METHOD FOR THE HYBRIDATION OF NUCLEAN ACID

Abstract

Buffer formulations for nucleic acid hybridization and their applications are described, which provide improvements in hybridization specificity, rate, and efficiency. The buffer formulation composition contains a target nucleic acid, at least one polar, aprotic, organic solvent, and a pH buffer system, wherein the target nucleic acid is attached by hybridization with a surface-bound nucleic acid, and wherein the hybridization of the target nucleic acid and the surface-bound nucleic acid exhibits high stringency and melting rate.

Description

CROSS-REFERENCE

[0001] This application is a continuation-in-part of U.S. Application No. 16 / 543,351, filed August 16, 2019, which claims the benefit of U.S. Provisional Application No. 62 / 841,541, filed May 1, 2019, each of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] The present disclosure relates to the field of molecular biology, such as compositions, methods, and systems for nucleic acid hybridization. More particularly, it relates to hybridization compositions and methods for nucleic acid attached to a surface.

[0003] Nucleic acid hybridization protocols represent an important part of many different nucleic acid amplification and analysis techniques. The limited specificity and reaction rates achieved using existing nucleic acid hybridization protocols can have a detrimental effect on the throughput and accuracy of downstream nucleic acid analysis procedures. Stringency control procedures often involve conditions that result in a significant reduction in the number of hybridized complexes. Therefore, there is a need for an improved method to achieve high hybridization stringency during sequencing analysis. SUMMARY

[0004] Methods are provided herein for attaching a target nucleic acid molecule to a surface, the method comprising: contacting a mixture comprising the target nucleic acid molecule, at a concentration of 1 nanomolar or less, with a hydrophilic surface comprising a capture probe coupled thereto, under conditions sufficient for capture of the target nucleic acid molecule by the capture probe in a period of less than 30 minutes.

[0005] In some embodiments, the mixture comprises a polar, aprotic solvent. In some embodiments, the polar, aprotic solvent comprises formamide. In some embodiments, the capture probe is a nucleic acid molecule. In some embodiments, the concentration is 0.50 nanomolar or less. In some embodiments, the concentration is 250 picomolar or less. In some embodiments, the concentration is 100 picomolar or less. In some embodiments, the period is less than or equal to 20 minutes. In some embodiments, the period is less than or equal to 15 minutes. In some embodiments, the period is less than or equal to 10 minutes. In some embodiments, the period is less than or equal to 5 minutes.

[0006] In some embodiments, the temperature of the hydrophilic surface is maintained between about 30 degrees Celsius and about 70 degrees Celsius. In some embodiments, the temperature of the hydrophilic surface is maintained substantially constant. In some embodiments, the methods further comprise hybridizing the target nucleic acid molecule with the capture probe at a hybridization efficiency that is increased compared to a comparable hybridization reaction performed at 90 degrees Celsius for 5 minutes for 120 minutes, followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius in a buffer comprising saline sodium citrate. In some embodiments, the methods further comprise hybridizing the target nucleic acid molecule with the capture probe at a hybridization stringency of at least 80%.

[0007] In some embodiments, the hydrophilic surface has a level of nonspecific cyanine-3 dye adsorption of less than about 0.25 molecules per square micrometer. In some embodiments, the mixture further comprises a pH buffer comprising 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or a combination thereof. In some embodiments, the mixture further comprises a molecular compaction agent selected from a group consisting of polyethylene glycol, dextran, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the hydrophilic surface comprises one or more hydrophilic polymer layers.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0008] Provided herein are methods for hybridizing a target nucleic acid molecule with a nucleic acid molecule coupled to a hydrophilic polymer surface, the method comprising: (a) providing at least one nucleic acid molecule coupled to a hydrophilic polymer surface; and (b) contacting the at least one nucleic acid molecule bound to the hydrophilic polymer surface with a hybridization composition comprising a target nucleic acid molecule at a concentration of 1 nanomolar or less, under conditions sufficient for hybridization of the nucleic acid molecule bound to the hydrophilic polymer surface by the target nucleic acid molecule in 30 minutes or less. In some embodiments, the conditions are maintained at a substantially constant temperature.

[0009] In some embodiments, the hydrophilic polymer surface has a water contact angle of less than 45 degrees. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 0.50 nanomolar or less. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 250 picomolar or less.

[0010] In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 100 picomolar or less. In some embodiments, contacting the at least one nucleic acid molecule coupled to the polymer surface with the hybridization composition is performed within a period of less than 30 minutes. In some embodiments, the period is less than 20 minutes. In some embodiments, the period is less than 15 minutes. In some embodiments, the period is less than 10 minutes. In some embodiments, the period is less than 5 minutes.

[0011] In some embodiments, the methods further comprise hybridizing the target nucleic acid molecule with the at least one nucleic acid molecule coupled to the polymer surface, at a hybridization efficiency that is increased compared to a comparable hybridization reaction conducted at 90 degrees Celsius for 5 minutes for 120 minutes, followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius in a buffer comprising saline sodium citrate. In some embodiments, the temperature is between about 30 degrees Celsius and 70 degrees Celsius. In some embodiments, the temperature is about 50 degrees Celsius. In some embodiments, the methods further comprise hybridizing the target nucleic acid molecule with the at least one nucleic acid molecule with a hybridization stringency of at least 80%.In some embodiments, the hydrophilic polymer surface has a level of nonspecific cyanine-3 dye adsorption of less than about 0.25 molecules per square micrometer.

[0012] In some embodiments, the hybridization composition further comprises: (a) at least one organic solvent having a dielectric constant of no more than about 115 as measured at 68 degrees Fahrenheit; and (b) a pH buffer. In some embodiments, the hybridization composition further comprises: (a) at least one organic solvent that is polar and aprotic; and (b) a pH buffer. In some embodiments, the at least one organic solvent comprises at least one functional group selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the at least one organic aprotic solvent comprises formamide. In some embodiments, the at least one organic aprotic solvent is miscible with water.In some embodiments, the at least one organic solvent is at least about 5% by volume based on the total volume of the hybridization composition. In some embodiments, the at least one organic solvent is at most 95% by volume based on the total volume of the hybridization composition.

[0013] In some embodiments, the pH buffer is at most about 90% by volume of the total volume of the hybridization composition. In some embodiments, the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or a combination thereof. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer is present in the hybridization composition in an amount effective to maintain the pH of the hybridization composition in a range of about 3 to about 10.

[0014] In some embodiments, the hybridization composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol, dextran, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol. In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of the molecular compaction agent is at least approximately 5% by volume based on the total volume of the hybridization composition.In some embodiments, the amount of the molecular compaction agent is at most approximately 50% by volume based on the total volume of the hybridization composition. In some embodiments, the at least one nucleic acid molecule is coupled to the polymer surface by covalent bonding.

[0015] In some embodiments, the hydrophilic polymer surface comprises one or more hydrophilic polymer layers, and the at least one nucleic acid molecule is coupled to the one or more hydrophilic polymer layers. In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0016] Provided herein are methods for attaching a target nucleic acid to a surface, comprising: (a) providing at least one surface-bound nucleic acid attached to a polymer surface with a water contact angle of less than 45 degrees; and (b) contacting the surface-bound nucleic acid with a hybridization composition under isothermal conditions, wherein the hybridization composition comprises: (i) the target nucleic acid; (ii) at least one organic solvent having a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit; and (iii) a pH buffer.

[0017] In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent having a dielectric constant no greater than 40 as measured at 68 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic, aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the hybridization composition.In some embodiments, the amount of organic solvent ranges between approximately 5 to 95% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG).In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the hybridization composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid is at least approximately 2% by volume based on the total volume of the hybridization composition.In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between approximately 2 to 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (e.g., [tris(hydroxymethyl)methylamino]propanesulfonic acid), Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES (e.g., 24[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), EPPS (e.g., 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid)), and MOPS (e.g., 3-(N-morpholino)propanesulfonic acid). In some embodiments, the pH buffer further comprises a second organic solvent.In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, the amount of pH buffer is effective to maintain the pH of the hybridization composition in the range between about 3 and about 10.

[0018] In some embodiments, the surface-bound nucleic acid is coupled to the surface by covalent or non-covalent bonding. In some embodiments, the polymer surface comprises one or more hydrophilic polymer layers, and wherein the surface-bound nucleic acid is coupled to the one or more hydrophilic polymer layers. In some embodiments, no more than 10% of the target nucleic acid is associated with the surface without hybridizing to the polymer surface of the nucleic acid. In some embodiments, the polymer surface has a level of non-specific cyanine-3 dye (Cy3) adsorption of less than about 0.25 molecules per square micrometer (µm 2). In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0019] In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of no more than 25 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of no more than 15 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of between 2 and 25 minutes. In some embodiments, the isothermal conditions are in a temperature range between approximately 30 and 70 degrees Celsius. Some embodiments comprise hybridizing the target nucleic acid with a surface-bound nucleic acid with a hybridization stringency of at least 80%.Some embodiments comprise hybridizing the target nucleic acid to the surface-bound nuclein with increased hybridization efficiency compared to a comparable hybridization reaction in which the organic buffer is saline sodium citrate and hybridization is carried out at 90 degrees Celsius for 5 minutes, followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the target nucleic acid is present in the hybridization composition at a concentration of 1 nanomolar or less. In some embodiments, the target nucleic acid is present in the hybridization composition at a concentration of 250 picomolar or less. In some embodiments, the target nucleic acid is present in the hybridization composition at a concentration of 100 picomolar or less.In some embodiments, the target nucleic acid is present in the hybridization composition at a concentration of 50 picomolar or less. In some embodiments, the methods further comprise hybridizing at least a portion of the surface-bound nucleic acid with at least a portion of the target nucleic acid in the hybridization composition, wherein the hybridization does not consist of cooling.

[0020] Methods for hybridization are provided herein, the method comprising: (a) providing at least one surface-bound nucleic acid molecule coupled to a surface; and (b) contacting the at least one surface-bound nucleic acid molecule with the hybridization composition comprising a target nucleic acid molecule, wherein the hybridization composition comprises: (i) at least one organic solvent; and (ii) a pH buffer. In some embodiments, the surface has a level of non-specific Cy3 dye adsorption that is less than about 0.25 molecules / µm 2 as measured by a fluorescence imaging system under non-signal saturating conditions. In some embodiments, no more than 5% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the surface-bound nucleic acid molecule.

[0021] In some embodiments, the surface-bound nucleic acid molecule is coupled to the surface by being bound to the surface. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the hydrophilic polymer surface has a water contact angle of less than 45 degrees. In some embodiments, the at least one organic solvent has a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit. In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent with a dielectric constant of no greater than 40 as measured at 68 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide.In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of organic solvent ranges between about 5 to 95% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the hybridization composition.In some embodiments, the hybridization composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG). In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of the molecular compaction agent is at least approximately 5% by volume based on the total volume of the hybridization composition.In some embodiments, the amount of the molecular compaction agent is less than 50% by volume based on the total volume of the hybridization composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid is at least about 2% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between about 2 to 50% by volume based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one of the buffering agents selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS.In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, the amount of pH buffer is effective to maintain the pH of the hybridization composition in the range between about 3 and about 10. In some embodiments, the surface-bound nucleic acid is coupled to the surface by covalent or non-covalent bonding. In some embodiments, the polymer surface comprises one or more hydrophilic polymer layers, and wherein the surface-bound nucleic acid is coupled to the one or more hydrophilic polymer layers. In some embodiments, no more than 10% of the target nucleic acid is associated with the surface without hybridizing with the nucleic acid bound to the polymer surface.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0022] In some embodiments, contacting the surface-bound nucleic acid molecule with the hybridization composition is performed for a period of no more than 25 minutes. In some embodiments, contacting the surface-bound nucleic acid molecule with the hybridization composition is performed for a period of no more than 15 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of between 2 and 25 minutes. In some embodiments, the isothermal conditions are in a temperature range between approximately 30 and 70 degrees Celsius. Some embodiments comprise hybridizing the target nucleic acid molecule with a surface-bound nucleic acid molecule with a hybridization stringency of at least 80%.Some embodiments comprise hybridizing the target nucleic acid molecule with the surface-bound nucleic acid molecule with an increased hybridization efficiency compared to a comparable hybridization reaction in which the organic buffer is saline sodium citrate and hybridization is carried out at 90 degrees Celsius for 5 minutes, followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 1 nanomolar or less. In some embodiments, the target nucleic acid is present in the hybridization composition at a concentration of 250 picomolar or less. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 100 picomolar or less.In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 50 picomolar or less. In some embodiments, the methods further comprise hybridizing at least a portion of the surface-bound nucleic acid molecule with at least a portion of the target nucleic acid molecule in the hybridization composition, wherein the hybridization does not consist of cooling. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition comprising the target nucleic acid is under stringency conditions that prevent the target nucleic acid molecule from hybridizing with a non-complementary nucleic acid molecule. In some embodiments, the stringency is at least or about 70%, 80%, or 90%. In some embodiments, the stringency is at least 80%.Provided herein are methods for attaching a target nucleic acid molecule to a surface, the method comprising: (a) providing at least one surface-bound nucleic acid molecule, wherein the at least one surface-bound nucleic acid molecule is coupled to a surface; and (b) contacting a hybridization composition comprising a target nucleic acid molecule with the at least one surface-bound nucleic acid molecule, wherein the hybridization composition comprises: (i) at least one organic solvent; and (ii) a pH buffer. In some embodiments, the polymer surface has a level of nonspecific Cy3 dye adsorption of less than about 0.25 molecules / µm. 2In some embodiments, no more than 5% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the surface-bound nucleic acid molecule. In some embodiments, contacting the hybridization composition with the at least one surface-bound nucleic acid molecule occurs under isothermal conditions. In some embodiments, the surface-bound nucleic acid molecule is coupled to the surface by binding to the surface. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the hydrophilic polymer surface has a water contact angle of less than 45 degrees.

[0023] In some embodiments, the at least one organic solvent has a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit. In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent having a dielectric constant of no greater than 40 as measured at 70 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic, aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid.In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of organic solvent is in the range between about 5 to 95% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof.In some embodiments, the molecular compaction agent is polyethylene glycol (PEG). In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the hybridization composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid molecule is at least approximately 2% by volume based on the total volume of the hybridization composition.In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between approximately 2 to 50% by volume based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, the amount of the pH buffer is effective to maintain the pH of the hybridization composition in the range between approximately 3 and approximately 10.

[0024] In some embodiments, the surface-bound nucleic acid molecule is coupled to the surface by covalent or non-covalent bonding. In some embodiments, the polymer surface comprises one or more hydrophilic polymer layers, and the surface-bound nucleic acid is coupled to the one or more hydrophilic polymer layers. In some embodiments, no more than 10% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the polymer surface-bound nucleic acid molecule.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer. In some embodiments, contacting the surface-bound nucleic acid molecule with the hybridization composition is performed for a period of no more than 25 minutes.In some embodiments, contacting the surface-bound nucleic acid molecule with the hybridization composition is performed for a period of no more than 15 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of between 2 and 25 minutes. In some embodiments, the isothermal conditions are in a temperature range between approximately 30 and 70 degrees Celsius. Some embodiments involve hybridizing the target nucleic acid molecule with the surface-bound nucleic acid molecule with a hybridization stringency of at least 80%.Some embodiments comprise hybridizing the target nucleic acid molecule with the surface-bound nucleic acid molecule with increased hybridization efficiency compared to a hybridization reaction in which the organic buffer is saline sodium citrate and hybridization is carried out at 90 degrees Celsius for 5 minutes, followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 1 nanomolar or less. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 250 picomolar or less. In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 100 picomolar or less.In some embodiments, the target nucleic acid molecule is present in the hybridization composition at a concentration of 50 picomolar or less. In some embodiments, the methods further comprise hybridizing at least a portion of the surface-bound nucleic acid molecule with at least a portion of the target nucleic acid molecule in the hybridization composition, wherein the hybridization does not consist of cooling.

[0025] Provided herein are methods for sequencing a target nucleic acid molecule, comprising: (a) contacting a surface-bound nucleic acid molecule coupled to a surface with a hybridization composition comprising a target nucleic acid molecule, the hybridization composition comprising: (i) at least one organic solvent; and (ii) a pH buffer; (b) amplifying the target nucleic acid molecule to form a plurality of clonally amplified clusters of the target nucleic acid; and (c) determining the identity of the target nucleic acid molecule, wherein a fluorescence image of the surface comprising the plurality of clonally amplified clusters of the target nucleic acid molecule exhibits a contrast-to-noise ratio (CNR) of at least 20 when the fluorescence image is acquired using a fluorescence imaging system under non-signal saturating conditions.In some embodiments, the methods further comprise hybridizing the target nucleic acid molecule with the at least one surface-bound nucleic acid coupled to a surface. In some embodiments, the CNR is at least 50. In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent having a dielectric constant of no greater than 40 as measured at 70 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic, aprotic solvent is miscible with water.In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of organic solvent ranges between about 5 to 95% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular compaction agent.In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG). In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the hybridization composition.In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid molecule. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid molecule is at least about 2% by volume based on the total volume of the hybridization composition. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid molecule ranges between about 2 to 50% by volume based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol.In some embodiments, the amount of pH buffer is effective to maintain the pH of the hybridization composition in the range between about 3 and about 10.

[0026] In some embodiments, the surface-bound nucleic acid molecule is coupled to the surface by covalent or non-covalent bonding. In some embodiments, the polymer surface comprises one or more hydrophilic polymer layers, and wherein the surface-bound nucleic acid molecule is coupled to the one or more hydrophilic polymer layers. In some embodiments, the polymer surface has a level of non-specific cyanine 3 dye (Cy3) adsorption of less than about 0.25 molecules per square micrometer (µm 2). In some embodiments, no more than 5% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the surface-bound nucleic acid molecule. In some embodiments, no more than 10% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the surface-bound nucleic acid molecule. In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition occurs under isothermal conditions. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed in a temperature range between 30 and 70 degrees Celsius. In some embodiments, contacting the surface-bound nucleic acid molecule with the hybridization composition is performed within a period of no more than 25 minutes. In some embodiments, the methods further comprise removing the hybridization composition from the surface after a period of no more than 25 minutes.In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of time between 2 and 25 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of time between 2 and 4 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is performed for a period of time of 2 minutes. In some embodiments, the at least one surface-bound nucleic acid molecule is circular. In some embodiments, the methods further comprise hybridizing at least a portion of the surface-bound nucleic acid molecule with at least a portion of the target nucleic acid in the hybridization composition, wherein the hybridization does not consist of cooling.In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition comprising the target nucleic acid occurs under stringency conditions that prevent the target nucleic acid from hybridizing with a non-complementary nucleic acid. In some embodiments, the stringency is at least or approximately 70%, 80%, or 90%. In some embodiments, the stringency is at least 80%.

[0027] Provided herein are compositions for hybridizing a target nucleic acid molecule with a surface-bound nucleic acid molecule, the composition comprising: (a) a target nucleic acid molecule; (b) at least one organic solvent; and (c) a pH buffer. In some embodiments, no more than 10% of the total number of the target nucleic acid molecule is associated with the surface without hybridizing with the surface-bound nucleic acid molecule. In some embodiments, no more than 5% of the total number of the target nucleic acid molecule is bound to the surface without hybridizing with the surface-bound nucleic acid molecule.

[0028] In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent having a dielectric constant no greater than 40 as measured at 70 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic, aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the composition.In some embodiments, the amount of organic solvent ranges between approximately 5 to 95% by volume based on the total volume of the composition. In some embodiments, the pH buffer system comprises a pH buffer. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the composition. In some embodiments, the composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG).In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid molecule. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid molecule is at least approximately 2% by volume based on the total volume of the composition.In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid molecule ranges between approximately 2 to 50% by volume based on the total volume of the composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, an amount of the pH buffer is effective to maintain the pH of the composition in the range between approximately 3 and approximately 10.

[0029] In some embodiments, the surface-bound nucleic acid molecule is coupled to a surface by covalent or non-covalent bonding. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the polymer surface comprises one or more hydrophilic polymer layers, and the surface-bound nucleic acid molecule is coupled to the one or more hydrophilic polymer layers.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer. In some embodiments, the target nucleic acid molecule is present in the composition at a concentration of 1 nanomolar or less. In some embodiments, the target nucleic acid molecule is present in the composition at a concentration of 250 picomolar or less.In some embodiments, the target nucleic acid molecule is present in the composition at a concentration of 100 picomolar or less. In some embodiments, the target nucleic acid molecule is present in the composition at a concentration of 50 picomolar or less.

[0030] Provided herein, in some embodiments, are microfluidic systems comprising the composition described herein. In some embodiments, the microfluidic systems comprise a flow cell device. In some embodiments, the flow cell device is a microfluidic chip flow cell. In some embodiments, the flow cell device is a capillary flow cell device.In some embodiments, at least one surface of the flow cell device comprises one or more hydrophilic polymer layers comprising a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the flow cell device comprises a composition described herein formulated as a fluid. In some embodiments, the flow cell device comprises one or more surface-bound nucleic acid molecules coupled to the at least one surface of the flow cell.In some embodiments, the target nucleic acid molecule in the composition is hybridized with one or more surface-bound nucleic acid molecules coupled to at least one surface of the flow cell. In some embodiments, the flow cell device is operatively coupled to an imaging system to capture the image of the at least one surface of the flow cell device comprising the hybridized target nucleic acid molecule and the one or more surface-bound nucleic acid molecules. The methods described herein include determining an identity of the target nucleic acid molecule using the microfluidic system described herein.

[0031] Provided herein are kits comprising: (a) a surface; and (b) a composition comprising: (i) at least one organic solvent; and (ii) a pH buffer. In some embodiments, the surface comprises one or more surface-bound nucleic acid molecules coupled to the surface. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the hydrophilic polymer surface has a water contact angle of less than 45 degrees. In some embodiments, the hydrophilic polymer surface comprises one or more hydrophilic polymer layers, and wherein the surface-bound nucleic acid is coupled to the one or more hydrophilic polymer layers.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran. In some embodiments, the kit further comprises instructions for hybridizing the one or more surface-bound nucleic acid molecules with one or more target nucleic acid molecules. In some embodiments, the kit further comprises instructions for determining the identity of one or more target nucleic acid molecules.

[0032] In some embodiments, the organic solvent is a polar, aprotic solvent. In some embodiments, the organic solvent is an organic solvent having a dielectric constant no greater than 40 as measured at 70 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic, aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the composition.In some embodiments, the amount of organic solvent ranges between approximately 5 to 95% by volume based on the total volume of the composition. In some embodiments, the pH buffer system further comprises a pH buffer. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the composition. In some embodiments, the composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG).In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the one or more target nucleic acid molecules. In some embodiments, the amount of the additive for controlling the melting temperature of the one or more target nucleic acid molecules is at least approximately 2% by volume based on the total volume of the composition.In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between approximately 2 to 50% by volume based on the total volume of the composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, the amount of the pH buffer is effective to maintain the pH of the composition in the range between approximately 3 and approximately 10.

[0033] Methods are provided herein for using the kits described herein. In some embodiments, surface-bound nucleic acid molecules are coupled to the surface by a covalent or non-covalent bond. In some embodiments, the methods comprise: (a) combining the one or more target nucleic acid molecules and the composition of the kit to form a master mix; and (b) contacting the master mix with one or more surface-bound nucleic acid molecules coupled to the surface provided in the kit. In some embodiments, the methods further comprise (c) hybridizing one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to a surface. In some embodiments, the polymer surface has a level of non-specific Cy3 dye adsorption of less than about 0.25 molecules / µm 2In some embodiments, no more than 10% of the total number of the one or more target nucleic acid molecules are associated with the surface without hybridizing with the surface-bound nucleic acid molecule. In some embodiments, no more than 5% of the total number of the one or more target nucleic acid molecules are associated with the surface without hybridizing with the one or more surface-bound nucleic acid molecules. In some embodiments, the hybridization of one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to a surface is performed under isothermal conditions. In some embodiments, the isothermal conditions are performed in a temperature range between 30 and 70 degrees Celsius.In some embodiments, the methods further comprise (d) amplifying the target nucleic acid hybridized with the surface-bound nucleic acid to form a plurality of clonally amplified clusters of one or more target nucleic acid molecules coupled to the surface; and (c) determining the identity of the one or more target nucleic acid molecules. In some embodiments, a fluorescence image of the surface comprising the plurality of clonally amplified clusters of the one or more target nucleic acid molecules exhibits a contrast-to-noise ratio (CNR) of at least 20 when the fluorescence image is acquired using a fluorescence imaging system under non-signal saturating conditions. In some embodiments, the CNR is at least 50.

[0034] In some embodiments, the hybridization of the surface-bound nucleic acid with the target nucleic acid is performed in a period of no more than 25 minutes. In some embodiments, the methods further comprise removing the composition from the surface after a period of no more than 25 minutes. In some embodiments, the hybridization of the surface-bound nucleic acid with the target nucleic acid is performed in a period of between 2 and 25 minutes. In some embodiments, the hybridization of the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed in a period of between 2 and 4 minutes. In some embodiments, the hybridization of the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed in a period of 2 minutes.In some embodiments, the at least one surface-bound nucleic acid is circular. In some embodiments, hybridization does not involve cooling. In some embodiments, contacting the master mix with the one or more surface-bound nucleic acid molecules occurs under stringency conditions that prevent the one or more target nucleic acid molecules from hybridizing with a non-complementary nucleic acid. In some embodiments, the stringency is at least or approximately 70%, 80%, or 90%. In some embodiments, the stringency is at least 80%.

[0035] Provided herein are systems comprising: (a) a surface comprising one or more surface-bound nucleic acid molecules coupled to the surface; (b) one or more nucleic acid molecules; and (c) a composition comprising: (i) at least one organic solvent; and (ii) a pH buffer. In some embodiments, the systems further comprise a fluorescence imaging device. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the hydrophilic polymer surface has a water contact angle of less than 45 degrees. In some embodiments, the hydrophilic polymer surface comprises one or more hydrophilic polymer layers, and wherein the one or more surface-bound nucleic acid molecules are coupled to the one or more hydrophilic polymer layers.In some embodiments, the one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), polyacrylic acid (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

[0036] In some embodiments, the organic solvent is an organic solvent having a dielectric constant no greater than 40 as measured at 70 degrees Fahrenheit. In some embodiments, the organic solvent is acetonitrile, alcohol, or formamide. In some embodiments, the organic solvent comprises at least one functionality selected from hydroxy, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic aprotic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature a double-stranded nucleic acid. In some embodiments, the amount of organic solvent is at least about 5% by volume based on the total volume of the composition.In some embodiments, the amount of organic solvent ranges between approximately 5 to 95% by volume based on the total volume of the composition. In some embodiments, the pH buffer system further comprises a pH buffer. In some embodiments, the amount of pH buffer is at most 90% by volume based on the total volume of the composition. In some embodiments, the composition further comprises a molecular compaction agent. In some embodiments, the molecular compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. In some embodiments, the molecular compaction agent is polyethylene glycol (PEG).In some embodiments, the molecular compaction agent has a molecular weight in the range between approximately 5,000 and 40,000 Daltons. In some embodiments, the amount of molecular compaction agent is at least approximately 5% by volume based on the total volume of the composition. In some embodiments, the amount of molecular compaction agent is less than 50% by volume based on the total volume of the composition. In some embodiments, the methods further comprise an additive for controlling a melting temperature of the target nucleic acid. In some embodiments, the amount of the additive for controlling the melting temperature of the one or more target nucleic acid molecules is at least approximately 2% by volume based on the total volume of the composition.In some embodiments, the amount of the additive for controlling the melting temperature of the one or more target nucleic acid molecules ranges between approximately 2 to 50% by volume based on the total volume of the composition. In some embodiments, the pH buffer comprises at least one buffering agent selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, BisTris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES, EPPS, and MOPS. In some embodiments, the pH buffer further comprises a second organic solvent. In some embodiments, the pH buffer comprises MOPS and methanol. In some embodiments, the amount of the pH buffer is effective to maintain the pH of the composition in the range between approximately 3 and approximately 10.

[0037] Methods for using the systems described herein are provided. In some embodiments, the one or more surface-bound nucleic acid molecules are coupled to the surface by a covalent or non-covalent bond. In some embodiments, the methods comprise: (a) combining the one or more target nucleic acid molecules and the composition of the system to form a master mix; (b) contacting the master mix with one or more surface-bound nucleic acid molecules coupled to the surface provided by the system; (c) hybridizing the one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to the surface;(d) amplifying the one or more target nucleic acid molecules hybridized with the one or more surface-bound nucleic acid molecules to form a plurality of clonally amplified clusters of the one or more surface-bound target nucleic acid molecules; and (e) determining the identity of the one or more target nucleic acid molecules by capturing an image of the surface with the fluorescence imaging device. In some embodiments, the polymer surface exhibits a level of nonspecific Cy3 dye adsorption of less than about 0.25 molecules / µm; 2. In some embodiments, the hybridization of one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to a surface is performed under isothermal conditions. In some embodiments, the isothermal conditions are performed in a temperature range between 30 and 70 degrees Celsius. In some embodiments, no more than 10% of a total number of the one or more target nucleic acid molecules are associated with the surface without hybridizing with the one or more surface-bound nucleic acid molecules. In some embodiments, no more than 5% of the total number of the one or more target nucleic acid molecules are associated with the surface without hybridizing with the one or more surface-bound nucleic acid molecules.In some embodiments, a fluorescence image of the surface comprising one or more amplified target nucleic acid molecules exhibits a contrast-to-noise ratio (CNR) of at least 20 when the fluorescence image is acquired using the fluorescence imaging device under non-signal saturating conditions. In some embodiments, the CNR is at least 50.

[0038] In some embodiments, hybridizing the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed in a period of no more than 25 minutes. In some embodiments, the methods further comprise removing the composition from the surface after a period of no more than 25 minutes. In some embodiments, hybridizing the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed in a period of between 2 and 25 minutes. In some embodiments, hybridizing the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed in a period of between 2 and 4 minutes.In some embodiments, the hybridization of the one or more surface-bound nucleic acid molecules with the one or more target nucleic acid molecules is performed within a period of 2 minutes. In some embodiments, the at least one surface-bound nucleic acid is circular. In some embodiments, the hybridization does not involve cooling. In some embodiments, contacting one or more surface-bound nucleic acid molecules with the hybridization composition comprising the one or more target nucleic acid molecules is performed under stringency conditions that prevent the one or more target nucleic acid molecules from hybridizing with a non-complementary nucleic acid molecule. In some embodiments, the stringency is at least or approximately 70%, 80%, or 90%. In some embodiments, the stringency is at least 80%. RECORDING BY REFERENCE

[0039] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application were specifically and individually incorporated by reference into this document. In the event of a conflict between a term used herein and a term in an incorporated document, the term used herein shall control. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and upon payment of the necessary fee.

[0041] Some novel features of the methods and compositions disclosed herein are set forth in the present disclosure. A better understanding of the features and advantages of the methods and compositions disclosed herein will be achieved by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the disclosed compositions and methods are utilized, and the accompanying drawings thereof: Fig. 1A-B: provide non-limiting examples of image data demonstrating improvements in hybridization stringency, speed, and efficiency by reformulating the hybridization buffer used for solid-phase nucleic acid amplification as described herein. Fig. Figure 1A provides examples of image data concerning two different hybridization buffer formulations and protocols. Fig.Figure 1B provides an example of corresponding image data acquired using a standard hybridization buffer and protocol. Fig. Figure 2 illustrates the workflow for sequencing nucleic acid on weakly binding surfaces using the disclosed hybridization methods, and non-limiting examples of achievable processing times. Fig. Figure 3 shows the surface pattern hybridization images (NASA results at 100pM) of the samples corresponding to the compositions used for hybridization. Fig. Figure 4 shows a table with the hybridization design for experimental spot counts. Fig. Figure 5 shows the PCR images of the samples after surface amplification of the nucleic acid. Fig. 6 shows the workflow according to the various embodiments disclosed herein. Fig.Figure 7 shows the workflow for a sequence reaction according to the various embodiments described herein. Fig. Figure 8 shows an exemplary workflow for nucleic acid hybridization according to the various embodiments described herein. Fig. 9A-9B show how sample nucleic acids hybridized to the nucleic acid molecules coupled to the surface with weak, nonspecific binding are visualized according to the various embodiments described herein ( Fig. 9A) or amplified ( Fig. 9B). Fig. Figure 10 schematically shows an example of a computer control system. Fig. 11 shows a workflow for purifying and isolating sample nucleic acids from a biological sample, creating a directory, and hybridizing using various embodiments disclosed herein. DETAILED DESCRIPTION

[0042] Disclosed herein are methods, compositions, systems, and kits for hybridizing nucleic acids with nucleic acid molecules coupled to a surface. The methods, compositions, systems, and kits described herein are particularly useful in nucleic acid amplification, nucleic acid sequencing, or a combination thereof. The methods, compositions, systems, and kits described herein enable improved nucleic acid hybridization performance and can be performed in a fraction of the time and at a fraction of the cost compared to standard nucleic acid hybridization methods. This is achieved by using optimized hybridization compositions (e.g., buffers, organic solvents) in combination with low, nonspecific binding surfaces that are hydrophilic.

[0043] Existing standard nucleic acid hybridization methods are complex, time-consuming, and lack the specificity and efficacy needed for cost-effective, high-throughput applications. Existing hybridization methods often require high temperatures (e.g., 90 degrees Celsius), long incubation times (e.g., 1-2 hours), and a large amount of nucleic acid as starting material (e.g., 10 nanomolar). At least one reason standard nucleic acid hybridization methods lack specificity and efficacy is that they use surfaces prone to nonspecific binding of proteins or nucleic acids, which contributes to increased background signal.

[0044] Compared to standard nucleic acid hybridization techniques, the methods, compositions, systems, and kits described herein provide improved hybridization specificity and efficiency for target nucleic acid molecules with surface-bound nucleic acid molecules. These methods and systems utilize a low-nonspecific binding surface to reduce background signal. The low-nonspecific binding surfaces described herein are designed to prevent proteins, nucleic acids, and other biomolecules from "sticking" to the surface substrate. The low-nonspecific binding surfaces described herein are hydrophilic. In some cases, the low-nonspecific binding surfaces have a water contact angle of less than or equal to 50 degrees.

[0045] In some embodiments, the methods comprise hybridizing a target nucleic acid with a nucleic acid molecule coupled to a hydrophilic surface (e.g., a surface with low, nonspecific binding) using the hybridization compositions described herein. The methods described herein are useful for hybridizing, amplifying, or sequencing nucleic acid, or a combination thereof. The methods described herein achieve improved hybridization performance on surfaces with low, nonspecific binding. In addition, the methods described herein achieve nonspecific cyanine-3 dye (Cy3) adsorption of less than 0.25 molecules / µm 2 .

[0046] Optimized hybridization compositions described here, particularly when used with a surface with low nonspecific binding, enable isothermal hybridization reactions performed at 60 degrees Celsius for only 2 minutes using starting nucleic acid with a concentration of no more than 50 picomolar.Compared to a comparable hybridization reaction using standard hybridization protocols and reagents, the methods described herein provide (i) improved hybridization rates, (ii) improved hybridization specificity, (iii) improved hybridization stringency, (iv) improved hybridization efficiency (or yield), (v) reduced requirements for the amount of starting material needed, (vi) lower temperature requirements for isothermal or thermal ramping amplification protocols, (vii) increased melting rates, and (viii) retention of a low percentage of the total number of target nucleic acid molecules (or amplified clusters of the target nucleic acid molecules) associated with the surface without hybridizing with the surface-bound nucleic acid.Due to the increased performance, reduced cost, and reduced time required to perform a hybridization reaction, the methods, compositions, systems, and kits are ideal for high-throughput nucleic acid hybridization, amplification, and sequencing applications.

[0047] Standard hybridization formulations (e.g., saline sodium citrate buffer) achieve poor hybridization specificity or efficacy when used in standard hybridization protocols using the nonspecific binding surfaces described here. The hybridization reaction, or melting interaction, between target nucleic acid molecules in solution and nucleic acid molecules coupled to the low nonspecific binding surface can be influenced by several factors, including the availability of hydrogen bonding partners in the solution and the polarity of the solution. In general, nucleic acids prefer solid solutions when possible to take advantage of the additional entropic stabilization resulting from the ability to achieve dynamic states in three, rather than two, dimensions, as would be available on a solid surface.At equilibrium, in a system comprising a nucleic acid, a solution, and a hydrophilic surface (e.g., surface with nonspecific binding), a nucleic acid molecule is preferentially stabilized in a solution rather than in a surface-bound state when the solvent is aqueous.

[0048] Existing hybridizations utilize protic solvents (e.g., saline sodium citrate buffer), which would be disadvantageous for nucleic acid hybridization reactions described here with the low-binding surfaces. Aprotic solvents provide a favorable environment for the target nucleic acid molecules to remain in solution rather than binding to the low-binding surface. This is due to the ability of the protic solvent to provide sufficient hydrogen bond partners of sufficient size and distribution to allow hydrogen bond interactions to occur between the exposed hydrogen bond donors and acceptors along the nucleic acid backbone or any exposed side-chain residues.

[0049] In contrast, the hybridization compositions described here drive the target nucleic acid molecule to the surface with low, nonspecific binding while in solution by using an aprotic organic solvent, such as formamide. The aprotic solvents described here reduce the proportion of solvent molecules capable of meeting the hydrogen bonding requirements of the nucleic acid chains and allow for an entropy loss in the solid solution, which will drive the system toward stabilization by placing the nucleic acid on the surface (e.g.,The entropy loss caused by forcing the solid solution to accommodate the unbound hydrogen bonding elements in the nucleic acid is greater than the entropy loss caused by the loss of the third dimension of dynamic freedom when the polymer is bound to the surface. Furthermore, introducing an aprotic organic solvent into the solution can help lower the entropy and, in turn, provide a more favorable environment for the nucleic acid to bind to the hydrophilic surface. For example, adding an aprotic solvent such as acetonitrile helps drive the nucleic acid in the solution toward a surface-bound state.

[0050] The hybridization compositions described herein also include concentrations of protic and aprotic organic solvents to prevent precipitation of the target nucleic acid from solution, which can be caused by high concentrations of aprotic solvent in the solution. Thus, the hybridization composition described herein causes the nucleic acids to selectively associate with hydrophilic surfaces (e.g., surfaces with low, nonspecific binding) while remaining essentially in solution.

[0051] The hybridization compositions described herein optionally include compaction agents capable of modulating interactions of the nucleic acids with the solid solution. In some cases, the hybridization compositions include relaxing agents, divalent cations, or intercalating agents capable of modulating the dynamics of the polymer itself and also modulating the interactions of the nucleic acids with surfaces in the presence of partially aprotic solid solvents. Providing such agents in combination with buffers containing a fraction of aprotic or non-hydrogen-bonding components can, in some cases, provide improved control over the interaction of the nucleic acid molecules with the hydrophilic surfaces.

[0052] Various aspects of the disclosed nucleic acid hybridization methods can be applied in solution-phase or solid-phase nucleic acid hybridization, as well as in any other type of nucleic acid amplification or analysis application (e.g., nucleic acid sequencing). It should be understood that various aspects of the disclosed methods, devices, and systems can be appreciated individually, collectively, or in combination with one another.

[0053] The methods, compositions, systems, and kits described herein are suitable for a wide range of applications beyond those involving nucleic acid-surface interactions, since the same thermodynamic parameters optimized by the methods and compositions described herein govern a number of polymer-biomolecule interactions, polymer-surface interactions, and biomolecule-surface interactions. Therefore, the methods, compositions, systems, and kits described herein can be applied to adjust the polarity or hydrogen bonding potential, or a combination thereof, of a solvent in other systems involving this interaction.

[0054] Solution-based hybridization is the basis of many applications in solution-based molecular biology and solution-phase DNA manipulation, particularly polymerase chain reaction (PCR) (L. Garibyan and N. Avashia, J. Invest. Dermatol., 2013, 133, e6; Z. Xiao, D. Shangguan, Z. Cao, X. Fang, and W. Tan, 2008, DNA guided drug delivery, Chemistry 14, 1769-75; and F. Wei, C. Chen, L. Zhai, N. Zhang, and XS Zhao, 2005, DNA based biosensors, J. Am. Chem. Soc., 127, 5306-5307; and S. Tyagi and FR Kramer, Nat. Biotechnol., 1996, 14, 303-308).The diffusion rates in many of these reactions are sufficient to drive efficient hybridization and the formation of a functional double-stranded form, which can be kinetically analyzed as a second-order reaction, whereby the progressive reaction of duplex formation is second-order and the reverse reaction, which involves the separation of the duplex structure to form the two single-stranded complements (strands A and B), is first-order (Han, C., Improvement of the Speed ​​and Sensitivity of DNA Hybridization Using Isotachophoresis, Stanford Thesis. 2015). These reactions can be written as: A+B→←koffkonAB d[AB]dt=kon[A][B]−koff[AB]

[0055] Various approaches have been pursued to not only increase the rate of the hybridization reaction but also the reaction specificity in the presence of interfering non-complementary DNA fragments. Such approaches include, but are not limited to, the addition of MgCl2 and higher salt concentrations and lower temperatures to accelerate the reactions (H. Kuhn, V. V. Demidov, J.M. Coull, M.J. Fiandaca, B.D. Gildea, and M.D. Frank-Kamenetskii, J. Am. Chem. Soc., 2002, 124, 1097-1103; N.A. Straus and T.I. Bonner, Biochim. Biophys. Acta, Nucleic Acids Protein Synth., 1972; 277, 87-95). The price for accelerated reactions often has to be paid in reaction specificity (JMS Bartlett and D. Stirling, PCR protocols, Humana Press, 2003; W. Rychlik, WJ Spencer, and RE Rhoads, Nucleic Acids Res., 1990, 18).Sometimes additional techniques are used that potentially improve reaction specificity, using volume exclusion or molecular packing techniques, or a combination of these, that employ inert polymers as hybridization buffer additives (R. Wieder and J.G. Wetmur, Biopolymers, 1981, 20, 1537-1547, J.G. Wetmur, Biopolymers, 1975, 14, 2517-2524). Additionally, organic solvents have been used as additives to accelerate hybridization kinetics while maintaining reaction specificity (N. Dave and J. Liu, J. Phys. Chem. B, 2010, 114, 15694-15699).

[0056] While hybridization improvements in solutions can be transferred to surface-based hybridization techniques, surface-based hybridization requirements include far-reaching implications for a variety of critical biological assays, such as gene expression analysis (DT Ross, U. Scherf, MB Eisen, CM Perou, C. Rees, P. Spellman, V. Iyer, SS Jeffrey, M. Van de Rijn, M. Waltham, A. Pergamenschikov, J.C. Lee, D. Lashkari, D. Shalon, TG Myers, J.N. Weinstein, D. Botstein, and PO Brown, Nat. Genet., 2000; 24, 227-235; A. Adomas, G. Heller; A. Olson, J. Osborne, M. Karlsson, J. Nahalkova, Van Zyl, R. Sederoff, J. Stenlid, R. Finlay, and FO Asiegbu, Tree Physiol., 2008, 28, 885-897; Schena, D. Shalon, RW Davis, and P. 0. Brown, Science, 1995, 270, 467-470), disease diagnosis (J. Marx, Science, 2000, 289, 1670-1672), genotyping and SNP detection (JG Hacia, JB Fan, O. Ryder, L. Jin, K. Edgemon, G. Ghandour, RA Mayer, B. Sun, L. Hsie, C.M. Robbins, LC Brody, D. Wang, ES Lander, R. Lipshutz, SP Fodor, and FS Collins, Nat. Genet., 1999, 22, 164-167), rapid pathogen screening based on pathogen nucleic acid, next-generation sequencing (NGS), and a host of other genome-based applications (M.J. Heller, Annu. Rev. Biomed. Eng., 2002, 4, 129-53). The common requirement of all these reactions is high reaction specificity in a highly multiplexed solution of target sequences, which can range from thousands to billions of different sequences, so that the targets are rapidly bound to the solid surface for later probing or amplification, or a combination thereof, to enable the interrogation of DNA (or other nucleic acid) for applications such as sequencing or array-based analysis. It was found that the efficiency of surface-based hybridization reactions is much smaller than that in solution reactions, e.g.approximately an order of magnitude less effective. In past attempts to find a hybridization method for solid surfaces that provides high specificity and accelerated hybridization reaction rates, considerable effort has been devoted (DY Zhang, SX Chen, and P. Yin, Nat. Chem., 2012, 4, 208-14).

[0057] Disclosed herein are innovative combinations of assays derived from studies of surface- and solution-based hybridization outlined above, as well as other fields of study including DNA hydrogenation and quadruplex studies, resulting in significant improvements in hybridization kinetics and specificity. The disclosed hybridization compositions provide highly accurate (e.g., an improvement of >2 orders of magnitude over traditional approaches) and accelerated (e.g., an improvement of >1-2 orders of magnitude over traditional approaches) hybridization when used with a surface with low, nonspecific binding for applications such as next-generation sequencing (NGS) and other biological assays requiring highly accurate nucleic acid hybridization in a multiplexed pool comprising a large number of target sequences. Hybridization process

[0058] Methods for nucleic acid hybridization between a sample nucleic acid molecule and a capture nucleic acid molecule are provided herein. Fig. 11, the sample nucleic acid molecule is isolated and purified from a biological sample derived from a subject 1110. A library of isolated and purified sample nucleic acid molecules is prepared in 1111. The library of sample nucleic acid molecules is hybridized in the presence of hybridization composition 1112 with nucleic acid molecules described herein bound to a surface with low, nonspecific binding.

[0059] Biological sample. The biological sample disclosed herein includes nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, lipids, and viruses. In one example, a biological sample is a nucleic acid sample including one or more nucleic acid molecules. Example biological samples may include polynucleotides, nucleic acids, oligonucleotides, cell-free nucleic acid (e.g., cell-free DNA (cfDNA)), circulating cell-free nucleic acid, circulating tumor nucleic acid (e.g., circulating tumor DNA (ctDNA)), circulating tumor cell nucleic acid (CTC), nucleic acid fragments, nucleotides, DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double-stranded DNA, chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), ribosomal RNA, cell-free DNA, cell-free fetal DNA (cffDNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, viral RNA, etc.

[0060] Any substance containing nucleic acid can be the source of the biological sample. The substance can be a fluid, such as a biological fluid. A fluid substance can include, but is not limited to, blood, umbilical cord blood, saliva, urine, sweat, serum, semen, vaginal fluid, gastric juice, digestive fluid, cerebrospinal fluid, placental fluid, body cavity fluid, ocular fluid, serum, breast milk, lymph fluid, or combinations thereof. The substance can be solid, such as biological tissue. The substance can contain normal, healthy tissue, diseased tissue, or a mixture of healthy and diseased tissue.

[0061] Biological samples described here are obtained from various subjects. A subject can be a living subject or a dead subject. Examples of subjects include, but are not limited to, humans, mammals, non-human mammals, rodents, amphibians, reptiles, canids, felids, bovines, equines, goats, sheep, chickens, avians, mice, rabbits, insects, snails, microbes, bacteria, parasites, or fish. The subject is, in some cases, a patient who has, is suspected of having, or is at risk of developing a disease or disorder. In some cases, the subject may be a pregnant woman. In some cases, the subject may be a normal, healthy, pregnant woman. In some cases, the subject may be a pregnant woman at risk of carrying a child with a certain birth defect.

[0062] A sample can be obtained from a subject using various approaches. For example, a sample can be obtained from a subject by establishing access to the circulatory system (e.g., intravenously or intra-arterially via a syringe or other device), collecting a secreted biological sample (e.g., saliva, sputum, urine, feces), obtaining a biological sample surgically (e.g., through a biopsy) (e.g., intraoperative samples, postoperative samples), by swabbing (e.g., buccal swab, nasal swab), or pipetting.

[0063] Processing of Biological Samples The biological sample described here is, in some cases, processed. The processing includes filtering the sample, binding a sample component containing an analyte, binding the analyte, stabilizing the analyte, purifying the analyte, or a combination thereof. Non-limiting examples of sample components include cells, viral particles, bacterial particles, exosomes, and nucleosomes. In some cases, blood plasma or serum is isolated from a whole blood sample. In some cases, the whole blood is derived from venous blood or capillary blood of a subject described here.

[0064] Library Preparation of Sample Nucleic Acids The sample nucleic acids described herein are, in some cases, converted into a library by labeling the sample nucleic acids with a barcode, or tag. The library of sample nucleic acids is amplified in some embodiments, for example, by isothermal amplification. Non-limiting examples of amplification methods include loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), single-strand break enzyme amplification reaction (NEAR), recombinase polymerase amplification (RPA), and ramification amplification (RAM) methods.

[0065] In some cases, isothermal amplification is used. In some cases, the amplification is isothermal, except for an initial heating step before the start of isothermal amplification. A number of isothermal amplification methods, each with different considerations and providing different advantages, are known in the art and have been reviewed in the literature, e.g., by Zanoli and Spoto, 2013, "Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices," Biosensors 3: 18-43, and Fakruddin, et al., 2013, "Alternative Methods of Polymerase Chain Reaction (PCR)," Journal of Pharmacy and Bioallied Sciences 5(4): 245-252, each of which is incorporated herein by reference in its entirety.

[0066] In some cases, the amplification method is rolling circle amplification (RCA). RCA is an isothermal method for nucleic acid amplification, which allows amplification of the sample DNA sequence by more than 10 9 -fold at a single temperature, typically approximately 30°C. Multiple rounds of isothermal enzymatic syntheses are performed by O29 DNA polymerase, which extends a circle-hybridized primer by continuously rotating around the circular DNA sample. In some cases, the amplification reaction is performed using RCA at approximately 28°C to 32°C. Suitable RCA methods are described in US Pat. No. 6,558,928.

[0067] In some cases, amplification comprises targeted amplification. In some cases, amplifying a nucleic acid comprises contacting a nucleic acid with at least one primer having a sequence corresponding to the target chromosome sequence. Amplification may be multiplexed, comprising contacting the nucleic acid with multiple sets of primers, each of a first pair in a first set and each of a second pair in a second set being different.

[0068] Hybridization. Methods described herein comprise contacting a sample nucleic acid molecule with a capture nucleic acid molecule, optionally coupled to a low-binding surface. In some cases, the capture nucleic acid molecule is coupled to the low-binding surface, and hybridization occurs on the surface. In some cases, the capture nucleic acid molecules are not coupled to the low-binding surface, and hybridization occurs in solution. Methods provided herein further comprise hybridizing the sample nucleic acid molecule with the capture nucleic acid molecule.

[0069] Methods comprise hybridizing at least a portion of the sample nucleic acid molecule comprising a nucleic acid sequence that is sufficiently complementary to a portion of the capture nucleic acid molecule. The portion of the capture nucleic acid molecule and the sample nucleic acid molecule may be at least or equal to approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. The portion of the capture nucleic acid molecule and the sample nucleic acid molecule can be between 4 and 50, 5 and 49, 6 and 48, 7 and 47, 8 and 46, 9 and 45, 10 and 44, 11 and 43, 12 and 42, 13 and 41, 14 and 40, 15 and 39, 16 and 38, 17 and 37, 18 and 36, 19 and 35, 20 and 34, 21 and 33, 22 and 32, 23 and 31, 24 and 30, 25 and 29, 26 and 28 nucleotides. The portion of the capture nucleic acid molecule and the sample nucleic acid molecule can be between 8 and 20 nucleotides.In some cases, at least 90% of the nucleic acids in a portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule completely hybridize. In some cases, at least 95% of the nucleic acids in a portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule completely hybridize. In some cases, between 95-100% of the nucleic acids in a portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule completely hybridize.

[0070] One in Fig.The non-limiting example shown in Figure 8 depicts one or more sample nucleic acid molecules 801 being circularized 802 using ligation (e.g., splint ligation) 802 and introduced into one or more nucleic acid molecules 808 coupled to a hydrophilic substrate 807 of a low nonspecific binding surface 806 in the presence of a hybridization composition 805. In this example, the low nonspecific binding surface is immersed in the hybridization composition. In alternative embodiments, the one or more sample nucleic acid molecules are introduced into the hybridization composition before being introduced into the one or more nucleic acid molecules 808 coupled to a hydrophilic substrate 807 of a low nonspecific binding surface 806.Hybridization occurs between the sample nucleic acid molecule and the surface-coupled nucleic acid molecule 809.

[0071] Sample nucleic acids. The one or more sample nucleic acid molecules described herein are obtained from a biological sample described herein. The sample nucleic acid molecules are a deoxyribonucleic acid molecule (DNA) or a ribonucleic acid molecule (RNA). In some cases, the DNA is selected from cell-free DNA (cfDNA), circulating cell-free nucleic acid, circulating tumor nucleic acid (e.g., circulating tumor DNA (ctDNA)), circulating tumor cell nucleic acids (CTC), nucleic acid fragments, nucleotides, DNA, complementary DNA (cDNA), double-stranded DNA (dsDNA), single-stranded DNA, chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial DNA). In some cases, the RNA is selected from ribosomal RNA, cell-free DNA, cell-free fetal DNA (cffDNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, viral RNA, etc.

[0072] Coupling of capture nucleic acids to the surface. The nucleic acid molecules coupled to the surface (e.g., capture molecule) can be coupled to the surface using a number of suitable options. In some cases, the nucleic acid molecules are coupled to the surface through covalent bonding. In some cases, the nucleic acid molecules are coupled to the surface through noncovalent bonding. In some cases, the nucleic acid molecules are attached to the surface through a biointeraction. Non-limiting examples of biointeraction surface chemistry include biotin / streptavidin interactions (or variations thereof), polyhistidine (his)-tag - Ni / NTA conjugation chemistry, methoxy-ether conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxides, azides, hydrazides, alkynes, isocyanates, and silanes. Compositions

[0073] Hybridization compositions are provided herein. The hybridization compositions in the present disclosure comprise at least one organic solvent, which in some cases is polar and aprotic (e.g., having a dielectric constant of less than or equal to about 115 as measured at 68 degrees F). The hybridization compositions comprise a pH buffer. Optionally, the hybridization compositions comprise one or more molecular compaction / volume exclusion agents, one or more additives that affect DNA melting temperatures, one or more additives that affect DNA hydration, or a combination thereof.The hybridization compositions described herein used with low, non-specific binding surfaces, such as silica coated with low binding polymers such as polyethylene glycol (PEG) for sequencing, genotyping, or sequencing-related technologies, can be achieved using any one or a combination of the following hybridization composition components.

[0074] Organic solvent: An organic solvent is a solvent or solvent system comprising carbon-based or carbon-containing substances that are capable of dissolving or dispersing other substances. An organic solvent can be miscible or immiscible with water.

[0075] Polar Solvent: A polar solvent, as contained in the hybridization composition described herein, is a solvent or solvent system comprising one or more molecules characterized by the presence of a permanent dipole moment, e.g., a molecule exhibiting a spatially uneven distribution of charge density. A polar solvent may be characterized by a dielectric constant of 20, 25, 30, 35, 40, 45, 50, 55, 60, or greater, or by a value or range of values ​​that include the aforementioned values. For example, a polar solvent may have a dielectric constant of greater than 100, greater than 110, greater than 111, or greater than 115.In some cases, the dielectric constant is measured at a temperature greater than or equal to approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 degrees Fahrenheit (F). In some cases, the dielectric constant is measured at a temperature less than or equal to approximately -20, -25, -30, -35, -40, -45, -50, -55, -60, 65, -70, -75, -80, -85, -90, -95, -100, -150, -200, -250, -300, -350, -400, -450, or -459 degrees F. In some cases, the dielectric constant is measured at a temperature of 68 degrees F. In some cases, the dielectric constant is measured at a temperature of 20 degrees F.

[0076] A polar solvent, as described herein, may include a polar aprotic solvent. A polar aprotic solvent, as described herein, may also contain no ionizable hydrogen in the molecule. Furthermore, preferably in the context of the presently disclosed compositions, polar solvents or polar aprotic solvents may be replaced with strong, polarizing functional groups such as nitrile, carbonyl, thiol, lactone, sulfone, and carbonate groups, so that the underlying solvent molecules exhibit a dipole moment. Polar solvents and polar aprotic solvents may exist in both aliphatic and aromatic or cyclic forms. In some embodiments, the polar solvent is acetonitrile.

[0077] The organic solvent described herein may have a dielectric constant that is the same as or close to that of acetonitrile. The dielectric constant of the organic solvent may range between about 20-60, about 25-55, about 25-50, about 25-45, about 25-40, about 30-50, about 30-45, or about 30-40. The dielectric constant of the organic solvent may be greater than or equal to about 20, 25, 30, 35, or 40. The dielectric constant of the organic solvent may be less than or equal to about 30, 40, 45, 50, 55, or 60. The dielectric constant of the organic solvent may be about 35, 36, 37, 38, or 39.

[0078] The dielectric constant can be measured using a test capacitor. Representative polar aprotic solvents with a dielectric constant between 30 and 120 can be used. Such solvents may include, but are not limited to, acetonitrile, diethylene glycol, N,N-dimethylacetamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, formamide, hexamethylphosphoramide, glycerin, methanol, N-methyl-2-pyrrolidinone, nitrobenzene, or nitromethane.

[0079] The organic solvent described herein may have a polarity index that is the same as or close to that of acetonitrile. The polarity index of the organic solvent may range between 2-9, 2-8, 2-7, 2-6, 3-9, 3-8, 3-7, 3-6, 4-9, 4-8, 4-7, or 4-6. The polarity index of the organic solvent may be greater than or equal to about 2, 3, 4, 4.5, 5, 5.5, or 6. The polarity index of the organic solvent may be less than about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 10. The polarity index of the organic solvent may be approximately 5.5, 5.6, 5.7, or 5.8.

[0080] The Snyder Polarity Index can be calculated according to the methods disclosed in Snyder, LR, Journal of Chromatography A, 92(2):223-30 (1974), which is incorporated herein by reference. Representative polar aprotic solvents with a Snyder Polarity Index between 6.2 and 7.3 can be used. Such solvents may include, but are not limited to, acetonitrile, dimethylacetamide, dimethylformamide, N-methylpyrrolidone, N,N-dimethyl sulfoxide, methanol, or formamide.

[0081] Relative polarity can be determined according to the methods given in Reichardt, C., Solvents and Solvent Effects in Organic Chemistry, 3rd Ed., 2003, which is incorporated by reference into this document, and especially in view of its disclosure of polarities and methods for determining or evaluating them for solvents and solvent molecules. Polar aprotic solvents with a relative polarity between 0.44 and 0.82 can be used. Such solvents may include, but are not limited to, dimethyl sulfoxide, acetonitrile, 3-pentanol, 2-pentanol, 2-butanol, cyclohexanol, 1-octanol, 2-propanol, 1-heptanol, i-butanol, 1-hexanol, 1-pentanol, acetylacetone, ethyl acetoacetate, 1-butanol, benzyl alcohol, 1-propanol, 2-aminoethanol, ethanol, diethylene glycol, methanol, ethylene glycol, glycerin, or formamide.

[0082] The solvent polarity (E T(30)) can be calculated according to the methods disclosed in Reichardt,C., Molecular Interactions, Volume 3, Ratajczak, H. and Orville, WJ, Eds (1982), which is incorporated by reference in its entirety into this document.

[0083] Some examples of organic solvents include, but are not limited to, acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetanilide, N-acetylpyrrolidone, 4-aminopyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadiene dioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloromaleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyltetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, diphenylsulfone, 1,2-Dinitrobenzene, 2,4-Dinitrotoluene, Dipheynylsulfone, Epsilon-Caprolactam, Ethanesulfonyl chloride, Ethyl-Ethylphosphinate, N-Ethyltetrazole, Ethylene carbonate, Ethylene trithiocarbonate, Ethylene glycol sulfate, Ethylene glycol sulfite, Furfural, 2-Furonitrile, 2-Imidazole, Isatin, Isoxazole, Malononitrile, 4-Methoxybenzonitrile, 1-Methoxy-2-Nitrobenzene, Methyl-alpha-Bromotetronate, 1-Methylimidazole,N-Methylimidazol, 3-Methylisoxazol, N-Methylmorpholin-N-Oxid, Methylphenylsulfon, N-methylpyrrolidinon, Methylsulfolan, Methyl-4-Toluenesulfonat, 3-Nitroanilin, Nitrobenzimidazol, 2-Nitrofuran, 1-Nitroso-2-Pyrolidinone, 2-Nitrothiophen, 2-Oxazolidinon, 9,10-Phenanthrenequinon, N-Phenylsydnon, Phthaanhydrid, Picolinonitril (2-Cyanopyridin), 1,3-Propansulton, 13-Propiolakton, Propylenkarbonat, 4H-Pyran-4-Thion, 4H-Pyran-4-one (y-pyrone), Pyridazin, 2-Pyrrolidon, Saccharin, Succinonitril, Sulfanilamid, Sulfolan, 2,2,6,6-Tetrachlorocyclohexanon, Tetrahydrothiapyranoxid, Tetramethylensulfon (sulfolan), Thiazol, 2-Thiouracil, 3,3,3-Trichloropropen, 1,1,2-Trichloropropen, 1,2,3-Trichloropropen, Trimethylensulfid-Dioxid, und Trimethylensulfit.,

[0084] Polar aprotic solvents with a solvent polarity between 44 and 60 can be used. Such solvents may include, but are not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol, triethyl phosphite, 3-pentanol, acetonitrile, nitromethane, cyclohexanol, 2-pentanol, 4-methyl-1,3-dioxolan-2-one, propylene carbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol, 2,6-dimethylphenol, 2,6-xylenol, 1-decanol, cyclopentanol, dimethylsulfone, 1-octanol, diethylene glycol mono-n-butyl ether, butyldigol, 1-heptanol, 3-phenyl-1-propanol, 1,3-dioxolane-2-one, ethylene carbonate, 1-hexanol, 4-Chlorobutyronitrile, 5-methyl-2-isopropylphenol, thymol, 3,5,5-trimethyl-1-hexanol, 3-methyl-1-butanol, isoamyl alcohol, 2-methyl-l-propanol, isobutyl alcohol, 2-(tert-butyl)phenol, 1-pentanol, 2-phenylethanol, 2-methylpentane-2,4-diol, dipropylene glycol, 2-isopropylphenol, 2-n-butoxyethanol, ethylene glycol mono-n-butyl ether, 1-butanol,2-Hydroxymethyl-tetrahydrofuran, Tetrahydrofurfurylalkohol, 2-Hydroxymethylfuran, Furfurylalkohol, 1-Propanol, 2,4-Dimethylphenol, 2,4-Xylenol, Benzylalkohol, 2-Ethoxyphenol, 2-Ethoxyethanol, 1,5-Pentanediol, 1-Bromo-2-propanol, 2-Methyl-5-isopropylphenol, Carvacrol, 2-Aminoethanol, Ethanol, n-Methylacetamid, 3-Chloropropionitril, 2-Propen-1-ol, Allylalkohol, 2-Methoxyethanol, 2-Methylphenol, o-Cresol, 1,3-Butanediol, 2-Propyn-1-ol, Propargylalkohol. 3-Methylphenol, m-Cresol, Triethylenglykol, Diethylenglykol, n-Methylformamid, 1,2-Propanediol, 1,3-Propanediol, 2-Chlorophenol, Methanol, 1,2-Ethanediol, Glykol, Formamid, 2,2,2-Trichloroethanol, 1,2,3-Propanetriol, Glycerol, 2,2,3,3-Tetrafluoro-1-propanol, 2,2,2-Trifluoroethanol, 4-n-Butylphenol, 4-Methylphenol, oder p-Cresol.,

[0085] Polar aprotic solvents with a dielectric constant in the range between 30-115 can be used. Such solvents may include, but are not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol, triethyl phosphite, 3-pentanol, acetonitrile, nitromethane, cyclohexanol, 2-pentanol, 4-methyl-1,3-dioxolan-2-one, propylene carbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol, 2,6-dimethylphenol, 2,6-xylenol, 1-decanol, cyclopentanol, dimethylsulfone, 1-octanol, diethylene glycol mono-n-butyl ether, butyldigol, 1-heptanol, 3-phenyl-1-propanol, 1,3-dioxolane-2-one, ethylene carbonate, 1-hexanol, 4-Chlorobutyronitrile, 5-methyl-2-isopropylphenol, thymol, 3,5,5-trimethyl-1-hexanol, 3-methyl-1-butanol, isoamylalcohol, 2-methyl-1-propanol, isobutyl alcohol, 2-(tert-butyl)phenol, 1-pentanol, 2-phenylethanol, 2-methylpentane-2,4-diol, dipropylene glycol, 2-isopropylphenol, 2-n-butoxyethanol, ethylene glycol mono-n-butyl ether,1-Butanol, 2-Hydroxymethyl-tetrahydrofuran, Tetrahydrofurfurylalkohol, 2-Hydroxymethylfuran, Furfurylalkohol, 1-Propanol, 2,4-Dimethylphenol, 2,4-Xylenol, Benzylalkohol, 2-Ethoxyphenol, 2-Ethoxyethanol, 1,5-Pentanediol, 1-Bromo-2-propanol, 2-Methyl-5-isopropylphenol, Carvacrol, 2-Aminoethanol, Ethanol, n-Methylacetamid, 3-Chloropropionitril, 2-Propen-1-ol, Allylalkohol, 2-Methoxyethanol, 2-Methylphenol, o-Cresol, 1,3-Butanediol, 2-Propyn 1-ol, Propargylalkohol, 3-Methylphenol, m-Cresol, Triethylenglykol, Diethylenglykol, n-Methylformamid, 1,2-Propanediol, 1,3-Propanediol, 2-Chlorophenol, Methanol, 1,2-Ethanediol, Glykol, Formamid, 2,2,2-Trichloroethanol, 1,2,3-Propanetriol, Glycerol, 2,2,3,3-Tetrafluoro-1-propanol, 2,2,2-Trifluoroethanol, 4-n-Butylphenol, 4-Methylphenol, oder p-Cresol.,

[0086] Addition of organic solvents: In some cases, the disclosed hybridization buffer formulations may contain the addition of an organic solvent. Examples of suitable solvents include, but are not limited to, acetonitrile, ethanol, DMF, and methanol, or any combination thereof at varying percentages (e.g., >5%). In some cases, the percentage (by volume) of organic solvent included in the hybridization buffer may range between about 1% and about 20%. In some cases, the percentage by volume of an organic solvent may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, or at least 20%.In some cases, the percentage by volume of an organic solvent may be at most 15%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. Any of the maximum and minimum values ​​described in this paragraph may be combined to form a range included in the present disclosure; for example, the percentage by volume of an organic solvent may be between about 4% and about 15%. The percentage by volume of an organic solvent may be any value within this range, e.g., about 7.5%.

[0087] If the organic solvent comprises a polar aprotic solvent, the amount of polar aprotic solvent is present in an amount effective to denature the double-stranded nucleic acid. In some embodiments, the amount of polar aprotic solvent is greater than or equal to about 10% by volume based on the total volume of the formulation. The amount of polar aprotic solvent is in volume % based on the total volume of the formulation approximately or greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. The amount of polar aprotic solvent is in volume % based on the total volume of the formulation, less than approximately 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or higher.In some embodiments, the amount of polar aprotic solvent ranges between approximately 10 to 90 volume percent based on the total volume of the formulation. In some embodiments, the amount of polar aprotic solvent ranges between approximately 25 to 75 volume percent based on the total volume of the formulation. In some embodiments, the amount of polar aprotic solvent in volume percent based on the total volume of the formulation ranges from approximately 10% to 95%, 10% to 85%, 20% to 90%, 20% to 80%, 20% to 75%, or 30% to 60%. In some embodiments, the polar aprotic solvent is formamide.

[0088] If the organic solvent comprises an aprotic solvent, the amount of polar aprotic solvent is present in an amount effective to denature the double-stranded nucleic acid. In some embodiments, the amount of aprotic solvent is greater than or equal to about 10% by volume based on the total volume of the formulation. The amount of aprotic solvent is in volume % based on the total volume of the formulation about or greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. The amount of aprotic solvent is less than about 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or higher by volume based on the total volume of the formulation. In some embodiments, the amount of aprotic solvent ranges between about 10 to 90% by volume based on the total volume of the formulation.In some embodiments, the amount of aprotic solvent ranges between approximately 25 and 75% by volume based on the total volume of the formulation. In some embodiments, the amount of aprotic solvent ranges from approximately 10% to 95%, 10% to 85%, 20% to 90%, 20% to 80%, 20% to 75%, or 30% to 60% by volume, or higher, based on the total volume of the formulation.

[0089] Addition of a Molecular Compaction / Volume Exclusion Agent The composition described herein may contain one or more compaction agents that promote molecular compaction. The compaction agent is selected from a group consisting of polyethylene glycol (PEG), dextran, hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose, or a combination thereof. An exemplary compaction agent may include one or more of polyethylene glycol (PEG), dextran, proteins such as ovalbumin or hemoglobin, or Ficoll.

[0090] A sufficient amount of a compacting agent in the composition enables, enhances, or supports molecular compaction. The amount of the compacting agent is approximately or greater than approximately 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more by volume based on the total volume of the formulation. In some cases, the amount of the molecular compacting agent is greater than or equal to approximately 5% by volume based on the total volume of the formulation. The amount of the active ingredient for compaction is less than approximately 3%, 5%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or more by volume based on the total volume of the formulation. In some cases, the amount of the active ingredient for molecular compaction may be less than or equal to approximately 30% by volume based on the total volume of the formulation.In some embodiments, the amount of organic solvent ranges between about 25 to 75% by volume based on the total volume of the formulation. In some embodiments, the amount of organic solvent ranges between about 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20% by volume based on the volume of the formulation. In some cases, the amount of molecular compaction agent can range between about 5 and about 20% by volume based on the total volume of the formulation. In some embodiments, the amount of organic solvent is in the range between about 1 to 30 volume percent based on the total volume of the formulation.

[0091] An example of the compaction agent in the composition is polyethylene glycol (PEG). In some embodiments, the PEG used may have a molecular weight sufficient to enhance or assist molecular compaction. In some embodiments, the PEG used in the composition has a molecular weight in the range between approximately 5,000-50,000 Da. In some embodiments, the PEG used in the composition has a molecular weight in the range between approximately 10,000-40,000 Da. In some embodiments, the PEG used in the composition has a molecular weight in the range between approximately 10,000-30,000 Da. In some embodiments, the PEG used in the composition has a molecular weight in the range between approximately 20,000 Da.

[0092] In some cases, the disclosed hybridization buffer formulations may include the addition of a molecular compaction or volume exclusion agent. Examples of molecular compaction or volume exclusion agents include macromolecules (e.g., proteins) that, when added to a solution at higher concentrations, can alter the properties of other molecules in the solution by reducing the volume of solvent available to the other molecules. In some cases, the percentage of the volume of the molecular compaction or volume exclusion agent in the hybridization buffer formulations may range from approximately 1% to approximately 50%.In some cases, the volume percentage of the molecular compaction agent or volume exclusion agent may be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. In some cases, the volume percentage of the molecular compaction agent or volume exclusion agent may be at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most 1%. Any of the maximum and minimum values ​​described in this paragraph may be combined to form a range included in the present disclosure, for example, the percentage of the volume of the molecular compaction or volume exclusion agent may be between about 5% and about 35%.The percentage of the volume of molecular compaction or volume exclusion agent can have any value within this range, e.g., approximately 12.5%.

[0093] pH Buffer System: The compositions described herein include a pH buffer system that maintains the pH of the compositions within a range suitable for the hybridization process. The pH buffer system may contain one or more buffering agents selected from the group consisting of Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, MES, and MOPS. The pH buffer system may further contain a solvent. An exemplary pH buffer system contains MOPS, MES, TAPS, phosphate buffer in combination with methanol, acetonitrile, ethanol, isopropanol, butanol, t-butyl alcohol, DMF, DMSO, or any combination thereof.

[0094] The amount of the pH buffer system effectively maintains the pH of the formulation within a range suitable for hybridization. In some cases, the pH may be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the pH may be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, or at most 3. Any of the maximum and minimum values ​​described in this paragraph may be combined to form a range included in the present disclosure; for example, the pH of the hybridization buffer may be between about 4 and about 8. The pH of the hybridization buffer may have any value within this range, e.g., about pH 7.8. In some cases, the pH range is from about 3 to about 10.In some cases, the disclosed hybridization buffer formulations may include pH adjustments ranging from approximately pH 3 to pH 10, with a narrower buffer range of 5-9.

[0095] Additives that affect DNA melting temperatures: The compositions described here may contain one or more additives that allow for better control of the nucleic acid melting temperature and improve the stringency control of the hybridization reaction. Hybridization reactions are typically performed under stringent conditions to achieve hybridization accuracy. In some cases, the additive for controlling the nucleic acid melting temperature is formamide.

[0096] The amount of the nucleic acid melting temperature control additive may vary depending on other active ingredients included in the compositions. The amount of the nucleic acid melting temperature control additive is about or greater than about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more by volume based on the total volume of the formulation. In some cases, the amount of the nucleic acid melting temperature control additive is greater than or equal to about 2% by volume based on the total volume of the formulation. In some cases, the amount of the nucleic acid melting temperature control additive is greater than or equal to about 5% by volume based on the total volume of the formulation. In some cases, the amount of the nucleic acid melting temperature control additive is less than about 3%, 5%, 10%, 12%.5%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or more by volume based on the total volume of the formulation. In some embodiments, the amount of nucleic acid melting temperature control additive is in the range of approximately 1% to 40%, 1% to 35%, 2% to 50%, 2% to 40%, 2% to 35%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 10%, 5% to 50%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20% by volume based on the total volume of the formulation. In some embodiments, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between approximately 2 to 20 volume percent based on the total volume of the formulation. In some cases, the amount of the additive for controlling the melting temperature of the target nucleic acid ranges between approximately 5 to 10 volume percent based on the total volume of the formulation.

[0097] In some cases, the disclosed hybridization buffer formulations may additionally contain an additive that influences the duplex melting temperature of the nucleic acid. A suitable additive that can be used to influence the melting temperature of nucleic acid is, for example, but not exclusively, formamide. In some cases, the percentage by volume of a melting temperature additive included in the hybridization buffer formulation may range from about 1% to about 50%. In some cases, the percentage by volume of a melting temperature additive may be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.In some cases, the volume percentage of melting temperature additive may be at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most 1%. Any of the maximum and minimum values ​​described in this paragraph may be combined to form a range included in the present disclosure. For example, the volume percentage of melting temperature additive may be between about 10% and about 25%. The volume percentage of melting temperature additive may be any value within this range, e.g., about 22.5%.

[0098] Additives that affect DNA hydration: In some cases, the disclosed hybridization buffer formulations may additionally contain an additive that affects the hydration of the nucleic acid. These may include, but are not limited to, betaine, urea, glycine betaine, or any combination thereof. In some cases, the percentage by volume of a hydration additive in the hybridization buffer formulation may range from approximately 1% to approximately 50%. In some cases, the percentage by volume of a hydration additive may be at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.In some cases, the volume percentage of a hydration additive may be at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most 1%. Any of the maximum and minimum values ​​described in this paragraph may be combined to form a range included in the present disclosure; for example, the volume percentage of the hydration additive may be between about 1% and about 30%. The volume percentage of the melting temperature additive may be any value within this range, e.g., about 6.5%. Systems

[0099] Provided herein are systems comprising the hybridization compositions described herein and a surface with low, nonspecific binding. The systems described herein, in some cases, comprise a flow cell device. Furthermore, in some cases, the systems include an imaging system (e.g., a camera and an inverted fluorescence microscope). Furthermore, the systems may comprise one or more computer control systems for performing computer-implemented methods for analyzing nucleic acid.

[0100] Low-binding surface: Also disclosed herein is a low-binding surface that enables improved nucleic acid hybridization and amplification performance. In general, the disclosed surface may comprise one or more covalently or non-covalently attached chemical modification layers with low binding, e.g., silane layers, polymer film, and one or more covalently or non-covalently attached primer sequences that can be used to attach single-stranded oligonucleotide templates to the surface. In some cases, the formulation of the surface, e.g.,the chemical composition of one or more layers, the coupling chemistry used to crosslink one or more layers at the surface or to each other or in a combination thereof, and the total number of layers are varied so that nonspecific protein binding, nucleic acid molecules, and other hybridization and amplification-enhancing components are minimized or reduced relative to a comparable monolayer at the surface. Often, the formulation of the surface can be varied so that nonspecific hybridization at the surface is minimized or reduced relative to a comparable monolayer. The formulation of the surface can be varied so that nonspecific amplification at the surface is minimized or reduced relative to a comparable monolayer.The formulation of the surface can be varied to maximize specific amplification rates or yields, or a combination of these, at the surface. Amplification levels suitable for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 30 amplification cycles, which in some cases are disclosed herein.

[0101] Non-limiting examples of low non-specific binding surfaces are provided in copending U.S. patent application Ser. No. 16 / 739,007, which is incorporated herein by reference in its entirety. The terms "low non-specific binding surface" and "low binding surface" are used interchangeably to refer to hydrophilic surfaces that exhibit a low level of non-specific binding of proteins or nucleic acid compared to non-hydrophilic surfaces. In some cases, the low non-specific binding surface is passivated, meaning it is coated with a substrate that is hydrophilic.

[0102] Materials from which the substrate or support structure can be made include, but are not limited to, glass, fused silica, silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cycloolefin polymer (COP), cycloolefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0103] The substrate or support structure can be rendered into a variety of geometries and dimensions and can comprise a variety of materials. In some cases, the substrate or support structure can be locally planar (e.g., comprising a microscope slide or the surface of a microscope slide). In general, the substrate or support structure can be cylindrical (e.g., comprising a capillary or the inner surface of a capillary), spherical (e.g., comprising the outer surface of a non-porous bead), or irregular (e.g., comprising the outer surface of an irregularly shaped, non-porous bead or particle). In some cases, the surface of the substrate or support structure utilized for nucleic acid hybridization and amplification can be a solid, non-porous surface.In some cases, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous, so that the coatings described therein can penetrate into the porous surface and the nucleic acid hybridization and amplification reactions performed thereon can take place within the pores.

[0104] The substrate or support structure comprising the one or more chemically modified layers, e.g., layers of a low and non-specific binding polymer, can be independent or integrated into another structure or assembly. In some cases, for example, the substrate or support structure can comprise one or more surfaces within an integrated or arrayed microfluidic flow cell. The substrate or support structure can comprise one or more surfaces within a microplate format, e.g., the bottom surface of a well in a microplate. As described above, in some embodiments, the substrate or support structure comprises the inner surface (such as the lumen surface) of a capillary. In alternative embodiments, the substrate or support structure comprises the inner surface (such as the lumen surface) of a capillary etched into a planar chip.

[0105] The chemical modification layers can be applied uniformly along the surface of the substrate or support structure. Alternatively, the surface of the substrate or support structure can be non-uniformly distributed or structured, so that the chemical modification layers are confined to one or more discrete areas of the substrate. The substrate surface can be structured, for example, using photolithographic techniques to create an ordered arrangement or a random pattern of the chemically modified areas of the surface. The substrate surface can be structured using contact printing or inkjet printing techniques, or a combination of these.In some cases, an ordered arrangement or random pattern of chemically modified discrete regions may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 or more discrete regions, or any number between the range recited herein.

[0106] To achieve surfaces with low, non-specific binding (here also referred to as “low binding” or “passivated” surfaces), hydrophilic polymers can be non-specifically adsorbed or covalently polymerized onto the substrate or support surface. The passivation can be carried out, for example, using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, dextran or other hydrophilic polymers with different molecular weights and end groups attached to the surface, such as silane chemistry.The end groups extended from the surface can include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In some cases, two or more layers of a hydrophilic polymer, e.g., a linear polymer, branched polymer, or multiply branched polymer, can be attached to the surface. In some cases, two or more layers can be covalently coupled or cross-linked to improve the stability of the resulting surface. In some cases, oligonucleotide primers with different base sequences or base modifications (or other biomolecules, e.g., enzymes or antibodies) can be attached to different surface densities of the resulting surface layer. In some cases, for example, both the surface functional group density and the oligonucleotide concentration can be varied to target specific primer density ranges.Additionally, primer density can be controlled by diluting oligonucleotides with other molecules that carry the same functional group. For example, oligonucleotides labeled as amines can be diluted with amine-labeled polyethylene glycol in a reaction with an NHS ester-coated surface to reduce the final primer density. Primers with different linker lengths between the hybridization region and the surface attachment functional group can also be used to control surface density. Examples of suitable linkers include poly-T and poly-A strands at the 5' end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be attached to the surface, and a fluorescence measurement can then be compared to a dye solution of known concentration.

[0107] As a result of the surface passivation processes disclosed therein, proteins, nucleic acids, and other biomolecules do not "stick" to the substrates, thus exhibiting low, nonspecific binding (nonspecific binding). Examples using standard monolayer surface preparations with various glass preparation states are shown below. Hydrophilic surfaces passivated to achieve ultralow, nonspecific binding for proteins and nucleic acids require new reaction conditions to improve primer deposition reaction efficiencies and hybridization performance and to induce effective amplification. All of these processes require oligonucleotide attachment, subsequent protein binding, and delivery to a low-binding surface.As described below, the combination of a novel primer surface conjugate formulation (Cy3 oligonucleotide grafting tritiation) and the resulting ultra-low nonspecific background (nonspecific binding functional assays performed with fluorescent red and green dyes) yielded results demonstrating the feasibility of the disclosed approaches. Some of the surfaces disclosed therein exhibit a ratio of specific (e.g., hybridization to a bound primer or probe) to nonspecific binding (e.g., B. inter) of a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1 or greater than 100:1, or include any value between the ranges recited herein. Some of the surfaces disclosed therein exhibit a ratio of specific to nonspecific fluorescent signals (e.g., for specifically hybridized to nonspecific, bound labeled oligonucleotides or for specifically amplified to nonspecific, bound (B inter ) or nonspecifically amplified (B intra ) labeled oligonucleotides or a combination thereof (B inter + B intra )) for a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1 on or any value therebetween.

[0108] To scale the primer surface density and add additional dimensionality to hydrophilic or amphiprotic surfaces, substrates comprising multilayer PEG coatings and other hydrophilic polymers have been developed. By using hydrophilic and amphiprotic surface layering approaches, including but not limited to the polymer / copolymer materials described below, it is possible to significantly increase the primer loading density at the surface. Conventional PEG coating approaches utilize monolayer primer deposition, which is generally intended for single-molecule applications but does not achieve high copy numbers for nucleic acid amplification applications.As described herein, "coating" can be achieved using conventional cross-linking approaches with any compatible polymer or monomer unit, allowing a surface comprising two or more highly cross-linked layers to be sequentially incorporated. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and the copolymers polylysine and PEG. In some cases, the different layers can be linked together through a variety of conjugation reactions, including, but not limited to, biotin-streptavidin bonding, azide-alkyne click reactions, amine-NHS-ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged polymer and negatively charged polymer.In some cases, the high-density primer materials can be constructed in a solution and then layered onto the surface in multiple steps.

[0109] The chemical bonding used to polymerize a chemically modified layer onto a support surface will fundamentally depend on both the material from which the support is made and the chemical nature of the layer. In some cases, the first layer may be covalently attached to the support surface. In some cases, the first layer may be non-covalently attached, e.g., adsorbed to the surface through non-covalent interactions, such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and the molecular components of the first layer. In any case, the substrate surface may be treated prior to applying or depositing the first layer. A variety of surface preparation techniques can be used to clean or treat the support surface.Glass or silicon surfaces can, for example, be acid cleaned with a piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) or cleaned with an oxygen plasma treatment method, or a combination thereof.

[0110] Silane chemistry includes a non-limiting approach to covalently modify the silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups) to the surface, which can then be used for the connection of linker molecules (e.g., linear hydrocarbon molecules of various lengths such as C6, C12, C18 hydrocarbon or linear polyethylene glycol molecules (PEG molecules)) or layered molecules (e.g., branched PEG molecules or other polymers). Examples of suitable silanes that can be used to prepare any of the disclosed low-binding support surfaces include, but are not limited to, 3-aminopropyltriethoxysilane (APTMS), 3-aminopropyltriethoxysilane (APTES), a variety of PEG silanes (e.g., comprising molecular weights of 1,000, 2,000, 5,000, 10,000, 20,000, etc.), amino-PEG silane (e.g.,comprising a free amino functional group), maleimide-PEG-silane, biotin-PEG-silane and the like.

[0111] A variety of molecules, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or a combination thereof, can be used to produce one or more chemically modified layers on the support surface, whereby the choice of components used can vary to influence one or more properties of the support surface, e.g. the surface density of functional groups, or bound oligonucleotide primers or a combination thereof, the hydrophilic / hydrophobic property of the support surface, or the three-dimensional nature (e.g. "thickness") of the support surface.Polymers from which one or more layers of low, nonspecific binding materials can be prepared in any of the disclosed support surfaces include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branching structures, streptavidin, polyacrylamide, polyester, dextran, polylysine, polylysine copolymer, or any combination thereof. Examples of conjugation chemistry that can be used to graft one or more layers of material (e.g., polymer layers) onto the support surface or to crosslink the layers together, or a combination thereof, include, but are not limited to, biotin-streptavidin interactions (or variations thereof), His-tagged Ni / NTA conjugation chemistry, methoxyether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimide, thiol, epoxide, azide, hydrazide, alkyne, isocyanate, and silane.

[0112] One or more layers of multilayer surfaces may comprise a branched polymer or be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

[0113] In some cases, the branched polymer used to make one or more layers of each multilayer surface disclosed therein may comprise at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, at least 40 branches. Molecules often have a power of 2 number of branches, such as 2, 4, 8, 16, 32, 64 or 128 branches.

[0114] PEG multilayers containing PEG (8,16,8) on PEG-amine-APTES exposed to two layers of 7 μM primer preload exhibit a surface concentration of 2,000,000 to 10,000,000. Similar concentrations were observed for 3-layer, multi-arm PEG (8,16,8) and (8,64,8) on PEG-amine-APTES exposed to an 8 μM primer and a 3-layer, multi-arm PEG (8,8,8), with star-shaped PEG-amine being used to replace dumbbell-shaped 16-mer and 64-mer PEGs. PEG multilayers with comparable first, second, and third PEG levels are also being considered.

[0115] Linear, branched, and multiply branched polymers used to prepare one or more layers of each of the multiply branched layers disclosed therein may have a molecular weight of at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 Daltons.

[0116] In some cases, for example, when at least one layer of a multiply branched layer comprises a branched polymer, the number of covalent bonds between a branched polymer molecule of the attached layer and the molecules of the underlying layer may be approximately between one covalent bond per molecule and about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between a branched polymer molecule of the new layer and molecules of the underlying layer may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, or more than 32 covalent bonds per molecule.

[0117] Any reactive functional group remaining after bonding a material layer to the support surface can optionally be blocked by attaching it to a small, inert molecule through powerful coupling chemistry. For example, when coupling chemistry with amines is used to attach new material layers to the underlying layer, remaining amine groups can then be acetylated or deactivated by coupling them to a small amino acid such as glycine.

[0118] The number of layers of low, non-specific binding materials, e.g., a hydrophilic polymer material, attached to the surface of the disclosed low-binding supports can range from 1 to about 10. In some cases, the number of layers can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of the maximum and minimum values ​​described in this paragraph can be combined to form a range included in the present disclosure; for example, the number of layers can range from about 2 to about 4. In some cases, all layers can comprise the same material.In some cases, each layer may comprise a different material. In some cases, the plurality of layers may comprise a variety of materials. In some cases, at least one layer may comprise a branched polymer. In some cases, all layers may comprise a branched polymer.

[0119] One or more layers of low, nonspecific binding materials can, in some cases, be attached or conjugated to the substrate surface, or a combination thereof, using a polar protic solvent, a polar aprotic solvent, a nonpolar solvent, or any combination thereof. In some cases, the solvent used for attaching or conjugating the layer, or a combination thereof, can include some type of alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, an aqueous buffered solution (e.g., phosphate buffer, phosphate-buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof.In some cases, an organic component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the total, or any percentage between the range mentioned here or adjacent thereto, with the remainder being water or an aqueous buffered solution. In some cases, an aqueous component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any percentage between the range recited herein or adjacent thereto, with the remainder consisting of an organic solvent. The pH of the solvent mixture used may be less than or equal to approximately 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any value between the range recited herein or adjacent thereto.The pH of the solvent mixture may be greater than or equal to approximately 10.

[0120] In some cases, one or more layers of low, non-specific binding materials can be attached or conjugated to the substrate surface, or a combination thereof, using an organic solvent mixture, wherein the dielectric constant of at least one component is less than 40 and constitutes at least 50% of the volume of the total mixture. In some cases, the dielectric constant of at least one component can be less than 10, less than 20, less than 30, or less than 40. In some cases, the component (at least one) can constitute at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, at least 70%, or at least 80% of the volume of the total mixture.

[0121] As described, the low nonspecific binding support of the present disclosure exhibits reduced nonspecific binding of proteins, nucleic acids, and other components of the hybridization or amplification formulation, or a combination thereof, used for amplifying the solid-phase nucleic acid. The degree of nonspecific binding exhibited by a given support surface can be assessed either qualitatively or quantitatively. In some cases, for example, the surface can be exposed to fluorescent dyes (e.g., Cy3, Cy5, etc.), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, or fluorescently labeled proteins (e.g.,polymerases) or a combination thereof under standardized conditions as a qualitative comparison of nonspecific binding on supports comprising different surface formulations, followed by a specified rinsing protocol and fluorescence imaging. In some cases, for example, the surface may contain fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, or fluorescently labeled proteins (e.g.polymerases) or a combination thereof under standardised conditions as a qualitative comparator of non-specific binding on supports comprising different surface formulations, followed by a specified rinsing protocol and fluorescence imaging - provided that care has been taken to ensure that fluorescence imaging takes place under conditions where the fluorescence signals are linearly related (or related in a predictable manner) to the number of fluorophores on the surface (e.g. under signal-saturating conditions or self-quenching of the fluorophore or a combination thereof is not a problem) and appropriate calibration standards are used.In some cases, other techniques, for example, radioisotope labeling and counting methods, may be used for quantitative assessment to the extent that nonspecific binding is exhibited by the various carrier surface formulations of the present disclosure.

[0122] Some of the surfaces disclosed herein have a ratio of specific to nonspecific binding of fluorophores such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more than 100, or include any value between the range recited herein. Some of the surfaces disclosed herein have a ratio of specific to nonspecific binding of fluorescence of a fluorophore such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more than 100, or include any value between the range recited herein.

[0123] As described, in some cases, the degree of nonspecific binding exhibited by the disclosed low-binding supports can be assessed using a standardized protocol for contacting a surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, a reverse transcriptase, a helicase, a single-strand binding protein (SSB), etc., or any combination thereof), a labeled nucleotide, a labeled oligonucleotide, etc., under standardized incubation and rinsing conditions, followed by determining the number of labels remaining on the surface and comparing the resulting signal with an appropriate calibration standard. In some cases, the label may comprise a fluorescent label. In some cases, the label may comprise a radioisotope.In some cases, the label may include any other recognizable label. Therefore, in some cases, the degree of nonspecific binding exhibited by a given support surface formulation may be evaluated in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some cases, the low-binding supports disclosed herein may exhibit nonspecific protein binding (or nonspecific binding of other specified molecules, e.g., Cy3 dye) of less than or equal to approximately 0.001 molecules per µm. 2 , less than or equal to approximately 0.01 molecules per µm 2 , less than or equal to approximately 0.1 molecules per µm 2 , less than or equal to approximately 0.25 molecules per µm 2 , less than or equal to approximately 0.5 molecules per µm 2 , less than or equal to approximately 1 molecule per µm 2 , less than or equal to approximately 10 molecules per µm 2, less than or equal to approximately 100 molecules per µm 2 or less than or equal to approximately 1,000 molecules per µm 2 A given support surface of the present disclosure may exhibit nonspecific binding that may be anywhere in this range, for example, less than 86 molecules per µm.

[0124] In some cases, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of a fluorophore such as Cy3 of at least or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or any value between the ranges recited herein. In some cases, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of fluorophores such as Cy3 that is greater than or equal to about 100. In some cases, the surfaces disclosed herein have a ratio of specific to non-specific fluorescent signals of a fluorophore such as Cy3 of at least or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or any value between the range recited herein.In some cases, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescent signals from a fluorophore such as Cy3 that is greater than or equal to approximately 100.

[0125] The low background surfaces in accordance with the present disclosure can have a ratio of specific dye compound (e.g., Cy3 compound) to non-specific dye adsorption (e.g., Cy3 dye adsorption) of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific attached dye molecule compounds per molecule that is non-specifically adsorbed. Similarly, when low background surfaces in accordance with the present disclosure, to which fluorophores such as Cy3 are attached and which are exposed to excitation energy, a ratio of specific fluorescent signals (which isfrom Cy3-labeled oligonucleotides attached to the surface) to non-specific, adsorbed dye fluorescence signals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1 or more than 50:1.

[0126] In some cases, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed support surface can be assessed, for example, by measuring water contact angles in which a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, a static contact angle can be determined. In some cases, an advancing contact angle or a receding contact angle can be determined. In some cases, the water contact angle for the low-binding hydrophilic support surface disclosed herein can be between 0 degrees and approximately 30 degrees. In some cases, the water contact angle for the low-binding hydrophilic support surface disclosed herein can be no more than 50 degrees, 45 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree.In many cases, the contact angle is no more than 40 degrees. A given low-bonding hydrophilic support surface disclosed herein may have a water contact angle value somewhere within this range.

[0127] In some cases, the hydrophilic surfaces disclosed herein support reduced wash times for biological assays, often by reducing nonspecific binding of biomolecules to the low-binding surfaces. In some cases, suitable washes can be performed in less than or equal to approximately 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. In some cases, suitable washes can be performed in less than 30 seconds, for example.

[0128] Some of the low-binding surfaces disclosed herein exhibit significant improvements in stability or durability to sustain extended exposure to solvents or elevated temperatures, or to repeated cycles of solvent exposure or temperature changes. In some cases, the stability of the disclosed surfaces can be tested, for example, by fluorescently labeling a functional group on the surface or a bound biomolecule (e.g., an oligonucleotide primer) on the surface and monitoring the fluorescent signal before, during, and after prolonged exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes.In some cases, the degree of change in fluorescence used to evaluate surface quality may be less than or equal to approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to solvents or elevated temperatures, or a combination thereof (or any combination of these percentages measured over these time periods).In some cases, the degree of change in fluorescence used to evaluate the quality of the surface may be less than or equal to approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles during which it repeatedly undergoes changes in solvent or temperature or a combination thereof (or any combination of these percentages over the multitude of cycles).

[0129] In some cases, the surfaces disclosed herein may exhibit a high ratio of specific signals to nonspecific signals or other background. For example, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent, unpopulated area of ​​the surface when used to amplify nucleic acids. Similarly, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent, nucleic acid-amplified, populated area of ​​the surface.

[0130] Fluorescence excitation energies vary among specific fluorophores and protocols and may vary in excitation wavelength, consistent with the selection of fluorophores or other usage parameters of a surface disclosed herein. In some cases, the wavelength is less than or equal to approximately 400 nanometers (nm). In some cases, the wavelength is greater than or equal to approximately 800 nm. In some cases, the wavelength is between 400 nm and 800 nm.

[0131] Accordingly, the surfaces disclosed herein exhibit low background fluorescence signals or a high contrast-to-noise ratio (CNR). In some cases, for example, at a location that is spatially distinct or separate from a labeled feature on the surface (e.g., a labeled spot, cluster, discrete region, subsection, or subset on the surface), the background fluorescence of the surface comprises a hybridized cluster of nucleic acid molecules or a clonally amplified cluster of nucleic acid molecules produced by 20 cycles of nucleic acid amplification via thermocycling, which may be no more than 20x, 10x, 5x, 2x, 1x, 0.5x, 0.1x, or less than 0.1x greater than the background fluorescence measured at the same location before the aforementioned hybridization or 20 cycles of nucleic acid amplification are performed.

[0132] In some cases, the fluorescence images of the disclosed low background surfaces have a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or more than 250 when used for nucleic acid hybridization or amplification applications or for clustering of hybridized or clonally amplified nucleic acid molecules (e.g., those directly or indirectly labeled with a fluorophore).

[0133] The surface comprising one or more chemically modified layers, e.g., layers of a low- and non-specifically binding polymer, can be independent or integrated into another structure or arrangement. The chemical modification layers can be applied uniformly along the surface. Alternatively, the surface can be patterned so that the chemical modification layers are confined to one or more discrete regions of the substrate. The surface can be patterned, for example, using photolithographic techniques to create an ordered arrangement or a random pattern of the chemically modified regions of the surface. The substrate surface can be patterned, for example, using contact printing or inkjet printing techniques, or a combination of these.In some cases, an ordered arrangement or random pattern of chemically modified regions may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 or more discrete regions.

[0134] To achieve surfaces with low, nonspecific binding (also referred to herein as “low binding” or “passivated” surfaces), hydrophilic polymers can be nonspecifically adsorbed or covalently grafted onto the surface. Passivation can be achieved, for example, using a poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene) or other hydrophilic polymers with different molecular weights and end groups attached to the surface, such as silane chemistry. The end groups extended from the surface can include, but are not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In some cases, two or more layers of a hydrophilic polymer, e.g., a linear polymer, branched polymer, or multiply branched polymer, can be attached to the surface.In some cases, two or more layers can be covalently coupled or cross-linked to improve the stability of the resulting surface. In some cases, oligonucleotide primers with different base sequences or base modifications (or other biomolecules, e.g., enzymes or antibodies) can be bound to the resulting surface layer at different surface densities. In some cases, for example, both the surface functional group density and the oligonucleotide concentration can be varied to target a specific primer density range. Additionally, the primer density can be controlled by diluting the oligonucleotide with other molecules that carry the same functional group.For example, oligonucleotides termed amines can be diluted with polyethylene glycol termed amines in a reaction with an NHS ester-coated surface to reduce the final primer density. Primers with different linker lengths between the hybridization region and the surface attachment functional group can also be applied to control surface density. Examples of suitable linkers include poly-T and poly-A strands at the 5' end of the primer (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers can be attached to the surface, and a fluorescence measurement can then be compared to a dye solution of known concentration.

[0135] To alter the primer surface density and add additional dimensionality to hydrophilic or amphiprotic surfaces, surfaces comprising multilayer PEG coatings or other hydrophilic polymers have been developed. By using hydrophilic and amphiprotic surface layering approaches, including but not limited to the polymer / copolymer materials described below, it is possible to significantly increase the primer loading density at the surface. Conventional PEG coating approaches utilize monolayer primer deposition, which is generally intended for single-molecule applications but does not achieve high copy numbers for nucleic acid amplification applications.As described here, "coating" can be achieved using conventional cross-linking approaches with any compatible polymer or monomer unit, allowing a surface having two or more highly cross-linked layers to be sequentially incorporated. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and the copolymers polylysine and PEG. In some cases, the different layers can be linked together through a variety of conjugation reactions, including, but not limited to, biotin-streptavidin bonding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged polymer and negatively charged polymer. In some cases, the primer materials can be engineered to high density in solution and subsequently layered onto the surface.

[0136] The chemical bonding used to graft a chemically modified layer onto a surface will fundamentally depend on both the material from which the surface is made and the chemical nature of the layer. In some cases, the first layer may be covalently attached to the surface. In some cases, the first layer may be non-covalently attached, e.g., adsorbed to the surface through non-covalent interactions such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and the molecular components of the first layer. In either case, the substrate surface may be treated prior to applying or depositing the first layer. A variety of surface preparation techniques can be used to clean or treat the surface.For example, glass or silicon surfaces can be acid cleaned with a piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)), base treated with KOH and NaOH, or cleaned with an oxygen plasma treatment method, or a combination thereof.

[0137] Silane chemistry includes a non-limiting approach to covalently modify the silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g. amine or carboxy groups) to the surface, which can then be used for the connection of linker molecules (e.g. linear hydrocarbon molecules of various lengths such as C6, C12, C18 hydrocarbon or linear polyethylene glycol molecules (PEG molecules)) or layered molecules (e.g. branched PEG molecules or other polymers). Examples of suitable silanes that can be used to prepare any of the disclosed low-bonding surfaces include, but are not limited to, 3-aminopropyltriethoxysilane (APTMS), 3-aminopropyltriethoxysilane (APTES), a variety of PEG silanes (e.g., comprising molecular weights of 1,000, 2,000, 5,000, 10,000, 20,000, etc.), amino-PEG silanes (e.g.,comprising a free amino functional group), maleimide-PEG-silanes, biotin-PEG-silanes and the like.

[0138] A variety of molecules, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or a combination thereof, can be used to produce one or more chemically modified layers on the surface, whereby the choice of components used can vary to influence one or more properties of the surface, e.g. the surface density of functional groups, or bound oligonucleotide primers or a combination thereof, the hydrophilic / hydrophobic property of the support surface, or the three-dimensional nature (e.g., "thickness") of the support surface.Polymers from which one or more layers of low, nonspecific binding materials in any of the disclosed surfaces may be made include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branching structures, streptavidin, polyacrylamide, polyester, dextran, polylysine, polylysine copolymer, or any combination thereof. Examples of conjugation chemistry that may be used to graft one or more layers of material (e.g., polymer layers) onto the surface or to crosslink the layers together, or a combination thereof, include, but are not limited to, biotin-streptavidin interactions (or variations thereof), His-tagged Ni / NTA conjugation chemistry, methoxyether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiol, epoxide, azide, hydrazide, alkyne, isocyanate, and silane.

[0139] One or more layers of multilayer surfaces may comprise a branched polymer or be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

[0140] In some cases, the branched polymer used to make one or more layers of each multilayer surface disclosed therein may comprise at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, at least 40 branches.

[0141] Linear, branched, and multiply branched polymers used to prepare one or more layers of each of the multiply branched layers disclosed therein may have a molecular weight of at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 Daltons.

[0142] In some cases, for example, when at least one layer of a multiply branched layer comprises a branched polymer, the number of covalent bonds between a branched polymer molecule of the attached layer and the molecules of the underlying layer may be approximately between one covalent bond per molecule and about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between a branched polymer molecule of the new layer and molecules of the underlying layer may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32 covalent bonds per molecule.

[0143] Any reactive functional group remaining after a material layer is bonded to the surface can optionally be blocked by attaching it to a small, inert molecule through powerful coupling chemistry. For example, when coupling chemistry with amines is used to attach new material layers to the underlying layer, remaining amine groups can then be acetylated or deactivated by coupling them to a small amino acid such as glycine.

[0144] The number of layers of low, non-specific binding materials, e.g., a hydrophilic polymer material, attached to the surface can be between 1 and about 10. In some cases, the number of layers can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. All of the maximum and minimum values ​​described in this paragraph can be combined to form a range included in the present disclosure; for example, the number of layers can be between about 2 and about 4. In some cases, all layers can comprise the same material. In some cases, each layer can comprise a different material.In some cases, the plurality of layers may comprise a variety of materials. In some cases, at least one layer may comprise a branched polymer. In some cases, all layers may comprise a branched polymer.

[0145] One or more layers of low, nonspecific binding materials can, in some cases, be attached or conjugated to the substrate surface, or a combination thereof, using a polar protic solvent, an organic solvent, a nonpolar solvent, or any combination thereof. In some cases, the solvent used for attaching or conjugating the layer, or a combination thereof, can include some type of alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, an aqueous buffered solution (e.g., phosphate buffer, phosphate-buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof.In some cases, an organic component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the total, with the remainder being water or an aqueous buffered solution. In some cases, an aqueous component of the solvent mixture used may comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, with the remainder being an organic solvent. The pH of the solvent mixture used may be less than or equal to about 6, about 6, 6.5, 7, 7.5, 8, 8.5, or 9. The pH of the solvent mixture used may be greater than or equal to about 9.

[0146] As described, the low and nonspecific binding support exhibits reduced nonspecific binding of nucleic acids and other components of the hybridization or amplification formulation, or a combination thereof, used for the amplification of the solid-phase nucleic acid. The degree of nonspecific binding exhibited by a given support surface can be assessed either qualitatively or quantitatively. In some cases, for example, exposure of the surface to fluorescent dyes (e.g., Cy3, Cy5, etc.), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, or fluorescently labeled proteins (e.g.,Polymerases) or a combination thereof under standardized conditions, followed by a specific rinsing protocol and fluorescence imaging, can be used as a qualitative comparison of nonspecific binding on supports comprising different surface formulations. In some cases, for example, exposure of the surface to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, or fluorescently labeled proteins (e.g.Polymerases) or a combination thereof under standardised conditions, followed by a specific rinsing protocol and fluorescence imaging, can be used as a qualitative comparator of non-specific binding on surfaces comprising different surface formulations - provided that care has been taken to ensure that fluorescence imaging takes place under conditions where the fluorescence signal is linearly (or predictably related) to the number of fluorophores on the surface (e.g. under signal-saturating conditions or self-quenching of the fluorophore or a combination thereof is not a problem) and appropriate calibration standards are used.In some cases, other techniques, for example, radioisotope labeling and counting methods, may be used for quantitative assessment of the degree to which nonspecific binding is exhibited by the different surface formulations of the present disclosure.

[0147] As described, in some cases, the degree of nonspecific binding exhibited by the disclosed low-binding surfaces can be assessed using a standardized protocol for contacting a surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, a reverse transcriptase, a helicase, a single-strand binding protein (SSB), etc., or any combination thereof), a labeled nucleotide, a labeled oligonucleotide, etc., under standardized incubation and rinsing conditions, followed by determining the number of labels remaining on the surface and comparing the resulting signal with an appropriate calibration standard. In some cases, the label may comprise a fluorescent label. In some cases, the label may comprise a radioisotope. In some cases, the label may comprise any other detectable label.In some cases, the degree of nonspecific binding exhibited by a given surface formulation can therefore be evaluated in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some cases, the low-binding surfaces disclosed herein can exhibit nonspecific protein binding (or nonspecific binding of other specified molecules, e.g., Cy3 dye) of less than or equal to approximately 0.001 molecules per µm. 2 , less than or equal to approximately 0.01 molecules per µm 2 , less than or equal to approximately 0.1 molecules per µm 2 , less than or equal to approximately 0.25 molecules per µm 2 , less than or equal to approximately 0.5 molecules per µm 2 , less than or equal to approximately 1 molecule per µm 2 , less than or equal to approximately 10 molecules per µm 2 , less than or equal to approximately 100 molecules per µm 2or less than or equal to approximately 1,000 molecules per µm 2 A given surface of the present disclosure may exhibit nonspecific binding that may be anywhere in this range, for example, less than or equal to about 86 molecules per µm 2 For example, some of the modified surfaces disclosed herein exhibit nonspecific protein binding of less than or equal to approximately 0.5 molecules / µm 2 after exposure to a solution of 1µM bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 minutes, followed by a 10-minute PBS rinse. Another example of the modified surfaces disclosed herein exhibits nonspecific protein binding of less than or equal to approximately 0.5 molecules / µm 2after exposure to a solution of 1 µM cyanine 3 dye-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) for 15 minutes, followed by a 3-minute rinse with deionized water. Some of the modified surfaces disclosed herein exhibit nonspecific binding of Cy3 dye molecules of less than or equal to approximately 0.25 molecules per µm 2 on.

[0148] The low background surfaces consistent with the present disclosure may have ratios of specific dye adsorption (e.g., Cy3 adsorption) to nonspecific dye adsorption (e.g., Cy3 dye adsorption) of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific attached dye molecules per molecule nonspecifically adsorbed. Likewise, low background surfaces consistent with the present disclosure to which fluorophores such as Cy3 are attached and which are exposed to excitation energy exhibit a ratio of specific fluorescent signals (resulting, for example, from Cy3-labeled oligonucleotides attached to the surface) to nonspecific adsorbed dye fluorescent signals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1.

[0149] In some cases, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed surfaces can be assessed, for example, by measuring water contact angles in which a small drop of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, a static contact angle can be determined. In some cases, an advancing contact angle or a receding contact angle can be determined. In some cases, the water contact angle for the low-binding hydrophilic surfaces disclosed herein can be between about 0 degrees and about 30 degrees. In some cases, the water contact angle for the low-binding hydrophilic surface disclosed herein can be no more than 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree.In many cases, the contact angle is no more than 40 degrees. A given low-bonding hydrophilic surface disclosed here may have a water contact angle somewhere within this range.

[0150] In some cases, the low-binding surfaces disclosed herein may exhibit significant improvements in stability or durability to extended exposure to solvents or elevated temperatures, or to repeated cycles of solvent exposure or temperature changes. In some cases, the stability of the disclosed surfaces may be tested, for example, by fluorescently labeling a functional group on the surface or a bound biomolecule (e.g., an oligonucleotide primer) on the surface and monitoring the fluorescent signal before, during, and after prolonged exposure to solvents and elevated temperatures or repeated cycles of solvent exposure or temperature changes.In some cases, the degree of change in fluorescence used to evaluate surface quality may be less than or equal to approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to solvents or elevated temperatures, or a combination thereof (or any combination of these percentages measured over these time periods).In some cases, the degree of change in fluorescence used to evaluate the quality of the surface may be less than or equal to approximately 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles during which it repeatedly undergoes changes in solvent or temperature or a combination thereof (or any combination of these percentages over the multitude of cycles).

[0151] In some cases, the surfaces disclosed herein may exhibit a high ratio of specific signals to nonspecific signals or other background. For example, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent, unpopulated area of ​​the surface when used to amplify nucleic acids. Similarly, some surfaces may exhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or more than 100 times greater than a signal from an adjacent, nucleic acid-amplified, populated area of ​​the surface.

[0152] Accordingly, the low-background surfaces disclosed herein exhibit low-background fluorescence signals or a high contrast-to-noise ratio (CNR).

[0153] Flow Cell Devices: In some aspects, the low nonspecific binding surfaces are surfaces of a flow cell device disclosed herein. The flow cell devices described herein may include a first reservoir receiving a first solution and having an inlet end and an outlet end, wherein the first active agent flows from the inlet end to the outlet end in the first reservoir; a second reservoir receiving a second solution and having an inlet end and an outlet end, wherein a second active agent flows from the inlet end to the outlet end in the second reservoir; and a central region having an inlet end fluidly coupled to the outlet end of the first reservoir and the outlet end of the second reservoir by at least one valve.In a flow cell device, the volume of the first solution flowing from the outlet of the first reservoir to the inlet of the central region is less than the volume of the second solution flowing from the outlet of the second reservoir to the inlet of the central region.

[0154] The reservoirs described in the device can be used to store various reagents. In some aspects, the first solution stored in the first reservoir is different from the second solution stored in the second reservoir. The second solution comprises at least one reagent common to a variety of reactions occurring in the central region. In some aspects, the second solution comprises at least one reagent from a list consisting of a solution, a polymerase, and a dNTP. In some aspects, the second solution comprises low-cost reagents. In some aspects, the first reservoir is fluidly coupled to the central region by a first valve, and the second reservoir is fluidly coupled to the central region by a second valve. The valve may be a diaphragm valve or other suitable valve.

[0155] The central region may comprise a capillary tube or a microfluidic chip with one or more microfluidic channels. In some embodiments, the capillary tube is a ready-to-use product. The capillary tube or microfluidic chip may also be removable from the device. In some embodiments, the capillary tube or microfluidic chip comprises an oligonucleotide population designed to sequence a eukaryotic genome. In some embodiments, the capillary tube or microfluidic channel may be removable in the central region.

[0156] Disclosed herein are single-capillary flow cell devices comprising a single capillary vessel and one or two fluid adapters attached to one or both ends of the capillary vessel, wherein the capillary vessel provides a fluid flow channel of a specific cross-sectional area and length, and wherein the fluid adapters are designed to mate with standardized tubing to provide convenient, interchangeable fluid connections to an external fluid flow control system. In general, the capillary vessels used in the disclosed flow cell devices (and flow cell cartridges to be described below) have at least one internal, axially aligned fluid flow channel (or "lumen") extending the entire length of the capillary vessel. In some aspects, the capillary vessel may have two, three, four, five, or more than five internal, axially aligned fluid flow channels (or "lumens").

[0157] A number of specific cross-sectional geometries for an individual capillary vessel (or lumen thereof) are consistent with the present disclosure, including, but not limited to, round, elliptical, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some aspects, the individual capillary vessel (or lumen thereof) may have any specific cross-sectional dimension or set of dimensions. For example, in some aspects, the largest cross-sectional dimension of the capillary vessel lumen (i.e., the diameter if the lumen has a round shape, or the diagonal if the lumen has a square or rectangular shape) may be between about 10 μm and about 10 mm.The length of the one or more capillary vessels used to create the disclosed single-capillary flow cell devices or flow cell cartridges can be between about 5 mm and about 5 cm or more. Capillary vessels, in some cases, have a gap height of about or exactly 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, or 500 μm, or any value falling within the range defined therein.

[0158] The present disclosure also includes flow cell devices comprising one or more microfluidic chips and one or two fluid adapters attached to one or both ends of the microfluidic chip, wherein the microfluidic chip provides one or more fluid flow channels of a particular cross-sectional area and length, and wherein the fluid adapters are configured to mate with the microfluidic chip to provide convenient, interchangeable fluid connections to an external fluid flow control system.

[0159] The microfluidic chip described herein includes one or more microfluidic channels etched into the surface of the chip. The microfluidic channels are defined as fluid channels with at least a minimum dimension of <1 nm to 1000 µm. The system of microfluidic channels, created on a glass or silicone substrate, has channel heights and widths in the range of <1 nm to 1000 µm. The channel length can be in the micrometer range.

[0160] The capillary vessels or microfluidic chips used to construct the disclosed flow cell devices can be made from any of a variety of materials known to those skilled in the art, including, but not limited to, glass (e.g., borosilicate glass, soda-lime glass, etc.), fused silica (quartz), a polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate material (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclo-olefin polymer (COP), cyclo-olefin copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluorocarbon rubber (FFKM) as less chemically reactive alternatives. PEI lies somewhere between polycarbonate and PEEK in terms of cost and compatibility. FFKM is also referred to as Kalrez, or any combination thereof.

[0161] The flow cell device (i.e., microfluidic chip or capillary flow cell) is operatively coupled to an imaging system described herein to capture or detect signals from DNA bases for applications such as nucleotide acid sequencing, analyte capture and detection, and the like.

[0162] Oligonucleotide primers and adapter sequences: In general, at least one layer of the one or more layers of low, nonspecific binding materials may comprise functional groups for covalently or noncovalently attaching oligonucleotide adapter or primer sequences, or the at least one layer may already contain covalently or noncovalently attached oligonucleotide adapter or primer sequences at the time of its application to the support surface. In some cases, the oligonucleotides bound to the polymer molecules of the at least one third layer may be distributed at a variety of depths throughout the layer.

[0163] One or more types of oligonucleotide primers can be attached to or bound to the support surface. In some cases, the one or more types of oligonucleotide adapters or primers can include spacer sequences, adapter sequences for hybridization to adapter-ligated matrix nucleic acid sequences, forward amplification primers, reverse amplification primers, sequencing primers, or molecular barcoding sequences, or any combination thereof. In some cases, one primer or adapter sequence can be bound to at least one layer of the surface. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adapter sequences can be bound to at least one layer of the surface.

[0164] In some cases, the bound oligonucleotide adapter, or the primer sequences, or a combination thereof, may have a length ranging from about 10 nucleotides to about 100 nucleotides. In some cases, the bound oligonucleotide adapter, or the primer sequences, or a combination thereof, may have a length of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some cases, the bound oligonucleotide adapter, or the primer sequences, or a combination thereof, may have a length of at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides.All of the maximum and minimum values ​​described in this paragraph can be combined to form a range included in the present disclosure. For example, in some cases, the length of the linked oligonucleotide adapter or primer sequences, or combinations thereof, can range from about 20 nucleotides to about 80 nucleotides. The length of the linked oligonucleotide adapters, or primer sequences, or combinations thereof can have any value within this range, e.g., about 24 nucleotides.

[0165] In some cases, the annealed primer sequences may include modifications designed to improve the specificity and efficiency of nucleic acid amplification performed on the low-binding supports. For example, in some cases, the primer may include polymerase cutoff points, so that the portion of the primer sequence between the surface conjugation point and the modification site is always single-stranded and functions as loading sites for 5' or 3' helicases in some helicase-dependent isothermal amplification methods. Other examples of primer modification that can be used to create polymerase cutoff points may include, but are not limited to, inserting an AEG chain into the backbone of the primer between two nucleotides towards the 5' end, inserting an abatic nucleotide (e.g.,a nucleotide that has neither a purine nor a pyrimidine base) or a lesion site that can be bypassed by the helicase.

[0166] As will be discussed in more detail in the examples below, the surface density of bound primers at the support surface, or the distance of the bound primers away from the support surface (e.g., by varying the length of a linker molecule used to attach the primers to the surface), or a combination thereof, can be varied to "tune" the support for optimal performance when using a particular amplification method. As noted below, adjusting the surface density of bound primers can affect the degree of specific or nonspecific amplification, or a combination thereof, observed on the support in a manner that varies depending on the amplification method chosen.In some cases, the surface density of the bound oligonucleotide primers can be varied by varying the ratio of the molecular components used to create the support surface. For example, in the case where an oligonucleotide primer-PEG conjugate is used to form the final layer of a low-binding support, the ratio of oligonucleotide primer-PEG conjugate to unconjugated PEG molecule can be varied. The resulting surface density of the bound primer molecules can then be estimated or measured using a variety of techniques. Examples include, but are not limited to, the use of radioisotope labeling and counting methods, covalent attachment of a cleavable molecule containing an optically detectable label (e.g.,a fluorescent label) that can be cleaved from a support surface of defined area, collected in a defined amount of an appropriate solvent, and then quantified by comparing fluorescent signals with those for a calibration solution containing a known concentration of labels, or using fluorescence imaging techniques, provided that care has been taken in the labeling reaction conditions and image acquisition settings to ensure that the fluorescence signals are linearly related to the number of fluorophores on the surface (e.g., that there is no self-quenching of the fluorophores on the surface).

[0167] In some cases, the resulting surface density of oligonucleotide primers on the low binding support surfaces of the present disclosure may be in the range of approximately 1,000 primer molecules per µm 2up to approximately 1,000,000 primer molecules per µm 2 In some cases, the surface density of oligonucleotide primers can be at least 1,000, at least 10,000, at least 100,000 or at least 1,000,000 molecules per µm 2 In some cases, the surface density of oligonucleotide primers may be at most 1,000,000, at most 100,000, at most 10,000 or at most 1,000 molecules per µm 2 All of the maximum and minimum values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases the surface density of the primers can be in a range of approximately 10,000 molecules per µm 2 up to approximately 100,000 molecules per µm 2 The surface density of primer molecules can have any value within this range, e.g., approximately 455,000 molecules per µm 2In some cases, the surface density of matrix directory nucleic acid sequences originally hybridized to adapter or primer sequences on the support surface may be less than or equal to that specified for the surface density of bound oligonucleotide primers. In some cases, the surface density of clonally amplified matrix directory nucleic acid sequences hybridized to adapter or primer sequences on the support surface may cover the same area specified for the surface density of bound oligonucleotide primers.

[0168] Local densities, as listed above, do not preclude variation in density across the surface, so that a surface may encompass an area having an oligo density of, for example, 500,000 µm 2 while also encompassing a second area that has a substantially different local density.

[0169] Imaging systems. Imaging systems described herein are used to detect hybridization between one or more sample nucleic acid molecules and to capture nucleic acid molecules coupled to a surface with low, nonspecific binding. In some cases, the imaging systems include a camera. In some cases, the imaging systems include a microscope, such as a fluorescence microscope. An inverted fluorescence microscope combined with a camera can be used to acquire an image of the surface with low, nonspecific binding and to detect hybridization between one or more sample nucleic acid molecules and capture nucleic acid molecules. A non-limiting example of an imaging system described herein is an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with a total internal reflection fluorescence (TIRF) microscope objective (100X, 1.5 NA, Olympus), a CCD camera (e.g.,A suitable camera (e.g., an Olympus EM-CCD monochrome camera, an Olympus XM-10 monochrome camera, or an Olympus DP80 color and monochrome camera), a light source (e.g., an Olympus 100W Hg lamp, an Olympus 75W Xe lamp, or an Olympus U-HGLGPS fluorescent light source), and excitation wavelengths of 532 nm or 635 nm were used. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), e.g., 405, 488, 532, or 633 nm dichroic reflectors / beamsplitters, and bandpass filters were chosen as 532 LP or 645 LP, according to the corresponding excitation wavelength.

[0170] Computer control systems. The present disclosure provides computer systems programmed or otherwise configured to perform the methods described herein, such as methods for sequencing nucleic acid, storing comparison nucleic acid sequences, performing sequence analysis, and / or comparing reference nucleic acid sequences, as described herein. An example of such a computer system is described in Fig. 10. As shown in Fig.10, the computer system 1001 includes a central processing unit (CPU, also referred to herein as a "processor" or "computer processor") 1005, which may be a single-core or multi-core processor, or a plurality of processors for parallel processing. The computer system 1001 also includes a memory or storage location 1010 (e.g., random access memory, read-only memory, flash memory), an electronic storage device 1015 (e.g., hard disk), a communications interface 1020 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1025 such as cache, other memory, data storage, and / or electronic display adapters. The memory 1010, the storage device 1015, the interface 1020, and the peripheral devices 1025 communicate with the CPU 1005 via a communications bus (solid lines), such as a motherboard.The storage unit 1015 may be a data storage unit (or data repository) for storing data. The computer system 1001 may be operatively coupled to a computer network ("network") 1030 via the communication interfaces 1020. The network 1030 may be the Internet, an intranet, or an extranet, and / or an intranet and / or extranet in communication with the Internet. In some cases, the network 1030 is a telecommunications and / or data network. The network 1030 may include one or more computer servers that may enable distributed computing, such as cloud computing. The network 1030 may, in some cases with the help of the computer system 1001, implement a peer-to-peer network that may allow devices coupled to the computer system 1001 to act as clients to a server.

[0171] CPU 1005 may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 1010. Examples of operations performed by CPU 1005 include fetch, decode, execute, and write-back.

[0172] Storage unit 1015 may store files, such as drivers, libraries, and stored programs. Storage unit 1015 may store user data, such as user settings and user programs. In some cases, computer system 1001 may include one or more additional data storage units located external to computer system 1001, for example, located on a remote server that communicates with computer system 1001 via an intranet or the Internet.

[0173] Computer system 1001 can communicate with one or more remote computer systems via network 1030. For example, computer system 1001 can communicate with a remote computer system of a user (e.g., operator). Examples of a remote computer system include personal computers (e.g., portable PCs), slate or tablet computers (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, smartphones (e.g., Apple® iPhone, Android-enabled devices, Blackberry®), or personal digital assistants. The user can access computer system 1001 via network 1030.

[0174] The methods described herein may be performed using machine-executable code (e.g., computer processor) stored in an electronic storage location of computer system 1001, such as memory 1010 or electronic storage device 1015. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by processor 1005. In some cases, the code may be obtained from storage device 1015 and stored in memory 1010 for easy access by processor 1005. In some situations, electronic storage device 1015 may be excluded, and machine-executable instructions are stored in memory 1010.

[0175] The code may be pre-compiled and configured for use with a machine including a processor configured to execute the code, or it may be compiled at runtime. The code may be provided in a programming language that can be selected to enable the code to be executed in a pre-compiled or as-compiled manner.

[0176] Aspects of the systems and methods provided herein, such as computer system 1001, may be embodied by programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture," typically in the form of machine-executable code (or processor) and / or associated data residing on or embodied in a machine-readable medium. Machine-executable code may be stored on an electronic storage device, such as memory (e.g., read-only memory, random access memory, flash memory) or on a hard disk.The "storage" type media may include some or all of the physical memory of computers, processors, or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives, and the like, which may provide non-volatile storage for software programming at any given time. All or part of the software may be communicated at times over the Internet or various other telecommunications networks. Such communications may, for example, enable the loading of software from one computer or processor to another, such as from a management server or host computer into the computing platform of an application server.Another type of media may also carry the software elements, such as optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical fixed networks, and over various air connections. The physical elements that carry such waves, such as wired or wireless connections, or the like, may also be considered software-carrying media. As used herein, and unless limited to non-transitory, physical "storage" media, terms such as computer- or machine-readable medium may refer to any medium involved in providing instructions to a processor for execution.

[0177] A machine-readable medium, such as computer-executable code, can therefore take many forms, including, but not limited to, a physical storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases shown in the drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Physical transmission media include coaxial cable, copper wire, and fiber optics, including the cables that comprise a bus within a computer system.Carrier wave transmission media can take the form of electrical or electromagnetic signals, or acoustic waves or light waves, such as those generated during radio frequency (RF) or infrared (IR) data communications. Common forms of computer-readable media therefore include, for example: a floppy disk, a diskette, a hard disk, magnetic tape or any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punched cards, paper tape, any other physical storage medium with hole patterns, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, and any other memory chip or cartridge, a carrier wave carrying data or instructions, cables or interconnects carrying such a carrier wave, or any other medium from which a computer can read programming code and / or data.Many of these types of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0178] Computer system 1001 may include, or be in communication with, an electronic display 1035 that includes a user interface (UI) for providing, for example, an output or readout from a nucleic acid sequencing instrument connected to computer system 1001. Such a readout may include a nucleic acid sequencing readout, such as a sequence of nucleic acid bases comprising a particular nucleic acid sample. The UI may also be used to present the results of an analysis using such a readout. Examples of UIs include, but are not limited to, a graphical user interface (GUI) and web-based user interfaces. Electronic display 1035 may be a computer monitor or a capacitive or resistive touchscreen. Performance of compositions and systems

[0179] Improvements in hybridization rate. In some cases, the use of the buffer formulations disclosed herein (optionally used in conjunction with a low-binding nonspecific surface) provides relative hybridization rates that are between approximately 2x and approximately 20x faster than those of a standard hybridization protocol. In some cases, the relative hybridization rate may be at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least 10x, at least 12x, at least 14x, at least 16x, at least 18x, or at least 20x that of a standard hybridization protocol.

[0180] The method and composition described herein can help shorten the time required to complete hybridization. In some embodiments, the hybridization time can range from about 1 second (s) to about 2 hours (h), about 5 s to about 1.5 h, about 15 s to about 1 h, or about 15 s to about 0.5 h. In some embodiments, the hybridization time can range from about 15 s to about 1 h. In some embodiments, the hybridization time may be shorter than 15 s, 30 s, 1 minute (min), 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min. In some embodiments, the hybridization time may be longer than 1 s, 5 s, 10 s, 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, or 5 min.

[0181] The melting methods described herein can significantly reduce melting time. In some embodiments, at least 90% of the target nucleic acid melts to the surface-bound nucleic acid in less than or equal to approximately 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min. In some embodiments, at least 80% of the target nucleic acid melts to the surface-bound nucleic acid in less than or equal to about 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min.In some embodiments, at least 90% of the target nucleic acid melts to the surface-bound nucleic acid in greater than or equal to about 1 s, 5 s, 10 s, 15 s, 30 s, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 4 min, or 5 min. In some embodiments, at least 90% of the target nucleic acid melts to the surface-bound nucleic acid in between about 10 s and about 1 hour, between about 30 s and about 50 min, between about 1 min and about 50 min, or between about 1 min and about 30 min. In some embodiments, at least 90% of the target nucleic acid melts to the surface-bound nucleic acid in between 2-25, 3-24, 4-23, 5-23, 6-22, 7-21, 8-20, 9-19, 10-18, 11-17, 12-16, or 13-15 min.

[0182] Improvements in hybridization efficiency. As used herein, hybridization efficiency (or yield) is a measure of the percentage of the total available tethered adapter sequences on a solid surface, primer sequence, or oligonucleotide sequence in general that are hybridized to complementary sequences. In some cases, the use of the optimized buffer formulations disclosed herein (optionally used in conjunction with a surface with low, nonspecific binding) provides improved hybridization efficiency over that of a standard hybridization protocol. In some cases, the achieved hybridization efficiency is better than 80%, 85%, 90%, 95%, 98%, or 99% in any of the above-mentioned hybridization reaction times.

[0183] The method and composition described herein can be used under isothermal melting conditions. In some embodiments, the methods described herein can eliminate the cooling required for most hybridizations. In some embodiments, the melting methods described herein can be performed at a temperature in the range of about 10°C to 95°C, about 20°C to 80°C, about 30°C to 70°C. In some embodiments, the temperature can be lower than about 40°C, 50°C, 60°C, 70°C, 80°C, or 90°C.

[0184] Improvements in hybridization specificity. Compared to a comparable hybridization reaction performed using standard hybridization conditions and reagents, the methods, systems, compositions, and kits described herein provide improved hybridization specificity. In some cases, the comparable hybridization reaction is performed on a low-binding surface described herein at 90 degrees Celsius for 5 minutes, followed by 120 minutes of cooling to a final temperature of 37 degrees Celsius in a buffer comprising saline sodium citrate. In some cases, the hybridization specificity that can be achieved is better than 1 base mismatch per 10 hybridizations, 1 base mismatch per 100 hybridizations, 1 base mismatch per 1,000 hybridizations, or 1 base mismatch per 10.000 hybridization events, The hybridization specificity can be measured using the techniques described in.

[0185] In some cases, at least or approximately 70%, 80%, or 90% of the sample nucleic acid molecules hybridize correctly to the capture nucleic acid molecules (e.g., adapter sequences, primer sequences, or oligonucleotide sequences) with a complementary sequence. In some cases, more than 90% of the sample nucleic acid molecules hybridize correctly to the capture nucleic acid molecules. In some cases, between 90% and 99% of the sample nucleic acid molecules hybridize correctly to the capture nucleic acid molecules. In some cases, 100% of the sample nucleic acid molecules hybridize correctly to the capture nucleic acid molecules.

[0186] Hybridization specificity can be measured by hybridizing labeled (e.g., Cy3) complementary oligos to surface-bound nucleic acid molecules immobilized to the surface, dehybridizing and collecting the hybridized oligos, and measuring a fluorescence signal from the collected oligos using a fluorescence plate reader at the appropriate excitation and emission wavelengths (e.g., 532, peak 570 / 30). The results are used to develop standard curves, and precise concentrations are measured. This assay can be repeated with oligos exhibiting varying degrees of complementarity and respective specificities.

[0187] Hybridization specificity, as measured on the surface, can be determined by dividing the nonspecific background counts (e.g., calculated using the methods outlined in Example 3) by the nonspecific sample hybridization / nonspecific background counts (also calculated using the methods outlined in Example 3). Calibration curves can be constructed, and experiments with oligos with varying degrees of complementarity can be added to more accurately calculate the respective specificities.

[0188] The specificity of a given nucleic acid sample, p, can be quantified by the relative sensitivity when a p spot is exposed to an exactly matching target, t, or a mismatch, m.

[0189] The specificity of the assay can be quantified by determining the proportion of falsely hybridized samples, P m, is considered In this case,

[0190] Improvements in hybridization sensitivity. "Hybridization sensitivity" refers to a concentration range of sample (or target) nucleic acid molecules in which hybridization occurs with a target hybridization specificity. In some cases, the target hybridization specificity is 90% or greater. In some cases, the methods, compositions, systems, and kits described herein use a concentration of less than 10 nanomolar of sample nucleic acid molecules to hybridize the sample nucleic acid molecules to capture nucleic acid molecules with high specificity. In some cases, a concentration of sample nucleic acid molecules between 10 nanomolar and 50 picomolar is used. In some cases, between 9 nanomolar and 100 picomolar sample nucleic acid molecules are used. In some cases, between 9 nanomolar and 150 picomolar sample nucleic acid molecules are used.In some cases, between 7 nanomolar and 200 picomolar sample nucleic acid molecules are used. In some cases, between 6 nanomolar and 250 picomolar sample nucleic acid molecules are used. In some cases, between 5 nanomolar and 250 picomolar sample nucleic acid molecules are used. In some cases, between 4 nanomolar and 300 picomolar sample nucleic acid molecules are used. In some cases, between 3 nanomolar and 350 picomolar sample nucleic acid molecules are used. In some cases, between 2 nanomolar and 400 picomolar sample nucleic acid molecules are used. In some cases, between 1 nanomolar and 500 picomolar sample nucleic acid molecules are used. In some cases, less than or equal to approximately 1 nanomolar sample nucleic acid molecules are used. In some cases, less than or equal to approximately 250 picomolar sample nucleic acid molecules are used.In some cases, less than or equal to approximately 200 picomolar sample nucleic acid molecules are used. In some cases, less than or equal to approximately 150 picomolar sample nucleic acid molecules are used. In some cases, less than or equal to approximately 100 picomolar sample nucleic acid molecules are used. In some cases, less than or equal to approximately 50 picomolar sample nucleic acid molecules are used.

[0191] In some cases, the hybridization sensitivity, if calculated using the International Union of Pure and Applied Chemistry (IUPAC), agrees with the sensitivity S e Slope of the calibration curve. The calibration curve describes the measured response, R, to a target concentration, c t , R(c t ), and

[0192] The quantitative resolution of the assay, Δc t , is then replaced by , where it is the measurement uncertainty based on its standard deviation. The detection limit, the lowest detectable ct, is determined by Δc t (c t = 0), because if the concentration c t is lower than Δc t (c t = 0), the uncertainty is larger than the signal, and if one assumes that R(ct) is proportional to the equilibrium hybridization fraction on surface x; i.e., R(ct) = κx + const where κ is a constant. This assumption is justified if the following conditions are met: (1) non-specific adsorption is negligible and R is solely due to hybridization at the surface; (2) the duration of the experiment is sufficiently long to allow hybridization to reach equilibrium; and (3) the measured signal is linearly dependent on the amount of oligonucleotides at the surface. Nucleic acid sequence in 2 applications

[0193] Nucleic acid sequencing is one of the many applications for which the methods, compositions, systems, and kits described here can be useful. With reference to Fig. 2 In some embodiments, the methods described herein include preparing a library of sample nucleic acid molecules for sequencing, hybridizing the library of sample nucleic acids to nucleic acid molecules coupled to a low, nonspecific binding surface in the presence of the hybridization compositions described herein, amplifying the library of sample nucleic acids in situ, dehybridizing the linearized and amplified sample nucleic acids from the nucleic acid molecules coupled to the low, nonspecific binding surface, hybridizing a primer sequence to the sample nucleic acids, and sequencing the sample nucleic acids.

[0194] With regard to Fig.6, a library of sample nucleic acid molecules is created 601, for example using a split ligation protocol, the library of sample nucleic acid molecules is hybridized 602 in the presence of a hybridization composition described herein with the nucleic acid molecules coupled to a surface with low, non-specific binding, the hybridization of the sample nucleic acid molecules with the nucleic acid molecules coupled to a surface with low, non-specific binding takes place 603, sequencing primers are hybridized 604 with complementary primer-binding sequences on nucleic acids, and the sequencing of the sample nucleic acids is carried out 605.

[0195] Fig.Figure 7 provides an exemplary sequencing workflow whereby a labeled deoxyribonucleotide triphosphate (dNTP) binds to the sample nucleic acid molecule to determine the identity of the complementary nucleotide in the nucleic acid sequence of the sample nucleic acid molecule 701. In some cases, the dNTP is labeled with a fluorophore (e.g., Cy3), either directly or through interaction with a labeled detection reagent. Optionally, the surface is washed to remove the unbound labeled dNTP. The surface is imaged to detect the presence of labeled dNTP 702. The labeled dNTP is detached from the sample nucleic acid molecule, and a blocked unlabeled dNTP is incorporated into the sample nucleic acid molecule 703. The blocked unlabeled nucleotide is cleaved 704. Steps 701-704 are repeated for the next nucleotide in the sample nucleic acid molecule 705.

[0196] The methods, compositions, systems, and kits described herein offer at least the following advantages, particularly in a nucleic acid sequencing process: (i) reduced fluid wash times (due to reduced nonspecific binding and thus faster sequencing cycle times), (ii) reduced imaging times (and thus faster turnaround times for assay readout and sequencing cycles), (iii) reduced overall workflow time requirements (due to reduced cycle times), (iv) reduced detection equipment costs (due to improvements in contrast-to-noise ratio), (v) improved readout accuracy (base calling) due to improvements in contrast-to-noise ratio), (vi) improved reagent stability and reduced reagent usage requirements (and thus reduced reagent costs),and (vii) fewer runtime failures due to failures in nucleic acid amplification. Definitions

[0197] Unless otherwise defined, all technical terms used herein have the exact meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0198] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference to "or" throughout this specification shall include "and / or" unless otherwise specified.

[0199] As used herein, the term "approximately" with respect to a number means that number plus or minus 10% of that number. When used in connection with a range, the term "approximately" means that range minus 10% of its lowest value and plus 10% of its highest value.

[0200] As used herein, the terms "DNA hybridization" and "nucleic acid hybridization" are used interchangeably and are intended to cover any type of nucleic acid hybridization, e.g., DNA hybridization, RNA hybridization, unless otherwise specified.

[0201] As used herein, the term "isothermal" means a condition in which the temperature remains essentially constant. A temperature that is "essentially constant" cannot deviate (e.g., become higher or lower) by more than 0.25 degrees, 0.50 degrees, 0.75 degrees, or 1.0 degrees over a period of time.

[0202] The terms "fusion" and "hybridization" are used interchangeably here to refer to the ability of two nucleic acid molecules to combine with each other. In some cases, "combining" refers to Watson-Crick base pairing between the bases in each of the nucleic acid molecules.

[0203] As used herein, “hybridization specificity” refers to a measure of the ability of nucleic acid molecules (e.g., adapter sequences, primer sequences, or oligonucleotide sequences) to correctly hybridize to a region of a nucleic acid molecule having a nucleic acid sequence that is fully complementary to the nucleic acid molecule.

[0204] As used herein, "hybridization sensitivity" refers to a concentration range of sample (or target) nucleic acid molecules in which hybridization occurs with high specificity. In some cases, the methods, compositions, systems, and kits described herein can achieve a concentration as low as 50 picomolar of nucleic acid molecules in which hybridization with high specificity is achieved. In some cases, the concentration range is between approximately 1 nanomolar and approximately 50 picomolar of sample nucleic acid molecules.

[0205] As used herein, “hybridization efficiency” refers to a measure of the percentage of the total available nucleic acid molecules (e.g., adapter sequences, primer sequences, or oligonucleotide sequences) that are hybridized to the region of the nucleic acid molecule containing the nucleic acid sequence that is fully complementary to the nucleic acid molecule.

[0206] As used herein, the term "hybridization stringency" refers to a percentage of nucleotide bases within at least a portion of a nucleic acid sequence that undergoes a complementary hybridization reaction by standard Watson-Crick base pairing (e.g., a hybridization region). As a non-limiting example, a hybridization stringency of 80% means that a stable duplex can be formed in which 80% of the hybridization region undergoes Watson-Crick base pairing. A higher hybridization stringency means that, within a given hybridization reaction, a higher degree of Watson-Crick base pairing is required to form a stable duplex.

[0207] As used herein, the terms “isolate” and “clean” are used interchangeably unless otherwise specified. Abbreviations Dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), 3-(N-morpholino)propanesulfonic acid (MOPS), Acetonitrile (ACN) 2-(N-Morpholino)ethanesulfonic acid (MES) saline sodium citrate (SSC) Formamide (Form.) Tris(hydroxymethyl)aminomethane (Tris) EXAMPLES

[0208] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. Example 1 - DNA hybridization on a surface with low, nonspecific binding

[0209] Fig. 1A-B provide examples of optimized hybridization achieved on a low binding surface using the disclosed hybridization method ( Fig.1A) with lower concentrations of hybridization reporter probe and shortened hybridization times compared to the results achieved with a traditional hybridization protocol on the same low-binding surface ( Fig. 1B).

[0210] Fig. Figure 1A shows low-binding surface hybridization reactions according to the embodiments described herein. The rows provide two test hybridization conditions, Hybridization Condition 1 (“Hyb 1”) and Hybridization Condition 2 (“Hyb 2”). Hyb 1 refers to hybridization buffer composition C10 from Table 1. Hyb 2 refers to hybridization buffer composition D18 from Table 1. A hybridization reporter probe (complementary oligonucleotide sequences labeled at the 5'-end with a CyTM3 fluorophore) with Fig.The concentrations reported in Figure 1A (10 nM, 1 nM, 250 pM, 100 pM, and 50 pM) were hybridized in the buffer preparations at 60 degrees Celsius for 2 minutes.

[0211] Fig. Figure 1B shows low-binding surface hybridization reactions according to a standard hybridization protocol with standard hybridization conditions (“Standard-Hyb-Conditions”). A standard hybridization buffer of 2X-5X saline sodium citrate (SSC) was used with the same hybridization reporter probe as above at the same concentrations as described above, as in Fig. 1A. The standard hybridization reaction was performed at 90 degrees Celsius with a slow process (2 hours) to reach 37 degrees Celsius.

[0212] For each of the Fig. 1A and Fig.In the hybridization reactions shown in Figure 1B, the top row for each hybridization reaction is test (“T”), which is the complementary oligos (e.g., CY3™-5'-ACCCTGAAAGTACGTGCATTACATG-3'), and the bottom row for each hybridization region is a control (“C”), which is non-complementary (e.g., CY3™-5'-ATGTCTATTACGTCACACTATTATG-3').

[0213] The surfaces used for all test conditions were surfaces with extremely low non-specific binding with a level of non-specific Cy3 dye adsorption corresponding to less than or equal to approximately 0.25 molecules / µm 2 In this example, the low-binding surfaces used were a glass substrate functionalized with silane-PEG-5K-COOH (Nanocs Inc.).

[0214] After completion of the hybridization reactions, the wells were washed with 50 mM Tris pH 8.0 and 50 mM NaCl.

[0215] Images were acquired using an inverted microscope (Olympus IX83) equipped with a 100 X TIRF objective, NA = 1.4 (Olympus), a dichroic mirror optimized for 532 nm light (Semrock, Di03-R5324-tl-25x36), a bandpass filter optimized for Cy3 emission (Semrock, FF01-562 / 40-25), and a camera (sCMOS, Andor Zyla) under non-signal saturating conditions for 1 s (Laser Quantum, Gern 532, < 1 W / cm 2 at the sample) while the sample was immersed in a buffer (25 mM ACES, pH 7.4 buffer). Images were taken as described above and the results in Fig. 1A (optimized) and Fig. 1B (standard) shown.

[0216] A significant signal was observed in both the Hyb 1 and Hyb 2 hybridization reactions ( Fig.1A), compared to the negative control, from the reaction with 250 picomolar (mP). In contrast, no signal was observed from the reaction with 250 mP under standard Hyb conditions, compared to the negative control. The same signal was observed for lower starting concentrations (e.g., 100 pM, 50 pM) of the hybridization reporter probe. Fig. Figure 1A shows a reduction of more than a factor of 200 in the starting DNA (labeled oligo) needed for a given DNA capture on surfaces with low, nonspecific binding, a reduction of more than 50-fold in hybridization times, and a reduction of half in hybridization temperatures, compared to standard hybridization procedures and reagents on the same substrates with low, nonspecific binding ( Fig.1B). The buffer compositions and methods described here exhibit improved hybridization specificity, reduced workflow times, and higher hybridization sensitivity. Example 2

[0217] Buffer compositions according to various embodiments described herein have been optimized to facilitate hybridization of single-matrix oligonucleotide fragments to the surfaces described herein with low, nonspecific binding.

[0218] Preparing the surface with low, nonspecific binding. Glass substrates (175 μm, 22 × 60 mm 2Corning Glass) were cleaned with KOH and ethanol. Low-binding glass surfaces were prepared by incubating silane-PEGSK-NHS (Nanocs) in ethanol at 65 degrees Celsius for 30 minutes. Oligonucleotides with 5'-modified NH2 were grafted onto these surfaces in a mixture of 1 micromolar (µM), 5.1 µM, and 46 µM oligonucleotides in methanol / phosphorus buffer for 20 minutes to form immobilized oligonucleotides coupled to the glass substrates.

[0219] Circularization of single-matrix oligonucleotide fragments into the directory. Single-matrix oligonucleotide fragments (approximately 100 base pairs in length) were circularized using a splint ligation protocol containing fragments complementary to surface-grafted primers.

[0220] Hybridization of the circularized directory to immobilized oligonucleotides. After circularization of the directory, circular directory fragments were added at a concentration of 100 picomolar (pM) in various test hybridization mixtures represented by columns BF. Individual buffer / directory hybridization mixtures were added to a 384-well plate with an attached functionalized surface at 50°C for 4 minutes.

[0221] Visualization of hybridization using assay buffer compositions. An intercalating DNA stain was added to the buffer / directory hybridization mixtures after the hybridization reaction to visualize hybridization of the circularized directories. An image of the 384-well plate was acquired using a fluorescence microscope and 488 nanometer (nm) excitation with a 60X water immersion objective (1.2 NA, Olympus) (see Fig. 3). A number of buffer compositions were tested for hybridization of the target nucleic acid (e.g., circularized directory) with surface-bound nucleic acid (e.g., immobilized oligonucleotides). Table 1 provides the buffer compositions and concentrations of immobilized oligonucleotides for each Fig. 3 apparent reaction, where columns 10-21 in Table 1 correspond to columns 10-21 of the Fig.3 and the lines BF correspond to the lines BF in Fig. 3. F10 and F11 are negative controls using standard hybridization conditions, with no background signal detected, demonstrating both the validity of the negative control and the property of low, nonspecific binding of the tested surfaces.

[0222] "Polymerization" concentration refers to the concentration of surface-bound oligos. Spot counts for each of the hybridization conditions were tabulated, with higher counts indicating more efficient hybridization buffer formulations, as in Fig. 4. Table 1 provides the buffer compositions and concentrations of immobilized oligonucleotides for each Fig. 4 apparent reaction, where columns 10-21 in Table 1 correspond to columns 10-21 of the Fig.4 and the lines BF correspond to the lines BF in Fig. 4 correspond.

[0223] Amplification of the hybridized target nucleic acid with surface-bound nucleic acid. After hybridization, the nucleic acids were amplified to quantify the hybridization efficiency. Rolling circle amplification (RCA) was performed using mixtures with Bst according to the manufacturer's instructions (New England Biolabs®). These amplified target nucleic acid colonies were further amplified using an RCA / PCR amplification strategy, with PCR cycles performed on the RCA multimer nanosphere to improve the detection sensitivity of the assay and more stringently quantify the hybridized directory.

[0224] The resulting surface-amplified products were again stained with intercalating DNA stains and imaged to verify hybridization specificity and efficiency (see Fig. 5). Table 1 provides the buffer compositions and concentrations of immobilized oligonucleotides for each Fig. 5 apparent reaction, where columns 10-21 in Table 1 correspond to columns 10-21 of the Fig. 5 and the lines BF correspond to the lines BF in Fig. 5 correspond.

[0225] Analysis of hybridization buffers and conditions. Hybridization conditions were determined based on the correlation of maximum spot counts from Fig. 3, Fig. 4 and Fig. 5. The hybridization buffers C10, D18 and E21 showed the highest spot count compared to the negative controls shown in F10 and F11, in which water was used instead of a hybridization buffer, in Fig. 4. This result was Fig. 5 validated after amplification. Example 3

[0226] In this example, the nonspecific binding of cyanine 3 (Cy3) dye-labeled molecules on the low nonspecific binding surfaces disclosed herein was measured. In independent, non-specific binding assays, 1 μM labeled Cy3 dCTP (GE Amersham), 1 μM Cy5 dGTP dye (Jena Biosciences), 10 μM aminoallyl-dUTP - ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP - ATTO-Rholl (Jena Biosciences), 10 μM aminoallyl-dUTP - ATTO-Rholl (Jena Biosciences), 10 μM cCTP - Cy3.5 (GE Amersham), and 10 μM 7-propargylamino-7-deaza-dGTP - Cy3 (Jena Biosciences) were individually applied to the low, non-specific binding surface described in Example 2 (glass substrates treated with silane-PEG5K, Nanocs) at 37°C for 15 minutes in a Incubated in a 384-well plate format. Each well was rinsed 2-3 times with 50 μl of deionized RNase / DNase-free water and 23 times with 25 mM ACES buffer, pH 7.4.An image of the 384-well plates was acquired at single-molecule resolution on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) equipped with a TIRF objective (100X, 1.4 NA, Olympus), an sCMOS camera (Zyla 4.2, Andor), and a light source with excitation wavelengths of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), e.g., 405, 488, 532, or 633 nm dichroic reflectors / beamsplitters, and bandpass filters were selected as 532 LP or 645 LP, matching the corresponding excitation wavelength. 5.

[0227] The imaging setup allowed the visualization of individual stained molecules bound to the substrates. Individual fluorescent spots were counted, and the total number of spots was divided by the respective area of ​​the ROI. For example, with a 100X objective and an Andor sCMOS camera with a pixel size of 6.5 micrometers, it is possible to calculate the area of ​​a region of interest (ROI).

[0228] Low, non-specific binding of the dye molecules above or below or equal to approximately 0.50 molecules per µm 2 was observed. In some cases, nonspecific binding of the dye molecules of less than or equal to 0.25 molecules per µm 2 observed. Example 4

[0229] A nucleic acid sequencing reaction is carried out under the Fig.2, using the disclosed hybridization compositions and methods of Example 1 and Example 2 on the surfaces used in Examples 1-3. In this non-limiting example, the processing times achieved are also in Fig. 2 shown.

[0230] While preferred embodiments of the compositions and methods disclosed herein have been shown and described, it will be apparent to those skilled in the art that such embodiments are presented only by way of example. Numerous variations, modifications, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It should be understood that in practicing the methods and compositions of the present disclosure, various alternatives to the methods and compositions described herein may be employed in any combination. This is followed by a sequence listing as an electronic document. This can be accessed both in DEPATISnet and in the DPMA register.

Claims

[1] A method for nucleic acid hybridization, the method comprising: (a) providing a surface comprising a hydrophilic polymer coating layer coupled thereto, wherein the hydrophilic polymer coating layer comprises a first nucleic acid molecule coupled thereto, and wherein the hydrophilic polymer coating layer has a water contact angle of less than 45 degrees; and (b) contacting the first nucleic acid molecule coupled to the hydrophilic polymer coating layer with a hybridization composition comprising a second nucleic acid molecule at a concentration of one nanomolar or less under conditions sufficient for hybridization of the second nucleic acid molecule with the first nucleic acid molecule bound to the hydrophilic polymer coating layer in 30 minutes or less. [2] The method of claim 1, wherein the conditions are maintained at a temperature between about 30 degrees Celsius and 70 degrees Celsius. [3] The method of claim 1 or claim 2, further comprising performing a nucleotide binding reaction on the surface between the first nucleic acid molecule and the second nucleic acid molecule. [4] The method of any one of claims 1-3, wherein the second nucleic acid molecule is present in the hybridization composition at a concentration of 0.50 nanomolar or less. [5] The method of any one of claims 1-3, wherein the second nucleic acid molecule is present in the hybridization composition at a concentration of 250 picomolar or less. [6] The method of any one of claims 1-3, wherein the second nucleic acid molecule is present in the hybridization composition at a concentration of 100 picomolar or less. [7] The method according to any one of claims 1-6, wherein contacting the first nucleic acid molecule coupled to the hydrophilic polymer coating layer with the hybridization composition is carried out in a period of less than 30 minutes. [8] The method of any one of claims 1-7, further comprising hybridizing the second nucleic acid molecule with the first nucleic acid molecule coupled to the hydrophilic polymer coating layer at a hybridization efficiency that is increased compared to a comparable hybridization reaction carried out for 120 minutes at 90 degrees Celsius for 5 minutes followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius in a buffer comprising saline sodium citrate. [9] The method of any one of claims 1-8, further comprising hybridizing the second nucleic acid molecule with the first nucleic acid molecule with a hybridization stringency of at least 80%. [10] The method of any one of claims 1-9, wherein the hydrophilic polymer coating layer has a level of non-specific cyanine-3 dye adsorption of less than about 0.25 molecules per square micrometer. [11] The method of any one of claims 1-10, wherein the hybridization composition further comprises: (a) at least one organic solvent which is polar and aprotic; and (b) a pH buffer. [12] The method of claim 11, wherein the at least one organic solvent has a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit. [13] A process according to any one of claims 11-12, wherein the at least one organic solvent comprises at least one functional group selected from nitrile, lactone, sulfone, sulfite and carbonate. [14] The method of any one of claims 11-13, wherein the hybridization composition further comprises formamide. [15] A process according to any one of claims 11-14, wherein the at least one organic solvent is miscible with water. [16] The method of any one of claims 11-15, wherein the at least one organic solvent is at least about 5% by volume based on the total volume of the hybridization composition. [17] The method of claim 16, wherein the at least one organic solvent is at most about 95% by volume based on the total volume of the hybridization composition. [18] A method according to any one of claims 11-17, wherein the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol or a combination thereof. [19] The method of any one of claims 1-18, wherein the first nucleic acid molecule coupled to the hydrophilic polymer coating layer is coupled to the hydrophilic polymer coating layer via a covalent bond. [20] The method of any one of claims 11-19, wherein the hybridization composition further comprises a molecular densification agent selected from a group consisting of polyethylene glycol, dextrans, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethylcellulose, hydroxypropylcellulose, methylcellulose, and hydroxymethylcellulose or a combination thereof.

Images