Polymer coatings
The present disclosure relates to polymer coatings covalently attached to the surface of a substrate and the preparation of the polymer coatings, such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), in the formation and manipulation of substrates, such as molecular arrays and flow cells. The present disclosure also relates to methods of preparing a substrate surface by using beads coated with a covalently attached polymer, such as PAZAM, and the method of determining a nucleotide sequence of a polynucleotide attached to a substrate surface described herein.
1 . A substrate comprising a polymer coating covalently attached to a surface of the substrate, wherein the polymer coating comprises recurring units of Formula (I):
wherein each R 1 and R 1′ is independently hydrogen or alkyl;
R 2 is H; and
R 3 is substituted alkylamido, or substituted alkylthio wherein R 3 comprises a reactive group selected from the group consisting of optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxyl, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxyl, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol.
2 . The substrate of claim 1 , wherein the polymer coating is covalently attached to the surface via a series of functional groups attached to the surface, wherein the series of functional groups are selected from the group consisting of acrylamide, alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine, maleimide, and benzoyl ester with a phosphine substituent in the ortho position, and combinations thereof.
3 . The substrate of claim 2 , wherein the series of functional groups on the surface comprise alkene, alkyne, or acrylamide, and the polymer coating comprises azido reactive groups.
4 . The substrate of claim 2 , wherein the series of functional groups are components of functionalized silanes on the surface.
5 . The substrate of claim 1 , further comprises a plurality of oligonucleotides covalently attached to the polymer coating through the reactive groups of the polymer coating.
6 . The substrate of claim 1 , further comprises a first plurality of oligonucleotides and a second plurality of oligonucleotides covalently attached to the polymer coating through the reactive groups of the polymer coating.
7 . The substrate of claim 6 , wherein the first plurality of oligonucleotides each comprises a P5 primer sequence, and the second plurality of oligonucleotides each comprises a P7 primer sequence.
8 . A method of preparing an array of polynucleotides on a surface of a substrate, comprising:
contacting the substrate of claim 6 with templates to be amplified, each template comprising at a 3′ end of the template a sequence capable of hybridizing to the first plurality of oligonucleotides, and at a 5′ end of the template a sequence the complement of which is capable of hybridizing to the second plurality of oligonucleotides; and
amplifying the templates using the first oligonucleotides and the second oligonucleotides.
9 . The method of claim 8 , wherein the polymer coating on the surface of the substrate comprises azido reactive groups.
10 . The method of claim 8 , wherein each of the first plurality of oligonucleotides comprises P5 primer sequence and each of the second plurality of oligonucleotides comprises P7 primer sequence.
11 . The method of claim 8 , wherein the surface comprises both polymer coated regions and inert regions.
12 . The substrate of claim 1 , wherein each of R 1 and R 1′ is H or alkyl.
13 . The substrate of claim 1 , wherein polymer coating further comprises recurring units of
14 . The substrate of claim 1 , wherein the surface comprises both polymer coated regions and inert regions.
15 . The substrate of claim 1 , wherein the substrate comprises a flow cell or a bead.
16 . A method of preparing an array of polynucleotides on a surface of a substrate, comprising reacting a plurality of first oligonucleotides and a plurality of second oligonucleotides with the polymer of the substrate of claim 1 .
17 . The method of claim 16 , wherein the first plurality of oligonucleotides each comprises a P5 primer sequence, and the second plurality of oligonucleotides each comprises a P7 primer sequence.
18 . A method of immobilizing a polymer to a surface of a substrate, comprising:
contacting a polymer with a surface of a substrate, wherein the surface comprising a series of functional groups attached thereto, and the polymer comprises recuring-recurring units of Formula (I):
wherein each R 1 and R 1′ is independently hydrogen or alkyl;
R 2 is H;
R 3 is substituted alkylamido, or substituted alkylthio, wherein R 3 comprises a reactive group selected from the group consisting of optionally substituted alkene, optionally substituted alkyne, oxo-amine, azido, formyl, hydroxyl, hydrazinyl, hydrazonyl, cyanuric chloride, thiocyanate, carboxyl, glycidyl, activated ester, epoxy, aziridine, triazoline, and thiol; and
reacting the reactive groups of the polymer with the series of functional groups attached to the surface of the substrate.
19 . The method of claim 18 , wherein the series of functional groups attached to the surface of the substrate are selected from the group consisting of acrylamide, alkene, alkyne, nitrene, aldehyde, hydrazine, activated ester, glycidyl ether, amine, maleimide, and benzoyl ester with a phosphine substituent in the ortho position, and combinations thereof.
20 . The method of claim 19 , wherein the series of functional groups on the surface comprise alkene, alkyne, or acrylamide, and the polymer comprises azido reactive groups.