De novo surface preparation and uses thereof
Methods and formulations for preparing low non-specific binding surfaces are described, and the prepared surface can provide improved performance for nucleic acid detection and base calling applications. The surface provides more accurate nucleic acid detection, enhanced contrast to noise ratio, and better data collection.
1. A method of performing nucleic acid sequence determination, the method comprising:
(a) providing:
(i) a substrate comprising a surface;
(ii) at least one hydrophilic polymer coating layer on the surface; and
(iii) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer, wherein the plurality of oligonucleotide molecules is present with a surface density of at least 1,000 molecules per squared micrometer (μm 2 ); and
(b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer; and
(c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof, wherein an image of the surface exhibits a contrast-to-noise ratio of at least 20.
2. The method of claim 1 , wherein at least one hydrophilic polymer coating layer comprises a polymer having a molecular weight of at least 1,000 Daltons.
3. The method of claim 1 , wherein the at least one hydrophilic polymer coating layer comprises branched hydrophilic polymer molecules having at least 4 branches.
4. The method of claim 1 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 100 nanomolar (nM).
5. The method of claim 1 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 10 nM.
6. A method of performing nucleic acid sequence determination, the method comprising:
(a) providing:
(i) a substrate comprising a surface;
(ii) at least one hydrophilic polymer coating layer on the surface; and
(iii) a plurality of oligonucleotide molecules comprising a sequence that includes a polymerase stop point, such plurality attached to the at least one hydrophilic polymer coating layer, and present with a surface density of at least 1,000 molecules/μm 2 ;
(b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer; and
(c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules.
7. The method of claim 6 , further comprising amplifying the plurality of sample nucleic acid molecules.
8. The method of claim 7 , wherein amplifying comprises a bridge amplification reaction.
9. The method of claim 7 , wherein amplifying comprises a rolling circle amplification (RCA) reaction.
10. The method of claim 7 , wherein amplifying comprises a helicase-dependent amplification reaction or a recombinase-dependent amplification reaction.
11. The method of claim 6 , wherein the at least one hydrophilic polymer coating layer exhibits a water contact angle of less than 50 degrees.
12. The method of claim 6 , wherein the at least one hydrophilic polymer coating layer comprises a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
13. The method of claim 6 , wherein the at least one hydrophilic polymer coating layer comprises PEG.
14. The method of claim 6 , wherein at least one hydrophilic polymer coating layer comprises a polymer having a molecular weight of at least 1,000 Daltons.
15. The method of claim 6 , wherein the at least one hydrophilic polymer coating layer comprises branched hydrophilic polymer molecules having at least 4 branches.
16. The method of claim 6 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 100 nanomolar (nM).
17. The method of claim 6 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 10 nM.
18. The method of claim 6 , wherein the plurality of sample nucleic acid molecules are clonally-amplified prior to contacting the plurality of oligonucleotide molecules.
19. The method of claim 6 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
20. The method of claim 6 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a double-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
21. The method of claim 6 , wherein a surface density of the plurality of sample nucleic acid molecules is greater than the surface density of the plurality of oligonucleotide molecules.
22. The method of claim 6 , wherein said substrate is positioned on an interior of a flow channel, flow cell, or capillary lumen that is configured for use in performing a nucleic acid hybridization, amplification, or sequencing reaction, or any combination thereof.
23. The method of claim 6 , wherein a background fluorescence intensity measured at a region of the surface that is laterally-displaced from at least one discrete region of the surface having the at least one hydrophilic polymer coating layer and the plurality of oligonucleotide molecules attached thereto is no more than twice of an intensity of said background fluorescence intensity measured at the at least one discrete region.
24. The method of claim 6 , further comprising extending an oligonucleotide of the plurality of oligonucleotide molecules by one nucleotide, wherein the oligonucleotide is coupled to the sample nucleic acid molecule.
25. The method of claim 6 , wherein in (c), the nucleotide is incorporated in the nucleotide binding reaction.
26. A method of performing nucleic acid sequence determination, the method comprising:
(a) providing:
(i) a substrate comprising a surface;
(ii) at least one hydrophilic polymer coating layer on the surface, wherein the at least one hydrophilic polymer coating layer comprises:
(1) a first layer comprising a first monolayer of polymer molecules tethered to the surface of the substrate;
(2) a second layer comprising a second monolayer of polymer molecules tethered to the polymer molecules of the first monolayer; and
(3) a third layer comprising a third monolayer of polymer molecules tethered to the polymer molecules of the second monolayer, wherein the polymer molecules of at least one of the first monolayer, the second monolayer, and the third monolayer comprises branched polymer molecules; and
(iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer, wherein the plurality of ogligonucleotide molecules is present with a surface density of at least 1,000 molecules per squared micrometer (μm 2 );
(b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer; and
(c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof.
27. The method of claim 26 , wherein oligonucleotides of the plurality of oligonucleotides are tethered to the polymer molecules of the second monolayer or third monolayer, and are distributed at a plurality of depths throughout the second layer or the third layer.
28. The method of claim 26 , wherein the plurality of the sample nucleic acid molecules is present at a surface density of at least 10,000 molecules per squared millimeter (mm 2 ).
29. The method of claim 26 , wherein the at least one hydrophilic polymer coating layer exhibits a water contact angle of less than 50 degrees.
30. The method of claim 26 , further comprising amplifying the plurality of sample nucleic acid molecules.
31. The method of claim 30 , wherein amplifying comprises a bridge amplification reaction.
32. The method of claim 30 , wherein amplifying comprises a rolling circle amplification (RCA) reaction.
33. The method of claim 30 , wherein amplifying comprises a helicase-dependent amplification reaction or a recombinase-dependent amplification reaction.
34. The method of claim 26 , wherein the at least one hydrophilic polymer coating layer comprises a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
35. The method of claim 26 , wherein the at least one hydrophilic polymer coating layer comprises PEG.
36. The method of claim 26 , wherein at least one hydrophilic polymer coating layer comprises a polymer having a molecular weight of at least 1,000 Daltons.
37. The method of claim 26 , wherein the branched polymer molecules comprise at least 4 branches.
38. The method of claim 26 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 100 nanomolar (nM).
39. The method of claim 26 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 10 nM.
40. The method of claim 26 , wherein the plurality of sample nucleic acid molecules are clonally-amplified prior to contacting the plurality of oligonucleotide molecules.
41. The method of claim 26 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
42. The method of claim 26 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a double-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
43. The method of claim 26 , wherein a surface density of the plurality of sample nucleic acid molecules is greater than the surface density of the plurality of oligonucleotide molecules.
44. The method of claim 26 , wherein said substrate is positioned on an interior of a flow channel, flow cell, or capillary lumen that is configured for use in performing a nucleic acid hybridization, amplification, or sequencing reaction, or any combination thereof.
45. The method of claim 26 , wherein a background fluorescence intensity measured at a region of the surface that is laterally-displaced from at least one discrete region of the surface having the at least one hydrophilic polymer coating layer and the plurality of oligonucleotide molecules attached thereto is no more than twice of an intensity of said background fluorescence intensity measured at the at least one discrete region.
46. The method of claim 26 , further comprising extending an oligonucleotide of the plurality of oligonucleotide molecules by one nucleotide, wherein the oligonucleotide is coupled to the sample nucleic acid molecule.
47. The method of claim 26 , wherein in (c), the nucleotide is incorporated in the nucleotide binding reaction.
48. A method of performing nucleic acid sequence determination, the method comprising:
(a) providing:
(i) a substrate comprising a surface;
(ii) at least one hydrophilic polymer coating layer on the surface; and
(iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer, wherein the plurality of oligonucleotide molecules is present with a surface density of at least 1,000 molecules/μm 2 ;
(b) contacting a plurality of sample nucleic acid molecules with the plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer; and
(c) performing at least a nucleotide binding reaction or a nucleotide incorporation reaction with a nucleotide that is labeled with a detectable tag and a sample nucleic acid molecule of the plurality of sample nucleic acid molecules or derivative thereof, wherein a fluorescence image of the surface exhibits a contrast-to-noise ratio of at least 20 when the detectable tag is Cyanine dye-3 (Cy3) and when the fluorescence image of the surface is acquired using an inverted fluorescence microscope and a camera under non-signal saturating conditions while the surface is immersed in a buffer.
49. The method of claim 48 , further comprising amplifying the plurality of sample nucleic acid molecules.
50. The method of claim 49 , wherein amplifying comprises a bridge amplification reaction.
51. The method of claim 49 , wherein amplifying comprises a rolling circle amplification (RCA) reaction.
52. The method of claim 49 , wherein amplifying comprises a helicase-dependent amplification reaction or a recombinase-dependent amplification reaction.
53. The method of claim 48 , wherein the at least one hydrophilic polymer coating layer exhibits a water contact angle of less than 50 degrees.
54. The method of claim 48 , wherein the at least one hydrophilic polymer coating layer, comprises a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.
55. The method of claim 48 , wherein the at least one hydrophilic polymer coating layer comprises PEG.
56. The method of claim 48 , wherein said at least one hydrophilic polymer layer comprises a second hydrophilic polymer coating layer.
57. The method of claim 48 , wherein said at least one hydrophilic polymer coating layer comprises a polymer having a molecular weight of at least 1,000 Daltons.
58. The method of claim 48 , wherein said at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least 4 branches.
59. The method of claim 48 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 100 nanomolar (nM).
60. The method of claim 48 , wherein contacting in (b) is performed using the plurality of sample nucleic acid molecules at a concentration of no greater than 10 nM.
61. The method of claim 48 , wherein the plurality of sample nucleic acid molecules are clonally-amplified prior to contacting the plurality of oligonucleotide molecules.
62. The method of claim 48 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a single-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
63. The method of claim 48 , wherein at least one sample nucleic acid of the plurality of sample nucleic acid molecules comprises a double-stranded multimeric nucleic acid molecule comprising repeats of a regularly occurring monomer unit.
64. The method of claim 48 , wherein a surface density of the plurality of sample nucleic acid molecules is greater than the surface density of the plurality of oligonucleotide molecules.
65. The method of claim 48 , wherein said substrate is positioned on an interior of a flow channel, flow cell, or capillary lumen.
66. The method of claim 65 , wherein the flow channel, flow cell, or capillary lumen are configured for use in performing a nucleic acid hybridization, amplification, or sequencing reaction, or any combination thereof.
67. The method of claim 48 , wherein a background fluorescence intensity measured at a region of the surface that is laterally-displaced from at least one discrete region of the surface having the at least one hydrophilic polymer coating layer and the plurality of oligonucleotide molecules attached thereto is no more than twice of an intensity of said background fluorescence intensity measured at the at least one discrete region.
68. The method of claim 48 , wherein the substrate comprises glass or plastic.
69. The method of claim 48 , further comprising extending an oligonucleotide of the plurality of oligonucleotide molecules by one nucleotide, wherein the oligonucleotide is coupled to the sample nucleic acid molecule.
70. The method of claim 48 , wherein in (c), the nucleotide is incorporated in the nucleotide binding reaction.