Amplification oligonucleotides

Patent metadata

Field	Value
Publication number	US-11859244-B2
Application number	US-202318185786-A
Country	US
Kind code	B2
Filing date	Mar 17, 2023
Priority date	Sep 3, 2021
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

Disclosed herein, inter alia, are polynucleotides, supports, kits, and methods of use thereof for amplifying, immobilizing, and sequencing polynucleotides.

First claim

What is claimed is:

1. A kit comprising
a first oligonucleotide comprising a first sequencing primer sequence and a second sequencing primer binding sequence;
a second oligonucleotide comprising from 5′ to 3′, a first platform primer sequence and a sequence complementary to the first sequencing primer sequence;
a third oligonucleotide comprising from 5′ to 3′, a second platform primer sequence and a sequence complementary to the second sequencing primer binding sequence;
wherein said first platform primer sequence comprises SEQ ID NO:92; and
said second platform primer sequence comprises SEQ ID NO:89.

2. The kit of claim 1 , wherein the first sequencing primer sequence is SEQ ID NO:152.

3. The kit of claim 2 , wherein the second sequencing primer binding sequence is SEQ ID NO:178.

4. The kit of claim 1 , further comprising a solid support.

5. The kit of claim 4 , wherein said solid support is a flow cell, particle, chip, slide, multi-well container, or unpatterned solid support.

6. The kit of claim 4 , wherein said solid support is a multi-well container.

7. The kit of claim 4 , wherein said solid support further comprises two or more wells, wherein each well comprises a first plurality of immobilized oligonucleotides, wherein each oligonucleotide of the first plurality comprises the first platform primer sequence, and a second plurality of immobilized oligonucleotides, wherein each oligonucleotide of the second plurality comprises the second platform primer sequence.

8. The kit of claim 7 , wherein said solid support comprises about 0.2 wells to about 4.0 wells per μm 2 .

9. The kit of claim 7 , wherein each of the wells is separated from each of the other wells by about 0.2 μm to about 2.0 μm.

10. The kit of claim 1 , further comprising a sequencing polymerase, and one or more amplification polymerases.

11. The kit of claim 10 , further comprising a plurality of native deoxynucleotides (dNTPs) and a plurality of modified dNTPs.

12. The kit of claim 11 , wherein said plurality of modified dNTPs comprises a reversible terminator moiety.

13. The kit of claim 1 , wherein said second oligonucleotide, said third oligonucleotide, or both said second and third oligonucleotides further comprise an index sequence.

14. The kit of claim 13 , wherein said index sequence comprises between 8 to 12 nucleotides.

15. The kit of claim 1 , further comprising a polynucleotide adapter, said polynucleotide adapter comprising a first strand comprising a first binding sequence and a first tail sequence; and a second strand comprising a second binding sequence and a second tail sequence, wherein at least a portion of the first binding sequence is hybridized to a portion of the second binding sequence; wherein the first tail sequence comprises SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:85, SEQ ID NO:92, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, or SEQ ID NO:123 and the second tail sequence comprises SEQ ID NO:4, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, or SEQ ID NO:140, and the first binding sequence comprises SEQ ID NO:152 and the second binding sequence comprises SEQ ID NO:178.

16. The kit of claim 15 , wherein the polynucleotide adapter is a Y polynucleotide adapter.

17. The kit of claim 15 , wherein the polynucleotide adapter is a hairpin adapter.

18. The kit of claim 17 , wherein the hairpin adapter comprises a cleavable site.

19. The kit of claim 17 , wherein the first tail sequence is covalently attached to the second tail sequence.

20. A kit comprising:
a first oligonucleotide comprising from 5′ to 3′, a first platform primer sequence and a first sequencing primer sequence;
a second oligonucleotide comprising from 5′ to 3′, a second platform primer sequence and a second sequencing primer sequence;
wherein said first platform primer sequence comprises SEQ ID NO:92; and
said second platform primer sequence comprises SEQ ID NO:89.

21. The kit of claim 20 , wherein the first sequencing primer sequence is SEQ ID NO:152.

Assignees

• Singular Genomics Systems Inc

Inventors

• Shultzaberger Ryan
• Lipson Allen
• Witters Daan

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6874

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

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6837

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

• B01L3/502707

  characterised by the manufacture of the container or its components · CPC title