Granted Patent B2

US 12,492,396 · App. 17/320,042 · Granted Dec 9, 2025

Inventors: Matthew Kellinger (San Diego, CA); Sinan Arslan (San Diego, CA); Michael Previte (San Diego, CA); Junhua Zhao (San Diego, CA)

Assignee: Element Biosciences, Inc.

C12N15/1068C12N9/12C12N9/1241C12Q1/6869

Abstract

Claims (23)

1 . A method comprising:

(a) providing a surface having coupled thereto at least one hydrophilic polymer layer, wherein said at least one hydrophilic polymer layer comprises a capture nucleic acid molecule coupled thereto;

(b) contacting said capture nucleic acid molecule with a labeled nucleic acid molecule comprising a fluorescent dye under conditions sufficient for hybridization of said labeled nucleic acid molecule to said capture nucleic acid molecule;

(c) coupling a circular nucleic acid molecule comprising an index site at one or more adaptors of individual circular nucleic acid molecules to at least a portion of said capture nucleic acid molecule; and

(d) obtaining an image of said surface using an inverted microscope and a camera under non-signal saturating conditions while said surface is immersed in a buffer,

wherein said image of said surface exhibits a contrast-to-noise ratio (CNR) of greater than or equal to about 10.

2 . The method of claim 1 , wherein said circular nucleic acid molecule further comprises one or more unique molecular identifiers (UMI).

3 . The method of claim 1 , further comprising, prior to (a), joining a 5′ end and a 3′ end of a linear nucleic acid molecule by an enzyme under conditions sufficient to circularize said linear nucleic acid molecule to form said circular nucleic acid molecule.

4 . The method of claim 3 , further comprising adding said one or more adaptors to said linear nucleic acid molecule.

5 . The method of claim 3 , further comprising generating a 5′ overhang and a 3′ overhang of said linear nucleic acid molecule.

6 . The method of claim 1 , wherein said circular nucleic acid molecule has a length comprising between 100 and 5,000 nucleotides.

7 . The method of claim 1 , wherein said circular nucleic acid molecule has a length comprising at least 10 nucleotides.

8 . The method of claim 3 , wherein said enzyme is a DNA ligase or a protelomerase.

9 . The method of claim 3 , further comprising, prior to (a), fragmenting DNA or RNA using a nucleic acid shearing enzyme or catalytic portion thereof to produce said linear nucleic acid molecule.

10 . The method of claim 1 , wherein said circular nucleic acid molecule comprises DNA.

11 . The method of claim 1 , wherein said circular nucleic acid molecule further comprises a second adaptor.

12 . The method of claim 11 , wherein said second adaptor comprises a P5 site or a P7 site.

13 . The method of claim 1 , wherein said circular nucleic acid molecule further comprises a universal primer binding site recognized by a primer sequence for use in a sequencing reaction to sequence at least a portion of said circular nucleic acid molecule or a derivative thereof.

14 . The method of claim 1 , further comprising performing an amplification reaction on said circular nucleic acid molecule.

15 . The method of claim 14 , wherein said amplification reaction comprises rolling circle amplification.

16 . The method of claim 15 , further comprising sequencing said circular nucleic acid molecule or a derivative thereof.

17 . The method of claim 16 , wherein said sequencing is paired-end sequencing.

18 . The method of claim 1 , wherein said circular nucleic acid molecule comprises a concatemer comprising repeating units of an identical sequence.

Assignments (1)

Continuity (3)

References Cited (206)