Substrates and methods useful in sequencing
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.
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.