Conjugation of carboxyl functional hydrophilic beads

Patent metadata

Field	Value
Publication number	US-10676790-B2
Application number	US-201816206957-A
Country	US
Kind code	B2
Filing date	Nov 30, 2018
Priority date	Jul 2, 2015
Publication date	Jun 9, 2020
Grant date	Jun 9, 2020

Abstract

A polymer substrate, such as a polymer coating or a polymer hydrogel network, includes carboxyl moieties that can be used as conjugation sites to which receptor or analyte molecules can be attached. In an example, the polymer substrate includes a polyacrylamide polymer network having alkanoic acid moieties or derivatives thereof, which can react with carboxyl activating compounds to provide an activated alkanoate moieties on the polyacrylamide network. Amine-terminated nucleic acids can react with the activated alkanoate moieties to capture the nucleic acid to the polymer network through an alkylamide moiety.

First claim

What is claimed is:

1. A method of conjugating a polyacrylamide substrate, the method comprising:
in a non-aqueous solvent, contacting the polyacrylamide substrate with a carboxyl activating compound, the polyacrylamide substrate including a carboxyl moiety, the carboxyl activating compound and the carboxyl moiety reacting to form an activated alkanoate moiety; and
in the non-aqueous solvent, contacting a lipophilic amine-terminated nucleic acid and the polyacrylamide substrate, an amine termination moiety of the liphophilic amine-terminated nucleic acid forming an amide moiety in place of the activated alkanoate moiety.

2. The method of claim 1 , wherein the polyacrylamide further includes an alkanol moiety.

3. The method of claim 1 , wherein the polyacrylamide substrate is a polyacrylamide coating.

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

5. The method of claim 1 , wherein the carboxyl activating compound is a succinimidyl compound.

6. The method of claim 5 , wherein the succinimidyl compound includes a succinimidyl uronium compound, a succinimidyl phosphonium compound, or a combination thereof.

7. The method of claim 6 , wherein the succinimidyl compound is a succinimidyl uronium compound.

8. The method of claim 7 , wherein the succinimidyl uronium is an O-type uronium compound.

9. The method of claim 1 , further comprising contacting a template nucleic acid with the nucleic acid, at least a portion of the template nucleic acid complementary to the nucleic acid.

10. The method of claim 9 , further comprising extending the nucleic acid complementary to the template nucleic acid.

11. The method of claim 10 , further comprising amplifying to make a plurality of nucleic acids complementary to the template nucleic acid.

12. The method of claim 10 , further comprising sequencing the extended nucleic acid.

13. The method of claim 12 , further comprising sequencing with pH-based sequencing.

14. The method of claim 1 , wherein the amine-terminated oligonucleotide includes the amine termination on a 5′ end.

15. The method of claim 1 , wherein the amine termination of the amine-terminated oligonucleotide includes an alkylamine termination moiety.

16. The method of claim 1 , wherein the non-aqueous solvent is a polar solvent.

17. The method of claim 1 , wherein the non-aqueous solvent is not reactive with the activated alkanoate moiety.

18. The method of claim 1 , wherein the non-aqueous solvent is an amide, a urea, a carbonate, an ether, a sulfoxide, a sulfone, a hindered alcohol, or a combination thereof.

19. The method of claim 1 , wherein the non-aqueous solvent is an amide or urea selected from formamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone, N,N,N′,N′-tetramethylurea, N,N′-dimethyl-N,N′-trimethyleneurea, or a combination thereof.

20. The method of claim 1 , wherein the non-aqueous solvent is tetrahydrofuran.

Assignees

• Life Technologies Corp
• Life Tech As

Inventors

• Fonnum Geir
• Menchen Steven M
• Lui Alfred
• Xiang Guobing
• Larsen Synne

Classifications

• C12Q1/6876

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

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C08J2333/26

  Homopolymers or copolymers of acrylamide or methacrylamide · CPC title

• C08J7/14

  with acids, their salts or anhydrides · CPC title