Granted Patent B2

US 12,331,356 · App. 17/156,452 · Granted Jun 17, 2025

Inventors: Sinan Arslan (San Diego, CA); Chunhong Zhou (San Diego, CA); Molly Min He (San Diego, CA); Matthew Kellinger (San Diego, CA); Hui Zhen Mah (San Diego, CA); Michael Previte (San Diego, CA); Lei Sun (San Diego, CA)

Assignee: ELEMENT BIOSCIENCES, INC.

C12Q1/6876C12N9/1241G01N21/6428G01N33/54306G01N2021/6439

Abstract

Claims (25)

1. A method for nucleic acid identification, the method comprising:

a) providing a surface having coupled thereto at least one polymer layer comprising a hydrophilic polymer coupled to a first nucleic acid molecule;

b) introducing a fluid composition comprising a plurality of nucleic acid molecules comprising a second nucleic acid molecule at a concentration of less than 1 nanomolar to said surface under conditions sufficient to hybridize at least a portion of said second nucleic acid molecule to at least a portion of said first nucleic acid molecule while reducing a nonspecific coupling of non-hybridized nucleic acids of said plurality of nucleic acid molecules to said surface as compared to a method performed without said surface or under said conditions, wherein said second nucleic acid molecule provides a template for a nucleotide binding reaction;

c) contacting said second nucleic acid molecule or derivative thereof with a detectable nucleotide moiety under conditions sufficient to cause the nucleotide binding reaction to occur between said detectable nucleotide moiety and a nucleotide of said second nucleic acid molecule or derivative thereof; and

d) detecting said detectable nucleotide moiety bound to said nucleotide of said second nucleic acid molecule or derivative thereof, thereby identifying said nucleotide,

wherein said method uses a reduced concentration of said second nucleic acid molecule and results in a shortened hybridization time as compared to a method that is not performed using said surface under said conditions.

2. The method of claim 1 , wherein an average length of said second nucleic acid is at least 10 kilobases (kb).

3. The method of claim 1 , further comprising: amplifying said second nucleic acid molecule prior to (c).

4. The method of claim 3 , wherein said amplifying comprises performing rolling circle amplification of said second nucleic acid molecule.

5. The method of claim 1 , wherein said second nucleic acid molecule is double stranded.

6. The method of claim 1 , wherein said nucleotide binding reaction comprises incorporation of said detectable nucleotide moiety into said second nucleic acid molecule or derivative thereof.

7. The method of claim 1 , further comprising preparing said second nucleic acid prior to (b) comprising circularizing a linear nucleic acid molecule to produce said second nucleic acid molecule that is circular.

8. The method of claim 7 , wherein said linear nucleic acid molecule is a concatemer.

9. The method of claim 1 , wherein a density of said first nucleic acid molecule is at least 10,000 molecules per square micrometer (μm 2 ).

10. The method of claim 9 , wherein said density is between about 10,000 μm 2 and about 1,000,000 per μm 2 .

11. The method of claim 1 , wherein said concentration of said second nucleic acid molecule in said fluid composition is 0.50 nanomolar or less.

12. The method of claim 1 , wherein said concentration of said second nucleic acid molecule in said fluid composition is 250 picomolar or less.

13. The method of claim 1 , wherein said concentration of said second nucleic acid molecule in said fluid composition is 100 picomolar or less.

14. The method of claim 1 , wherein said concentration of said second nucleic acid molecule in said fluid composition is 80 picomolar or less.

15. The method of claim 1 , wherein said second nucleic acid molecule is circular.

16. The method of claim 1 , wherein said fluid composition comprises at least 25% acetonitrile.

17. The method of claim 1 , wherein said fluid composition comprises at least 10% formaldehyde.

18. The method of claim 1 , wherein said fluid composition comprises two organic solvents, wherein one of said two organic solvents is polar and aprotic.

19. The method of claim 1 , wherein said fluid composition comprises a pH buffer with a pH of lower than 7.

20. The method of claim 1 , wherein said conditions comprise a temperature of about 50 degrees C.

Assignments (2)

Continuity (9)

References Cited (400)