IP Library › Granted Patent US 12,674,194
Granted Patent B2
US 12,674,194 · App. 17/704,895 · Granted Jul 7, 2026

In situ nucleic acid amplification

Inventors: Daan Witters (San Diego, CA); Allen Lipson (San Diego, CA); Eli N. Glezer (Del Mar, CA)
Assignee: Singular Genomics Systems, Inc.
C12Q1/6844C12Q1/6806
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Quick Facts
Patent No.
US 12,674,194
App. No.
17/704,895
Granted
Jul 7, 2026
Kind
B2
Abstract

Disclosed herein, inter alia, are novel methods pertaining to nucleic acid amplification and sequencing. Compositions for use in and produced by such methods are also provided.

Claims (25)

1 . A method of amplifying a circular template polynucleotide in situ, the method comprising:

(a) amplifying the circular template polynucleotide in a tissue section comprising a cell by extending a first amplification primer with a strand-displacing polymerase to generate a first extension product comprising one or more complements of the circular template polynucleotide, wherein extending occurs for about 30 seconds to 30 minutes; and

(b) contacting the first extension product with a second amplification primer in the tissue section, wherein said second amplification primer is attached to a polymer matrix or a cellular component in or on the cell, and extending said second amplification primer with a polymerase to generate a second immobilized extension product comprising one or more complements of the first extension product in situ.

2 . The method of claim 1 , wherein step (b) comprises (i) extension of a 3′ end of the first extension product hybridized to the second extension product, and/or (ii) extension of a 3′ end of an extension product hybridized to itself.

3 . The method of claim 1 , wherein each amplification primer comprises at least 17 nucleotides.

4 . The method of claim 1 , wherein the second amplification primer is attached to the cellular component.

5 . The method of claim 1 , wherein the polymer matrix is an exogenous polymer matrix.

6 . The method of claim 5 , wherein the second amplification primer is covalently bound to the exogenous polymer matrix.

7 . The method of claim 1 , wherein the cell forms part of a tissue section in situ, or wherein the cell is a prokaryotic cell or a eukaryotic cell.

8 . The method of claim 1 , wherein the cell is permeabilized and immobilized to a solid support.

9 . The method of claim 1 , wherein the circular template polynucleotide comprises single-stranded circular DNA.

10 . The method of claim 9 , wherein the circular template polynucleotide comprises one or more ribonucleotides.

11 . The method of claim 9 , wherein prior to step (a), forming the circular template polynucleotide comprises ligating ends of a linear polynucleotide together.

12 . The method of claim 1 , wherein step (a) comprises rolling circle amplification (RCA), exponential rolling circle amplification (eRCA), or hyperbranched rolling circle amplification (HRCA).

13 . The method of claim 1 , wherein step (a) comprises incubation with the strand-displacing polymerase at a temperature of about 20° C. to about 50° C.

14 . The method of claim 1 , wherein step (b) comprises a polymerase different than the polymerase of step (a).

15 . The method of claim 1 , wherein the circular template polynucleotide of step (a) is about 100 to about 1000 nucleotides in length, about 100 to about 300 nucleotides in length, about 300 to about 500 nucleotides in length, or about 500 to about 1000 nucleotides in length.

16 . The method of claim 1 , further comprising sequencing the first immobilized extension product and the second immobilized extension product.

17 . The method of claim 1 , wherein prior to step (a), forming the circular template polynucleotide comprises ligating ends of a linear polynucleotide together with the aid of a bridging oligonucleotide that is complementary with the two ends of the linear polynucleotide.

18 . The method of claim 1 , wherein step (b) further comprises contacting the second extension product with a third amplification primer in said cell, wherein said third amplification primer is attached to a polymer matrix or a cellular component, and extending said third amplification primer with a polymerase to generate an immobilized amplification product comprising one or more complements of the second extension product.

19 . The method of claim 1 , wherein the circular template polynucleotide comprises the complement of an endogenous nucleic acid sequence.

20 . The method of claim 1 , wherein the tissue section comprises a Formalin-Fixed Paraffin-Embedded (FFPE) tissue sample.

21 . The method of claim 1 , wherein the tissue section comprises bone tissue.

22 . The method of claim 1 , wherein prior to step (a), the method comprises hybridizing a first sequence of an oligonucleotide primer to a target nucleic acid molecule and hybridizing a second sequence of the oligonucleotide primer to the target nucleic acid molecule, and ligating the first sequence and second sequence together to form the circular template polynucleotide in the tissue.

23 . The method of claim 1 , wherein the first extension product is covalently attached to a first protein and wherein the second extension product is covalently attached to a second protein.

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 Sep 14, 2022
From: WITTERS, DAAN; LIPSON, ALLEN; GLEZER, ELI N.
To: SINGULAR GENOMICS SYSTEMS, INC
Reel/Frame 061097/0599 →
Continuity (3)
Continuation PCTUS2021031517 · May 10, 2021
Provisional Application 63023752 · May 12, 2020
Related Publication 20220235410A1 · Jul 28, 2022
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