# US 12,760,356 B2 · Compositions and methods for capturing and amplifying target polynucleotides using modified capture primers

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- Number: US 12,760,356 B2
- Document ID: US12760356B2
- Status: Granted
- Kind: B2
- Application number: 18/000,663
- Filed: 2021-12-20
- Published: 2024-01-11
- Granted: 2026-10-13
- Earliest claimed: 2020-12-21
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Hayden Black; Esteban Armijo; Mathieu Lessard-Viger
- Web page: https://patentgrep.com/patent/US12760356B2

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- 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 composition for capturing target polynucleotides at a surface of a substrate is provided. The composition may include a plurality of capture primers coupled to the surface of the substrate and including modified nucleic acids; and a plurality of orthogonal capture primers coupled to the surface of the substrate and including modified nucleic acids. The modified nucleic acids of the capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T. The modified nucleic acids of the orthogonal capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T.

## Claims

### Claim 1

A composition for capturing first target polynucleotides on a surface of a substrate, the composition comprising:
a plurality of capture primers coupled to the surface of the substrate, each of the capture primers comprising modified nucleic acids; and
a plurality of orthogonal capture primers coupled to the surface of the substrate, each of the orthogonal capture primers comprising modified nucleic acids,
first target polynucleotides, wherein each of the first target polynucleotides comprises a first adapter that is complementary to the capture primers, and a second adapter that is complementary to the orthogonal capture primers, wherein the first adapter and the second adapter are located in different locations of each of the first target polynucleotides and have different nucleotide sequences;
second target polynucleotides that are complementary to the first target polynucleotides,
wherein the first adapter of some of the first target polynucleotides is hybridized to the capture primers, thereby forming first duplexes on the surface of the substrate, and wherein the second adapter of some of the first target polynucleotides is hybridized to the orthogonal capture primers, thereby forming second duplexes on the surface of the substrate; and
wherein each of the first duplexes and each of the second duplexes has a melting temperature (Tm) that is greater than a Tm of third duplexes that are formed by hybridization of the second target polynucleotides to the first target polynucleotides.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the modified nucleic acids of the capture primers comprise locked nucleic acid (LNA), peptide nucleic acid (PNA), or 5-hydroxylbutynl-2′-deoxyuridine.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the capture primers further comprise deoxyribonucleic acid (DNA).

### Claim 4 (depends on 3)

The composition of claim 3, wherein the modified nucleic acids and the DNA are distributed between 5′ end and 3′ end of each of the capture primers.

### Claim 5 (depends on 3)

The composition of claim 3, wherein the modified nucleic acids are disposed at 5′ end of each of the capture primers and the DNA is disposed at 3′ end of each of the capture primers.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the modified nucleic acids of the orthogonal capture primers comprise locked nucleic acid (LNA), peptide nucleic acid (PNA), or 5-hydroxylbutynl-2′-deoxyuridine.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the orthogonal capture primers further comprise deoxyribonucleic acid (DNA).

### Claim 8 (depends on 7)

The composition of claim 7, wherein the modified nucleic acids and the DNA are distributed between 5′ end and 3′ end of each of the orthogonal capture primers.

### Claim 9 (depends on 7)

The composition of claim 7, wherein the modified nucleic acids are disposed at 5′ end of each of the orthogonal capture primers and the DNA is disposed at 3′ end of each of the orthogonal capture primers.

### Claim 10 (depends on 1)

The composition of claim 1, wherein each of the first duplexes has a melting temperature (Tm) between about 80° C. and about 110° C., and each of the second duplexes has a melting temperature (Tm) between about 80° C. and about 110° C.

### Claim 11 (depends on 1)

The composition of claim 1, wherein each of the first duplexes has a melting temperature (Tm) is between about 85° C. and about 105° C., and each of the second duplexes has a melting temperature (Tm) between about 85° C. and about 105° C.

### Claim 12 (depends on 1)

The composition of claim 1, wherein each of the first duplexes has a melting temperature (Tm) between about 90° C. and about 100° C., and each of the second duplexes has a melting temperature (Tm) between about 90° C. and about 100° C.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the hybridization of the first adapters of some of the first target polynucleotides to the capture primers, and the hybridization of the second adapters of some of the first target polynucleotides to the orthogonal capture primers occur under a condition that inhibits hybridization of the second target polynucleotides to the first target polynucleotides.

### Claim 14 (depends on 1)

The composition of claim 1, further comprising about 1% to about 100% formamide (% v/v).

### Claim 15 (depends on 1)

The composition of claim 1, further comprising about 5% to about 80% formamide (% v/v).

### Claim 16 (depends on 1)

The composition of claim 1, further comprising about 100 to about 800 mM Na+.

### Claim 17 (depends on 1)

The composition of claim 1, further comprising about 200 to about 800 mM Na+.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the capture primers are modified P5 capture primers, and wherein the orthogonal capture primers are modified P7 capture primers.

### Claim 19 (depends on 1)

The composition of claim 1, wherein each of the capture primers includes about five to about twenty of the modified nucleic acids, and wherein each of the orthogonal capture primers includes about five to about twenty of the modified nucleic acids.

### Claim 20 (depends on 1)

The composition of claim 1, wherein each of the capture primers includes at least about nine of the modified nucleic acids, and wherein each of the orthogonal capture primers includes at least about nine of the modified nucleic acids.

### Claim 21 (depends on 1)

The composition of claim 1, wherein each of the capture primers includes at least about twelve of the modified nucleic acids, and wherein each of the orthogonal capture primers includes at least about twelve of the modified nucleic acids.

### Claim 22 (depends on 1)

The composition of claim 1, wherein each of the capture primers includes at least about fifteen of the modified nucleic acids, and wherein each of the orthogonal capture primers includes at least about fifteen of the modified nucleic acids.

### Claim 23

A method for capturing and amplifying first target polynucleotides on a surface of a substrate, the method comprising:
providing a composition comprises:
a plurality of capture primers coupled to the surface of the substrate, each of the capture primers comprising modified nucleic acids; and
a plurality of orthogonal capture primers coupled to the surface of the substrate, each of the orthogonal capture primers comprising modified nucleic acids;
providing a fluid comprises:
first target polynucleotides, wherein each of the first target polynucleotides comprises a first adapter that is complementary to the capture primers and a second adapter that is complementary to the orthogonal capture primers; and
second target polynucleotides that are complementary to the first target polynucleotides;
contacting the composition with the fluid under a condition that inhibits hybridization of the second target polynucleotides to the first target polynucleotides
forming first duplexes on the surface of the substrate by hybridizing the first adapter of some of the first target polynucleotides to the capture primers;
forming second duplexes on the surface of the substrate by hybridizing the second adapter of some of the first target polynucleotides to the orthogonal capture primers; and then
generating amplicons of the first target polynucleotides by amplifying the first target polynucleotides of the first duplexes and/or the second duplexes,
wherein each of the first duplexes and each of the second duplexes has a melting temperature (Tm) that is greater than a Tm of third duplexes that are formed by hybridization of the second target polynucleotides to the first target polynucleotides.