Methods and compositions for sequencing double stranded nucleic acids using RCA and MDA

Patent metadata

Field	Value
Publication number	US-11608528-B2
Application number	US-202217814062-A
Country	US
Kind code	B2
Filing date	Jul 21, 2022
Priority date	Mar 3, 2020
Publication date	Mar 21, 2023
Grant date	Mar 21, 2023

Abstract

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

First claim

What is claimed is:

1. A method for determining a sequence, comprising
(a) providing a nucleic acid cluster comprising a concatemer attached to a solid support, wherein the concatemer comprises multiple copies of a sequence unit linked in series, and wherein the sequence unit comprises a target sequence and a primer binding site;
(b) hybridizing a sequencing primer to a primer binding site in a sequence unit of a strand of the concatemer, wherein the strand is covalently attached to the solid support;
(c) extending the sequencing primer along the strand to determine the target sequence from at least a portion of the target sequence in the strand;
(d) digesting the strand;
(e) hybridizing another sequencing primer to a primer binding site in a sequence unit of another strand of the concatemer, wherein the strand and the other strand are the reverse complements of one another, and wherein the strand and the other strand are formed by rolling circle amplification (RCA) and multiple displacement amplification (MDA); and
(f) extending the other sequencing primer along the other strand to determine the target sequence from at least a portion of the target sequence in the other strand.

2. The method of claim 1 , wherein the primer binding site to which the sequencing primer hybridizes to and the primer binding site to which the other sequencing primer hybridizes to are different.

3. The method of claim 1 , further comprising generating the other strand of the concatemer from the strand of the concatemer, optionally wherein generating the other strand comprises generating the other strand using an amplification primer that hybridizes to a primer binding site in a sequence unit of the strand.

4. The method of claim 1 , wherein the extending of the sequencing primer in step (c) and/or extending the other sequencing primer in step (f) comprises repeated cycles of (i) adding a reversibly terminated nucleotide to the primer and (ii) deblocking the reversibly terminated nucleotide on the primer.

5. The method of claim 4 , wherein the extending of the sequencing primer in step (c) and/or step (f) comprises at least 100 of the repeated cycles to determine the target sequence from at least 100 bases of the target sequence in the strand.

6. The method of claim 1 , wherein step (a) comprises
(i) providing a solid support comprising a capture primer;
(ii) hybridizing a nucleic acid template to the capture primer; and
(iii) generating the strand by extending the capture primer along the circular nucleic acid template by rolling circle amplification.

7. The method of claim 6 , wherein step (a) further comprises generating the other strand using an amplification primer that binds to a primer binding site in a sequence unit of the strand.

8. The method of claim 7 , wherein the primer binding site to which the amplification primer hybridizes to is different from the primer binding site to which the sequencing primer hybridizes to and the primer binding site to which the other sequencing primer hybridizes.

9. The method of claim 6 , wherein the nucleic acid template is a circular nucleic acid template.

10. The method of claim 6 , further comprises circularizing the nucleic acid template prior to step (a)(iii).

11. The method of claim 1 , wherein the cluster comprises multiple copies of the other strand of the concatemer.

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

13. The method of claim 1 , wherein the strand comprises one or more nucleotides that are modified or non-canonical and/or one or more nucleotides with bases that are modified or non-canonical, optionally wherein the bases that are modified or non-canonical are uracil.

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

15. The method of claim 14 , wherein the ratio of the modified or non-canonical deoxyribonucleotide trisphosphate in the strand to another deoxyribonucleotide triphosphate or all other deoxyribonucleotide triphosphates in the strand is from about 1:1000 to about 1:10.

16. The method of claim 14 , wherein the concentration of the modified or non-canonical deoxyribonucleotide trisphosphate is from about 0.01 mM to about 1 mM.

17. The method of claim 1 , wherein the digesting is performed using Uracil DNA glycosylase (UDG).

18. A method for determining a sequence, comprising
digesting a strand of a concatemer attached to a solid support, wherein the concatemer comprises multiple copies of a sequence unit linked in series, wherein the sequence unit comprises a target sequence and a primer binding site, wherein the strand is covalently attached to the solid support, wherein the concatemer is formed by rolling circle amplification (RCA) and multiple displacement amplification (MDA);
hybridizing a sequencing primer to a primer binding site in a sequence unit of another strand of the concatemer, wherein the strand and the other strand are the reverse complements of one another; and
extending the sequencing primer along the other strand to determine the target sequence from at least a portion of the target sequence in the other strand.

19. The method of claim 18 , further comprising determining the target sequence of at least a portion of the strand prior to digesting the strand.

Assignees

• Pacific Biosciences California Inc

Inventors

• Patterson Kurt
• Subramanian Hari K K
• Rohrman Brittany A
• Block Fabian

Classifications

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title