Nucleic acid circularization and amplification on a surface

Patent metadata

Field	Value
Publication number	US-12071665-B2
Application number	US-202318176616-A
Country	US
Kind code	B2
Filing date	Mar 1, 2023
Priority date	Oct 22, 2020
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

Abstract

Disclosed herein, inter alia, are compositions and methods for amplification of nucleic acid templates, including hybridizing a linear polynucleotide to an immobilized primer on a surface, circularizing the linear polynucleotide to form a circular polynucleotide, and extending the primer with a polymerase.

First claim

What is claimed is:

1. A method of amplifying a single-stranded polynucleotide, the method comprising:
contacting a solid support with the single-stranded polynucleotide comprising a first primer binding sequence and a second primer binding sequence, and hybridizing said first primer binding sequence and said second primer binding sequence to a splint oligonucleotide attached to the solid support;
extending the first primer binding sequence and ligating the extended first primer binding sequence to the second primer binding sequence together to form a circular polynucleotide;
extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and
hybridizing the amplification product to a second oligonucleotide attached to the solid support comprising a blocking group at a 3′ end that prevents polymerase extension.

2. The method of claim 1 , wherein said second oligonucleotide comprises the second primer binding sequence.

3. The method of claim 1 , wherein said single-stranded polynucleotide comprises a third primer binding sequence, and said second oligonucleotide comprises said third primer binding sequence.

4. The method of claim 1 , wherein said solid support is a bead or substantially planar.

5. The method of claim 1 , wherein said amplification product comprises a plurality of complements of the circular polynucleotide.

6. The method of claim 1 , wherein extending comprises incubating the splint oligonucleotide with a strand-displacing polymerase (a) for about 1 minute to about 2 hours, and/or (b) at a temperature of about 20° C. to about 50° C.

7. The method of claim 6 , wherein said strand-displacing polymerase is a phi29 polymerase, a phi29 mutant polymerase, or a thermostable phi29 mutant polymerase.

8. The method of claim 1 , wherein the circular polynucleotide is about 100 to about 1000 nucleotides or about 1000 to about 5000 nucleotides in length.

9. The method of claim 1 , wherein said amplification product comprises one or more cleavable sites.

10. The method of claim 1 , wherein said solid support comprises an array of wells.

11. The method of claim 1 , wherein said solid support comprises a plurality of splint oligonucleotides randomly distributed on the solid support.

12. The method of claim 1 , further comprising sequencing the amplification product.

13. The method of claim 1 , wherein said solid support comprises a polymer, wherein said splint oligonucleotide and second oligonucleotide are covalently attached to said polymer.

14. A method of forming a plurality of tethered amplification products on a solid support, said method comprising:
contacting a solid support with a sample comprising a plurality of single-stranded polynucleotides
hybridizing a first primer binding sequence and a second primer binding sequence of a single-stranded polynucleotide of said plurality of single-stranded polynucleotides to a splint oligonucleotide attached to the solid support, wherein said splint oligonucleotide comprises, from 5′ to 3′, a first primer sequence, a third sequence, and a second primer sequence, and;
extending the first primer binding sequence to form a complement of the third sequence and ligating the complement of the third sequence and the second primer binding sequence together to form a circular polynucleotide;
extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and hybridizing the amplification product to a second oligonucleotide attached to the solid support, wherein said second oligonucleotide is attached to the solid support at a 5′-end and comprises a blocking group at a 3′ end that prevents polymerase extension, thereby forming a plurality of tethered amplification products.

15. The method of claim 14 , wherein said solid support comprises a plurality of second oligonucleotides.

16. The method of claim 14 , wherein said solid support comprises an array of wells.

17. The method of claim 14 , wherein said solid support comprises a plurality of splint oligonucleotides randomly distributed on the solid support.

18. The method of claim 14 , wherein said solid support comprises a patterned surface.

19. The method of claim 14 , further comprising sequencing said tethered amplification products on said solid support.

20. The method of claim 19 , wherein sequencing comprises hybridizing one or more sequencing primers to the tethered amplification products and generating one or more sequencing reads by detecting a sequence of signals.

21. The method of claim 1 , wherein the splint oligonucleotide includes a first sequence complementary to a first primer sequence, a second primer sequence, and a third primer sequence, wherein the third primer sequence is between the first and the second primer sequence.

22. The method of claim 21 , wherein the third primer sequence is a sequencing primer binding sequence.

23. A method of amplifying a single-stranded polynucleotide, the method comprising:
contacting a solid support with the single-stranded polynucleotide comprising a first primer binding sequence and a second primer binding sequence, and hybridizing said first primer binding sequence and said second primer binding sequence to a splint oligonucleotide attached to the solid support;
ligating the first primer binding sequence and a second primer binding sequence together to form a circular polynucleotide;
extending the splint oligonucleotide with a polymerase to generate an amplification product comprising a complement of the circular polynucleotide; and
hybridizing the amplification product to a second oligonucleotide comprising a blocking group at a 3′ end that prevents polymerase extension, wherein said second oligonucleotide is attached to the solid support.

Assignees

• Singular Genomics Systems Inc

Inventors

• Witters Daan
• Glezer Eli N
• King Andrew
• Shore Sabrina
• Shultzaberger Ryan

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title