IP Library › Granted Patent US 12,680,133
Granted Patent B2
US 12,680,133 · App. 18/303,464 · Granted Jul 14, 2026

Methods, compositions, and solid supports for multi-dimensional sequencing

Inventors: Eli N. Glezer (Del Mar, CA); Daan Witters (San Diego, CA)
Assignee: Singular Genomics Systems, Inc.
C12Q1/6874
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Quick Facts
Patent No.
US 12,680,133
App. No.
18/303,464
Filed
Apr 19, 2023
Granted
Jul 14, 2026
Kind
B2
Art Unit
1681
USPC
435/6.12
Abstract

Disclosed herein, inter alia, are compositions and methods for detecting polynucleotides within a three-dimensional polymer matrix.

Claims (43)

1 . A method of amplifying a polynucleotide, said method comprising:

contacting a solid support comprising a multi-layer polymer with a polynucleotide, and amplifying the polynucleotide with a polymerase and a plurality of nucleotides to generate amplification products, wherein said multi-layer polymer comprises:

a first layer attached to said solid support, wherein said first layer comprises a first oligonucleotide within a first polymer layer, wherein said first oligonucleotide comprises a first amplification primer binding sequence and a first sequencing primer binding sequence;

a second layer, wherein said second layer does not comprise an amplification primer binding sequence within a second polymer layer; and

a third layer attached to said second layer, wherein said third layer comprises a second oligonucleotide within a third polymer layer, wherein said second oligonucleotide comprises a second amplification primer binding sequence and a second sequencing primer binding sequence,

wherein said second polymer layer is attached to said first and third polymer layer.

2 . The method of claim 1 , wherein the first amplification primer binding sequence and the second amplification primer binding sequence comprise the same sequence.

3 . The method of claim 1 , wherein amplifying comprises bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, emulsion PCR, or combinations thereof.

4 . The method of claim 1 , further comprising detecting the amplification products, wherein detecting comprises sequencing.

5 . The method of claim 4 , wherein sequencing comprises sequencing-by-synthesis, sequencing-by-binding, sequencing by ligation, or pyrosequencing, and generates a sequencing read.

6 . The method of claim 4 , wherein sequencing comprises contacting the amplification products with a first sequencing primer, extending the first sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the amplification products, detecting the detectable label, and repeating the extending and detecting steps, wherein said first sequencing primer is complementary to said first sequencing primer binding sequence of said first oligonucleotide.

7 . The method of claim 6 , wherein sequencing further comprises contacting the amplification products with a second sequencing primer, extending the second sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the amplification products, detecting the detectable label, and repeating the extending and detecting steps, wherein said second sequencing primer is complementary to said second sequencing primer binding sequence of said second oligonucleotide.

8 . A composition comprising:

(i) a first layer comprising a polymeric gel comprising a plurality of oligonucleotides attached to said polymeric gel;

(ii) a second layer comprising a polymeric gel, wherein said polymeric gel does not comprise a plurality of oligonucleotides attached to said polymeric gel; and

(iii) a third layer comprising a polymeric gel comprising a plurality of oligonucleotides attached to said polymeric gel.

9 . The composition of claim 8 , wherein said first layer and third layer each comprise a polymeric gel comprising said plurality of oligonucleotides covalently attached to said polymeric gel.

10 . The composition of claim 8 , wherein the first layer is immediately adjacent to the second layer, wherein the third layer is immediately adjacent to the second layer, and wherein the first layer and third layer are not immediately adjacent.

11 . The composition of claim 8 , wherein the first layer, second layer, and third layer are in fluidic contact.

12 . The composition of claim 8 , wherein the first, second, and third layers form a contiguous layered unit.

13 . The composition of claim 12 , further comprising two or more contiguous layered units.

14 . The composition of claim 8 , wherein each layer has a width of about 1-20 mm, a length of about 1-20 cm, and a depth of about 0.5-15 μm.

15 . The composition of claim 8 , wherein the first layer, the third layer, or both the first layer and the third layer are attached to a solid support.

16 . The composition of claim 8 , wherein the polymeric gel comprises a refractive index of about 1.3 when hydrated.

17 . A method of amplifying a template polynucleotide, the method comprising:

(a) annealing a template polynucleotide to a first oligonucleotide of the composition of claim 8 ;

(b) extending the first oligonucleotide with a polymerase to generate a complement template polynucleotide;

(c) contacting the complement template polynucleotide and the template polynucleotide with a chemical denaturant thereby separating the complement template polynucleotide from the template polynucleotide;

(d) removing the chemical denaturant and annealing the complement template polynucleotide to a second oligonucleotide on said composition; and

(e) extending the second oligonucleotide with the polymerase to generate a template polynucleotide, thereby amplifying the template polynucleotide.

18 . A method of amplifying a template polynucleotide, the method comprising:

(i) contacting the composition of claim 8 with an annealing solution, wherein one or more of the oligonucleotides anneals to the template polynucleotide;

(ii) contacting the composition with an extension solution;

(iii) contacting the composition with a chemical denaturant;

(iv) repeating steps (i)-(iii) to amplify the template polynucleotide.

19 . A method of sequencing a plurality of template polynucleotides, the method comprising:

(a) hybridizing the plurality of template polynucleotides to the plurality of oligonucleotides of the composition of claim 8 ;

(b) amplifying the template polynucleotides to produce discrete amplicon clusters, wherein (i) amplifying comprises extension of the oligonucleotides along the template polynucleotides within each first layer and third layer, (ii) each amplicon cluster originates from amplification of a single template polynucleotide, and (iii) the amplicon clusters are arranged at a plurality of depths in each first layer and third layer; and

(c) sequencing the amplicon clusters, wherein sequencing comprises detecting sequences of signals within each first layer and third layer of the composition at a first depth and a second depth.

20 . A solid support comprising a multi-layer polymer, wherein said multi-layer polymer comprises:

a first layer attached to said solid support, wherein said first layer comprises a first oligonucleotide within a first polymer layer, and said first oligonucleotide comprises a first amplification primer binding sequence and a first sequencing primer binding sequence;

a second layer, wherein said second layer does not comprise an amplification primer binding sequence within a second polymer layer, wherein said first polymer layer is attached to said second polymer layer; and

a third layer attached to said second layer, wherein said third layer comprises a second oligonucleotide within a third polymer layer, wherein said second oligonucleotide comprises a second amplification primer binding sequence and a second sequencing primer binding sequence.

Assignments (2)
SECURITY INTEREST Recorded Mar 7, 2025
From: SINGULAR GENOMICS SYSTEMS, INC.
To: FIRST-CITIZENS BANK & TRUST COMPANY
Reel/Frame 070440/0465 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 2, 2023
From: GLEZER, ELI N.; WITTERS, DAAN
To: SINGULAR GENOMICS SYSTEMS, INC
Reel/Frame 063505/0057 →
Continuity (3)
Provisional Application 63480409 · Jan 18, 2023
Provisional Application 63334067 · Apr 22, 2022
Related Publication 20230340591A1 · Oct 26, 2023
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