Methods for generating circular nucleic acid molecules

Patent metadata

Field	Value
Publication number	US-12134766-B2
Application number	US-202318153268-A
Country	US
Kind code	B2
Filing date	Jan 11, 2023
Priority date	Nov 15, 2018
Publication date	Nov 5, 2024
Grant date	Nov 5, 2024

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 for determining a sequence, comprising:
(a) providing a concatemer, wherein said concatemer comprises (i) an adapter sequence configured to couple to a single surface-bound oligonucleotide and (ii) a strand comprising two or more repeats of an identical sequence, and wherein said two or more repeats of said identical sequence comprise a target sequence and an enzyme recognition site;
(b) coupling said concatemer to a surface;
(c) hybridizing a primer sequence complementary to said target sequence, thereby producing a primed nucleic acid sequence;
(d) extending said primed nucleic acid sequence;
(e) digesting said strand;
(f) hybridizing another primer sequence to another target sequence in another strand of said concatemer, wherein said another strand is complementary to said strand, thereby producing another primed nucleic acid sequence, and wherein said strand and said another strand are formed by rolling circle amplification (RCA) and multiple displacement amplification (MDA); and
(g) extending said another primed nucleic acid sequence.

2. The method of claim 1 , wherein said target sequence and said another target sequence are different.

3. The method of claim 1 , further wherein said another strand is generated from said strand, optionally, wherein a generation of said another strand from said strand comprises using a primer that hybridizes to said target sequence of said strand.

4. The method of claim 1 , wherein step (a) further comprises:
(i) providing a support comprising an immobilized primer;
(ii) hybridizing a nucleic acid to said immobilized primer; and
(iii) performing a rolling circle amplification to extend said immobilized primer along said nucleic acid to generate said strand.

5. The method of claim 4 , further comprising generating said another strand using another immobilized primer that binds to a primer binding site of said strand.

6. The method of claim 5 , wherein said target sequence is different from said primer binding site.

7. The method of claim 1 , wherein said concatemer is a circular nucleic acid template.

8. The method of claim 4 , further comprising circularizing said concatemer prior to step (a)(iii).

9. The method of claim 1 , wherein said strand is generated from a nucleic acid template with isothermal amplification, further wherein said isothermal amplification is a rolling circle amplification.

10. The method of claim 1 , wherein a first portion of said target sequence is determined from said strand and a second portion of said target sequence is determined from said other strand.

11. The method of claim 1 , wherein said strand comprises one or more nucleotides that are modified or one or more nucleotides with bases that are modified.

12. The method of claim 11 , wherein said one or more nucleotides that are modified or said one or more nucleotides with said bases that are modified comprise non-canonical nucleotides.

13. The method of claim 12 , wherein said strand is generated by extending an immobilized primer in a presence of deoxyribonucleotide triphosphates comprising dATP, dTTP, dGTP, dCTP, and a modified or non-canonical deoxyribonucleotide triphosphate, optionally wherein said non-canonical deoxyribonucleotide trisphosphate is dUTP.

14. The method of claim 1 , wherein said concatemer further comprises one or more unique molecular identifiers (UMI).

15. The method of claim 1 , wherein said concatemer has a length comprising between about 100 and about 5,000 nucleotides.

16. The method of claim 1 , wherein said concatemer has a length comprising at least about 10 nucleotides.

17. A method for determining a sequence, comprising:
(a) digesting a strand of a concatemer attached to a solid support, wherein said concatemer comprises a strand comprising two or more repeats of an identical sequence, and wherein said two or more identical sequences comprise a target sequence and an enzyme recognition site;
(b) hybridizing a primer sequence to another target sequence in another strand of said concatemer, wherein said another strand is complementary to said strand, thereby producing another primed nucleic acid sequence; and
(c) extending said another primed nucleic acid sequence.

18. The method of claim 17 , further comprising determining said target sequence of at least a portion of said strand prior to said digesting said strand of (a).

19. The method of claim 17 , wherein said concatemer is a circular nucleic acid template.

20. The method of claim 19 , further comprising circularizing said concatemer.

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