayan; Corey M. Dambacher; Eugene Tu; Mark A. Bernard; Joseph Rokicki; Kerry Wilson
- Web page: https://patentgrep.com/patent/US10246744B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q)
- C12Q 2521/101: DNA polymerase
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 7)

The method of claim 7, further comprising, after step (c), removing the reversible terminator moiety on the 3′ terminal nucleotide of the primer.

### Claim 9 (depends on 1)

The method of claim 1, wherein each of the test nucleotides is an unlabeled nucleotide.

### Claim 10 (depends on 9)

The method of claim 9, wherein the polymerase comprises an exogenous label that is detected in step (c).

### Claim 11 (depends on 1)

The method of claim 1, wherein the next correct nucleotide comprises an exogenous label that is detected in step (c).

### Claim 12 (depends on 1)

The method of claim 1, further comprising (e) incorporating a nucleotide at the 3′ end of the primer after step (c).

### Claim 13 (depends on 12)

The method of claim 12, wherein the nucleotide that is incorporated is an unlabeled nucleotide.

### Claim 14 (depends on 12)

The method of claim 12, wherein the nucleotide that is incorporated is an unlabeled reversible terminator nucleotide.

### Claim 15 (depends on 12)

The method of claim 12, further comprising repeating steps (b) through (e) to sequence the primed template nucleic acid molecule.

### Claim 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.

### Claim 17 (depends on 16)

The method of claim 16, wherein the first combination comprises two types of test nucleotides.

### Claim 18 (depends on 17)

The method of claim 17, wherein the second combination comprises two types of test nucleotides.

### Claim 19 (depends on 18)

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.

### Claim 20 (depends on 18)

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.