Substrates and methods useful in sequencing

Patent metadata

Field	Value
Publication number	US-10941439-B2
Application number	US-201816176231-A
Country	US
Kind code	B2
Filing date	Oct 31, 2018
Priority date	Jul 6, 2015
Publication date	Mar 9, 2021
Grant date	Mar 9, 2021

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 sequencing a nucleic acid target, the method comprising:
applying a discrete substrate to a sequencing device, the discrete substrate conjugated to a 5′ terminal end of the nucleic acid target at a coupling site of the discrete substrate, a functional moiety coupled between the 5′ terminal end of the nucleic acid target and the discrete substrate, 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 7-deaza-purine base, wherein the functional moiety is attached to the 7-position of the 7-deaza-purine base;
applying a primer complementary to a portion of the nucleic acid target;
flowing a nucleotide into the sequencing device; and
detecting an incorporation event associated with the nucleotide.

2. The method of claim 1 , further comprising performing ion exchange on the nucleic acid target.

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

4. The method of claim 3 , wherein the dye moiety is a rhodamine moiety.

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

6. The method of claim 1 , wherein the functional moiety is a buffering moiety.

7. The method of claim 6 , wherein the buffering moiety is selected from the group consisting of 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, acetate, and any combination thereof.

8. The method of claim 1 , wherein the 7-deaza-purine base include guanosine or adenosine.

9. The method of claim 1 , further comprising:
forming the nucleic acid target by hybridizing a template nucleic acid to an oligonucleotide coupled to the discrete substrate, the functional moiety coupled between the 5′ terminal end of the oligonucleotide and the discrete substrate; and
extending the oligonucleotide.

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

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

12. The method of claim 11 , wherein the hydrogel network includes polyacrylamide.

Assignees

• Life Technologies Corp

Inventors

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

Classifications

• C12Q1/6834 Primary

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6874

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

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title