# US 11,078,526 B2 · Polyphenolic additives in sequencing by synthesis

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- Number: US 11,078,526 B2
- Document ID: US11078526B2
- Status: Granted
- Kind: B2
- Application number: 16/422,056
- Filed: 2019-05-24
- Published: 2019-10-31
- Granted: 2021-08-03
- Earliest claimed: 2016-02-11
- Assignees: IsoPlexis Corporation
- Applicants: IsoPlexis Corporation
- Inventors: Jerzy Olejnik; Michel Georges Perbost
- Web page: https://patentgrep.com/patent/US11078526B2

## CPC

- C12Q 1/6823: Release of bound markers
- C12Q 1/6869: Methods for sequencing
- 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

The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.

## Claims

### Claim 1

A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound gentisic acid, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
d) detecting said incorporated labeled nucleotide analogue; and
e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

### Claim 2 (depends on 1)

The method of claim 1, wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

### Claim 3 (depends on 1)

The method of claim 1, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′—OH group.

### Claim 4 (depends on 2)

The method of claim 2, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′—OH group.

### Claim 5 (depends on 4)

The method of claim 4, wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

### Claim 6 (depends on 1)

The method of claim 1, wherein said label is fluorescent.

### Claim 7

A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pyrocatechol, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
d) detecting said incorporated labeled nucleotide analogue; and
e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

### Claim 8 (depends on 7)

The method of claim 7, wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

### Claim 9 (depends on 7)

The method of claim 7, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′—OH group.

### Claim 10 (depends on 8)

The method of claim 8, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′—OH group.

### Claim 11 (depends on 10)

The method of claim 10, wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

### Claim 12 (depends on 7)

The method of claim 7, wherein said label is fluorescent.

### Claim 13

A method of incorporating labeled nucleotides, comprising:
a) providing
i) a plurality of nucleic acid primers and template molecules,
ii) a polymerase,
iii) a cleave reagent comprising a reducing agent and the polyphenolic compound pyrogallol, and
iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;
b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;
c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue;
d) detecting said incorporated labeled nucleotide analogue; and
e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

### Claim 14 (depends on 13)

The method of claim 13, wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

### Claim 15 (depends on 13)

The method of claim 13, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′—OH group.

### Claim 16 (depends on 14)

The method of claim 14, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′—OH group.

### Claim 17 (depends on 16)

The method of claim 16, wherein the method further comprises:
f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

### Claim 18 (depends on 13)

The method of claim 13, wherein said label is fluorescent.