Methods for generating circular nucleic acid molecules

Patent metadata

Field	Value
Publication number	US-12492396-B2
Application number	US-202117320042-A
Country	US
Kind code	B2
Filing date	May 13, 2021
Priority date	Nov 15, 2018
Publication date	Dec 9, 2025
Grant date	Dec 9, 2025

Abstract

Provided herein are methods for generating circular nucleic acid molecules and circular nucleic acid libraries. The methods can be used to generate clonal populations of target nucleic acid molecules for downstream applications such as sequencing.

First claim

What is claimed is:

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.

Assignees

• Element Biosciences Inc

Inventors

• Kellinger Matthew
• Arslan Sinan
• Previte Michael
• Zhao Junhua

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N9/12

  transferring phosphorus containing groups, e.g. kinases (2.7) · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2531/125

  Rolling circle · CPC title