Substrates and methods useful in sequencing

Patent metadata

Field	Value
Publication number	US-10150992-B2
Application number	US-201615202487-A
Country	US
Kind code	B2
Filing date	Jul 5, 2016
Priority date	Jul 6, 2015
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

A hydrogel network includes a hydrogel polymer having a coupling site, an oligonucleotide conjugated at a terminal end to the hydrogel polymer at the coupling site, and a functional moiety coupled between the terminal end of the oligonucleotide and the coupling site. Such a hydrogel network can be formed by a method including activating a coupling site of a substrate and binding a linker moiety coupled to a terminal end of an oligonucleotide to the activated coupling site, a functional moiety coupled between the terminal end of the oligonucleotide and the linker moiety.

First claim

What is claimed is:

1. A method of forming a nucleic acid substrate, the method comprising:
activating a coupling site of a substrate;
performing ion exchange on a modified oligonucleotide comprising a linker moiety coupled to a 5′ terminal end of an oligonucleotide, a functional moiety coupled between the 5′ terminal end of the oligonucleotide and the linker moiety, wherein the functional moiety is a dye moiety or a buffering moiety, wherein the functional moiety is attached to a modified nucleotide that includes a pyrimidine base, wherein the functional moiety is attached to the 5-position of the pyrimidine base; and
binding the linker moiety of the modified oligonucleotide to the activated coupling site.

2. The method of claim 1 , wherein the dye moiety is a rhodamine moiety.

3. The method of claim 1 , wherein the dye moiety is a fluorescein moiety.

4. The method of claim 1 , wherein the buffering moiety is morpholinoalkyl, triethanolamine, N-[tris(hydroxymethyl) methyl]-2-aminoethanesulfonic acid, 3-(N-tris[hydroxymethyl]methylamino)-2-hydroxypropanesulfonic acid, N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, imidazole, or acetate.

5. The method of claim 1 , wherein the pyrimidine base includes thymidine or cytidine.

6. The method of claim 1 , wherein the coupling site includes a carboxyl moiety and activating includes reacting with a succinimidyl compound.

7. The method of claim 1 , wherein the coupling site includes an amine moiety.

8. The method of claim 1 , further comprising hybridizing a template nucleic acid to the oligonucleotide and extending the oligonucleotide.

9. The method of claim 8 , further comprising sequencing the extended oligonucleotide.

10. The method of claim 1 , wherein the substrate is a hydrogel network.

11. The method of claim 10 , wherein the hydrogel network comprises polyacrylamide.

12. The method of claim 1 , wherein the linker moiety comprises a hydrocarbon, an ether, or polyether group.

13. The method of claim 12 , wherein the linker moiety comprises a hydrocarbon.

14. The method of claim 1 , further comprising generating the modified-oligonucleotide using phosphoramidite modified nucleotides.

Assignees

• Life Technologies Corp

Inventors

• Menchen Steven M
• Xiang Guobing
• Thwar Prasanna Krishnan
• Frediani Jeffrey
• Wong Allen
• Lui Alfred
• Lu Lily

Classifications

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title

• C12Q2525/117

  incorporating modified base · CPC title

• C12Q1/6874

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

• C12Q2565/518

  characterised by the immobilisation of the nucleic acid sample or target · CPC title