Nucleic acid hybridization method

Abstract

Nucleic acid hybridization buffer formulations and uses thereof that result in improvements in hybridization specificity, rate and efficiency are described. The buffer formulation composition comprises a target nucleic acid; at least one polar aprotic organic solvent and a pH buffer system wherein the target nucleic acid is attached to the surface by hybridization to a surface-binding nucleic acid bound to the surface, and wherein the hybridization of the target nucleic acid to the surface-binding nucleic acid has a higher severity and annealing rate.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of May 1, 2020, application number 202080043833.X, and invention name “Nucleic Acid Hybridization Method” (the corresponding PCT application with an application date of May 1, 2020 and application number PCT / US2020 / 031161).

[0002] Cross-references

[0003] This application is a continuation-in-part of U.S. Patent Application No. 16 / 543,351 filed on August 16, 2019, which claims the benefit of U.S. Provisional Application No. 62 / 841,541 filed on May 1, 2019, the entire contents of each of which are incorporated herein by reference. Background Art

[0004] The disclosure herein relates to the field of molecular biology, such as compositions, methods and systems for nucleic acid hybridization. In particular, it relates to hybridization compositions and methods for nucleic acids attached to surfaces.

[0005] Nucleic acid hybridization protocols constitute an important part of many different nucleic acid amplification and analysis techniques. The limited specificity and reaction rate achieved by using existing nucleic acid hybridization protocols may have an adverse effect on the throughput and accuracy of downstream nucleic acid analysis methods. Methods of stringency control generally involve conditions that cause a significant reduction in the number of hybridization complexes. Therefore, a method for improvement is needed to achieve high stringency hybridization during sequencing analysis. Summary of the invention

[0006] Provided herein is a method 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 the capture probe coupled thereto under conditions sufficient to allow the target nucleic acid molecule to be captured by the capture probe in a period of time less than 30 minutes.

[0007] 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 time period is less than or equal to 20 minutes. In some embodiments, the time period is less than or equal to 15 minutes. In some embodiments, the time period is less than or equal to 10 minutes. In some embodiments, the time period is less than or equal to 5 minutes.

[0008] In some embodiments, the hydrophilic surface is maintained at a temperature of about 30 degrees Celsius to about 70 degrees Celsius. In some embodiments, the hydrophilic surface is maintained at a substantially constant temperature. In some embodiments, the method further comprises hybridizing the target nucleic acid molecule to the capture probe with an increased hybridization efficiency compared to a comparable hybridization reaction, the comparable hybridization reaction being carried out in a buffer composition comprising saline-sodium citrate 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 some embodiments, the method further comprises hybridizing the target nucleic acid molecule to the capture probe with a hybridization stringency of at least 80%.

[0009] In some embodiments, the hydrophilic surface exhibits a non-specific cyanine 3 dye adsorption level of less than about 0.25 molecules / square micron. 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 crowding agent selected from polyethylene glycol, dextran, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethyl cellulose and any 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0010] Provided herein is a method for hybridizing a target nucleic acid molecule to 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 at least one nucleic acid molecule coupled to a polymer surface with a hybridization composition comprising a target nucleic acid molecule at a concentration of 1 nanomolar or less under conditions sufficient to allow the target nucleic acid molecule to hybridize to at least one nucleic acid molecule coupled to a polymer surface within 30 minutes or less. In some embodiments, the conditions are maintained at a substantially constant temperature.

[0011] 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. 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, at least one nucleic acid molecule coupled to the polymer surface is contacted with the hybridization composition for a period of less than 30 minutes. In some embodiments, the period of time is less than 20 minutes. In some embodiments, the period of time is less than 15 minutes. In some embodiments, the period of time is less than 10 minutes. In some embodiments, the period of time is less than 5 minutes.

[0012] In some embodiments, the method further comprises hybridizing the target nucleic acid molecule with at least one nucleic acid molecule coupled to the polymer surface with an increased hybridization efficiency compared to a comparable hybridization reaction, wherein the comparable hybridization reaction is carried out in a buffer comprising saline-sodium citrate for 120 minutes, at 90 degrees Celsius for 5 minutes, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the temperature is about 30 degrees Celsius to 70 degrees Celsius. In some embodiments, the temperature is about 50 degrees Celsius. In some embodiments, the method further comprises hybridizing the target nucleic acid molecule with at least one nucleic acid molecule with a hybridization stringency of at least 80%. In some embodiments, the hydrophilic polymer surface exhibits a non-specific cyanine 3 dye adsorption level of less than about 0.25 molecules / square micron.

[0013] In some embodiments, the hybridization composition further comprises: (a) at least one organic solvent having a dielectric constant of no greater than about 115 when 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 hydroxyl, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the at least one organic solvent comprises formamide. In some embodiments, the at least one organic 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 up to about 95% by volume based on the total volume of the hybridization composition.

[0014] In some embodiments, the pH buffer is at most about 90 volume percents of the cumulative 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 its combination. In some embodiments, the pH buffer further comprises the second organic solvent. In some embodiments, the pH buffer is present in the hybridization composition with an amount that effectively maintains the pH of the hybridization composition in the range of about 3 to about 10.

[0015] In some embodiments, the hybridization composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol, dextran, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol. In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 daltons. In some embodiments, based on the total volume of the hybridization composition, the amount of the molecular clustering agent is at least about 5 volume percents. In some embodiments, based on the total volume of the hybridization composition, the amount of the molecular clustering agent is at most about 50 volume percents. In some embodiments, at least one nucleic acid molecule coupled to the polymer surface is coupled to the polymer surface by covalent bonding.

[0016] In some embodiments, the hydrophilic polymer surface comprises one or more hydrophilic polymer layers, and wherein 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0017] 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 having 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 when measured at 68 degrees Fahrenheit; and (iii) a pH buffer.

[0018] 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 not more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount that effectively denatures double-stranded nucleic acids. In some embodiments, based on the total volume of the hybridization composition, the amount of the organic solvent is at least about 5 volume percents. In some embodiments, based on the total volume of the hybridization composition, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percents. In some embodiments, based on the total volume of the hybridization composition, the amount of the pH buffer is not more than 90 volume percents. In some embodiments, the hybridization composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the molecular clustering agent is less than 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the method further includes an additive for controlling the 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 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the additive for controlling the melting temperature of the nucleic acid is in the range of about 2 volume percent to 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (e.g., [tris(hydroxymethyl)methylamino]propanesulfonic acid), Tricine, Bicine, Bis-Tris, sodium hydroxide (NaOH), potassium hydroxide (KOH), TES (e.g., 2-[[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 the pH buffer is effective to maintain the pH of the hybridization composition in the range of about 3 to about 10.

[0019] 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 polymer surface-bound nucleic acid. In some embodiments, the polymer surface exhibits less than about 0.25 molecules / square micrometer (μm 2 ) of non-specific cyanine 3 (Cy3) dye adsorption levels. 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, polyglucosides, streptavidin, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer.

[0020] In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is carried out for a period of no more than 25 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is carried out for a period of no more than 15 minutes. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition is carried out for a period of 2-25 minutes. In some embodiments, the isothermal condition is at a temperature in the range of about 30 to 70 degrees Celsius. In some embodiments, the target nucleic acid is hybridized with the surface-bound nucleic acid with a hybridization stringency of at least 80%. In some embodiments, the target nucleic acid is hybridized with the surface-bound nucleic acid with an increased hybridization efficiency compared to a comparable hybridization reaction, in which the organic solvent is saline-sodium citrate and the hybridization is carried out for 120 minutes, 5 minutes at 90 degrees Celsius, and then cooled 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 method further comprises hybridizing at least a portion of the surface-bound nucleic acid to at least a portion of the target nucleic acid in the hybridization composition, the hybridization not comprising cooling.

[0021] Provided herein are hybridization methods comprising: (a) providing at least one surface-bound nucleic acid molecule coupled to a surface; and (b) contacting at least one surface-bound nucleic acid molecule with a 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 exhibits a relative humidity corresponding to less than about 0.25 molecules / μm when measured by a fluorescence imaging system under non-signal saturation conditions. 2 In some embodiments, no more than 5% of the total number of target nucleic acid molecules are associated with the surface without hybridizing to the surface-bound nucleic acid molecules.

[0022] In some embodiments, the surface-bound nucleic acid molecules are coupled to the surface by being bound to the surface. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the surface has a water contact angle of less than 45 degrees. In some embodiments, when measured at 68 degrees Fahrenheit, at least one organic solvent has a dielectric constant of not more than about 115. 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 not more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount that effectively denatures the double-stranded nucleic acid. In some embodiments, based on the total volume of the hybridization composition, the amount of the organic solvent is at least about 5 volume percent. In some embodiments, based on the total volume of the hybridization composition, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percent. In some embodiments, the amount of pH buffer is no more than 90 volume percent based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the molecular clustering agent is less than 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the method further comprises an additive for controlling the 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 volume percent based on the total volume of the hybridization composition. In some embodiments, based on the total volume of the hybridization composition, the amount of the additive for controlling the melting temperature of the nucleic acid is in the range of about 2 volume percent to 50 volume percent. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 of about 3 to 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 polymer surface-bound nucleic acid. 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, polyglucosides, streptavidin, and dextran. In some embodiments, one or more hydrophilic polymer layers comprise at least one dendritic polymer.

[0023] In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of no more than 25 minutes. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of no more than 15 minutes. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of 2-25 minutes. In some embodiments, isothermal conditions are at a temperature within the range of about 30 to 70 degrees Celsius. In some embodiments, the target nucleic acid molecules are hybridized with the surface-bound nucleic acid molecules with a hybridization stringency of at least 80%. In some embodiments, the target nucleic acid molecules are hybridized with the surface-bound nucleic acid molecules with an increased hybridization efficiency compared to comparable hybridization reactions, in which the organic solvent is saline-sodium citrate and the hybridization is carried out for 120 minutes, 5 minutes at 90 degrees Celsius, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the target nucleic acid molecules are 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 method further comprises 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, the hybridization not comprising cooling. In some embodiments, contacting the surface-bound nucleic acid with a hybridization composition comprising the target nucleic acid is performed under stringent 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 is a method 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 the target nucleic acid molecule with 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 surface exhibits less than about 0.25 molecules / μm 2 In some embodiments, no more than 5% of the total number of target nucleic acid molecules are associated with the surface without hybridizing with the surface-bound nucleic acid molecules. In some embodiments, contacting the hybridization composition with at least one surface-bound nucleic acid molecule is performed under isothermal conditions. In some embodiments, the surface-bound nucleic acid molecules are coupled to the surface by being bound to the surface. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the surface has a water contact angle of less than 45 degrees.

[0024] In some embodiments, at least one organic solvent has a dielectric constant of no more than about 115 when 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 more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature double-stranded nucleic acids. In some embodiments, the amount of the organic solvent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the pH buffer is no more than 90 volume percent based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the molecular clustering agent is less than 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the method further includes an additive for controlling the 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 about 2 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the additive for controlling the melting temperature of the nucleic acid molecule is in the range of about 2 volume percent to 50 volume percent based on the total volume of the hybridization composition. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 effectively maintained in the range of about 3 to about 10 with the pH of the hybridization composition.

[0025] In some embodiments, the surface-bound nucleic acid molecules are 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 total number of target nucleic acid molecules are associated with the surface without hybridizing with the polymer surface-bound nucleic acid molecules. 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, and dextran. In some embodiments, the one or more hydrophilic polymer layers comprise at least one dendrimer. In some embodiments, the surface-bound nucleic acid molecules are contacted with the hybridization composition for a period of no more than 25 minutes. In some embodiments, the surface-bound nucleic acid molecules are contacted with the hybridization composition for a period of no more than 15 minutes. In some embodiments, the surface-bound nucleic acid molecules are contacted with the hybridization composition for a period of 2-25 minutes. In some embodiments, the isothermal conditions are at a temperature within the range of about 30 to 70 degrees Celsius. In some embodiments, the target nucleic acid molecules are hybridized with the surface-bound nucleic acid molecules with a hybridization stringency of at least 80%. In some embodiments, the target nucleic acid molecules are hybridized with the surface-bound nucleic acid molecules with an increased hybridization efficiency compared to comparable hybridization reactions, in which the organic solvent is saline-sodium citrate and the hybridization is carried out for 120 minutes, 5 minutes at 90 degrees Celsius, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius. In some embodiments, the target nucleic acid molecules are present in the hybridization composition at a concentration of 1 nanomolar or less. In some embodiments, the target nucleic acid molecules are 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 method further comprises 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, and the hybridization does not include cooling.

[0026] Provided herein is a method for sequencing a target nucleic acid molecule, the method comprising: (a) contacting a surface-bound nucleic acid molecule coupled to a surface with a hybridization composition comprising a target nucleic acid molecule, wherein the hybridization composition comprises: (i) at least one organic solvent; and (ii) a pH buffer; (b) amplifying the target nucleic acid molecule to form a plurality of clonal amplification clusters of the target nucleic acid; and (c) determining the identity of the target nucleic acid molecule, wherein when a fluorescent image is captured using a fluorescent imaging system under non-signal saturation conditions, the fluorescent image of the surface comprising a plurality of clonal amplification clusters of the target nucleic acid molecule exhibits a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the method further comprises hybridizing the target nucleic acid molecule with at least one surface-bound nucleic acid coupled to the 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 more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite, and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount that effectively denatures the double-stranded nucleic acid. In some embodiments, the amount of the organic solvent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percent based on the total volume of the hybridization composition. In some embodiments, the amount of the pH buffer is not more than 90 volume percent based on the total volume of the hybridization composition. In some embodiments, the hybridization composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the hybridization composition. In some embodiments, based on the total volume of the hybridization composition, the amount of the molecule clustering agent is less than 50 volume percent. In some embodiments, the method further includes an additive for controlling the melting temperature of the target nucleic acid molecule. In some embodiments, based on the total volume of the hybridization composition, the amount of the additive for controlling the melting temperature of the target nucleic acid is at least about 2 volume percents. In some embodiments, based on the total volume of the hybridization composition, the amount of the additive for controlling the melting temperature of the nucleic acid molecule is in the range of about 2 volume percent to 50 volume percent.In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 effectively maintained in the range of about 3 to about 10 with the pH of the hybridization composition.

[0027] In some embodiments, the surface-bound nucleic acid molecules are 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 molecules are coupled to the one or more hydrophilic polymer layers. In some embodiments, the polymer surface exhibits less than about 0.25 molecules / square micrometer (μm 2). In some embodiments, no more than 5% of the total number of target nucleic acid molecules are associated with the surface without hybridizing with the surface-bound nucleic acid molecules. In some embodiments, no more than 10% of the total number of target nucleic acid molecules are associated with the surface without hybridizing with the surface-bound nucleic acid molecules. 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, polyglucosides, 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 molecules with the hybridization composition is performed under isothermal conditions. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out at a temperature in the range of about 30 to 70 degrees Celsius. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of no more than 25 minutes. In some embodiments, the method further includes 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 molecules with the hybridization composition is carried out for a period of 2-25 minutes. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of 2-4 minutes. In some embodiments, contacting the surface-bound nucleic acid molecules with the hybridization composition is carried out for a period of 2 minutes. In some embodiments, at least one surface-bound nucleic acid molecule is circular. In some embodiments, the method further includes hybridizing at least a portion of the surface-bound nucleic acid molecules with at least a portion of the target nucleic acid in the hybridization composition, and the hybridization does not include cooling. In some embodiments, contacting the surface-bound nucleic acid with the hybridization composition comprising the target nucleic acid is carried out under stringent conditions that prevent the target nucleic acid from hybridizing with non-complementary nucleic acids. In some embodiments, the stringency is at least or about 70%, 80% or 90%. In some embodiments, the stringency is at least 80%.

[0028] Provided herein are compositions for hybridizing a target nucleic acid molecule to 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 target nucleic acid molecules are associated with a surface without hybridizing with a surface-bound nucleic acid molecule. In some embodiments, no more than 5% of the total number of target nucleic acid molecules are associated with a surface without hybridizing with a surface-bound nucleic acid molecule.

[0029] 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 not more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount that effectively denatures double-stranded nucleic acids. In some embodiments, based on the total volume of the composition, the amount of the organic solvent is at least about 5 volume percents. In some embodiments, based on the total volume of the composition, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percents. In some embodiments, the pH buffer system comprises a pH buffer. In some embodiments, based on the total volume of the composition, the amount of the pH buffer is not more than 90 volume percents. In some embodiments, the composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the composition. In some embodiments, the amount of the molecular clustering agent is less than 50 volume percent based on the total volume of the composition. In some embodiments, the method further includes an additive for controlling the 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 volume percent based on the total volume of the composition. In some embodiments, the amount of the additive for controlling the melting temperature of the nucleic acid molecule is in the range of about 2 volume percent to 50 volume percent based on the total volume of the composition. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 of about 3 to about 10.

[0030] In some embodiments, the surface-bound nucleic acid molecules are coupled to the 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 wherein the surface-bound nucleic acid molecules are coupled to one or more hydrophilic polymer layers. In some embodiments, one or more hydrophilic polymer layers comprise molecules selected from the following: polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin and dextran. In some embodiments, one or more hydrophilic polymer layers comprise at least one dendritic polymer. 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.

[0031] In some embodiments, there is provided herein a microfluidic system, which includes a composition as described herein. In some embodiments, the microfluidic system includes 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 includes one or more hydrophilic polymer layers, and the hydrophilic polymer layer includes a molecule selected from: polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), poly (vinyl pyridine), poly (vinyl pyrrolidone) (PVP), poly (acrylic acid) (PAA), polyacrylamide, poly (N-isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2-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 includes a composition as described herein formulated as a fluid. In some embodiments, the flow cell device comprises one or more surface-bound nucleic acid molecules coupled to 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 operably coupled to an imaging system that is configured to capture an image of at least one surface of the flow cell comprising hybridized target nucleic acid molecules and one or more surface-bound nucleic acid molecules. The methods described herein include determining the identity of the target nucleic acid molecule using a microfluidic system as described herein.

[0032] Provided herein is a kit 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 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(vinyl pyridine), poly(vinyl pyrrolidone) (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, polyglucosides, streptavidin, and dextran. In some embodiments, the kit further comprises instructions for hybridizing one or more surface-bound nucleic acid molecules to 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.

[0033] 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 not more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount that effectively denatures double-stranded nucleic acids. In some embodiments, based on the total volume of the composition, the amount of the organic solvent is at least about 5 volume percents. In some embodiments, based on the total volume of the composition, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percents. In some embodiments, the pH buffer system comprises a pH buffer. In some embodiments, based on the total volume of the composition, the amount of the pH buffer is not more than 90 volume percents. In some embodiments, the composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the composition. In some embodiments, the amount of the molecular clustering agent is less than 50 volume percent based on the total volume of the composition. In some embodiments, the method further includes an additive for controlling the melting temperature of one or more target nucleic acid molecules. In some embodiments, the amount of the additive for controlling the melting temperature of one or more target nucleic acid molecules is at least about 2 volume percent based on the total volume of the composition. In some embodiments, the amount of the additive for controlling the melting temperature of nucleic acid is in the range of about 2 volume percent to 50 volume percent based on the total volume of the composition. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 of about 3 to about 10.

[0034] Provided herein are methods of using the kits described herein. In some embodiments, the surface-bound nucleic acid molecules are coupled to the surface by covalent or non-covalent bonds. In some embodiments, the method comprises: (a) combining one or more target nucleic acid molecules with a composition of the kit to form a master mixture; and (b) contacting the master mixture with one or more surface-bound nucleic acid molecules coupled to a surface provided in the kit. In some embodiments, the method further comprises (c) hybridizing the one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to the surface. In some embodiments, the surface exhibits less than about 0.25 molecules / μm 2 In some embodiments, no more than 10% of the total number of one or more target nucleic acid molecules are associated with the surface without hybridizing with the surface-bound nucleic acid molecules. In some embodiments, no more than 5% of the total number of 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, hybridization of one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to the surface is performed under isothermal conditions. In some embodiments, the isothermal conditions are performed at a temperature in the range of 30 to 70 degrees Celsius. In some embodiments, the method further includes (d) amplifying the target nucleic acid hybridized with the surface-bound nucleic acid to form a plurality of clonal amplification 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, when a fluorescent image is captured using a fluorescent imaging system under non-signal saturation conditions, a fluorescent image of a surface comprising a plurality of clonal amplification clusters of one or more target nucleic acid molecules exhibits a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the CNR is at least 50.

[0035] In some embodiments, the surface-bound nucleic acid is hybridized to the target nucleic acid for a period of no more than 25 minutes. In some embodiments, the method further includes removing the composition from the surface after a period of no more than 25 minutes. In some embodiments, the surface-bound nucleic acid is hybridized to the target nucleic acid for a period of 2-25 minutes. In some embodiments, the one or more surface-bound nucleic acid molecules are hybridized to one or more target nucleic acid molecules for a period of 2-4 minutes. In some embodiments, the one or more surface-bound nucleic acid molecules are hybridized to one or more target nucleic acid molecules for a period of 2 minutes. In some embodiments, at least one surface-bound nucleic acid is circular. In some embodiments, hybridization does not include cooling. In some embodiments, the master mixture is contacted with one or more surface-bound nucleic acid molecules under stringent conditions that prevent one or more target nucleic acid molecules from hybridizing with non-complementary nucleic acids. In some embodiments, stringency is at least or about 70%, 80% or 90%. In some embodiments, stringency is at least 80%.

[0036] Provided herein is a system comprising: (a) a surface comprising one or more surface-bound nucleic acid molecules, the one or more surface-bound nucleic acid molecules being coupled to a surface; (b) one or more target nucleic acid molecules; and (c) a composition comprising (i) at least one organic solvent; and (ii) a pH buffer. In some embodiments, the system further comprises a fluorescent imaging device. In some embodiments, the surface is a hydrophilic polymer surface. In some embodiments, the 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, one or more hydrophilic polymer layers comprise a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

[0037] In some embodiments, the organic solvent is an organic solvent having a dielectric constant of no more than 40 when 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 functional group selected from hydroxyl, nitrile, lactone, sulfone, sulfite and carbonate. In some embodiments, the organic solvent is miscible with water. In some embodiments, the organic solvent is present in an amount effective to denature double-stranded nucleic acids. In some embodiments, the amount of the organic solvent is at least about 5 volume percent based on the total volume of the composition. In some embodiments, the amount of the organic solvent is in the range of about 5 volume percent to 95 volume percent 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 the pH buffer is no more than 90 volume percent based on the total volume of the composition. In some embodiments, the composition further comprises a molecular clustering agent. In some embodiments, the molecular clustering agent is selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose and any combination thereof. In some embodiments, the molecular clustering agent is polyethylene glycol (PEG). In some embodiments, the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons. In some embodiments, based on the total volume of the composition, the amount of the molecular clustering agent is at least about 5 volume percents. In some embodiments, based on the total volume of the composition, the amount of the molecular clustering agent is less than 50 volume percents. In some embodiments, the method further includes an additive for controlling the melting temperature of the target nucleic acid. In some embodiments, based on the total volume of the composition, the amount of the additive for controlling the melting temperature of one or more target nucleic acid molecules is at least about 2 volume percents. In some embodiments, based on the total volume of the composition, the amount of the additive for controlling the melting temperature of one or more nucleic acid molecules is in the range of about 2 volume percent to 50 volume percents. In some embodiments, the pH buffer comprises at least one buffer selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, 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 of about 3 to about 10.

[0038] Provided herein are methods for using the systems described herein. In some embodiments, one or more surface-bound nucleic acid molecules are coupled to a surface via covalent or non-covalent bonds. In some embodiments, the method comprises: (a) combining one or more target nucleic acid molecules with a composition of the system to form a master mixture; (b) contacting the master mixture with one or more surface-bound nucleic acid molecules coupled to a surface provided in the system; (c) hybridizing one or more target nucleic acid molecules with one or more surface-bound nucleic acid molecules coupled to a surface; (d) amplifying one or more target nucleic acid molecules hybridized to one or more surface-bound nucleic acid molecules to form a plurality of clonal amplification clusters of one or more target nucleic acid molecules coupled to a surface; and (e) determining the identity of the one or more target nucleic acid molecules by capturing an image of the surface with a fluorescent imaging device. In some embodiments, the surface exhibits 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 the surface is carried out under isothermal conditions. In some embodiments, the isothermal conditions are carried out at a temperature in the range of 30 to 70 degrees Celsius. In some embodiments, no more than 10% of the total number of one or more target nucleic acid molecules are associated with the surface without hybridizing with one or more surface-bound nucleic acid molecules. In some embodiments, no more than 5% of the total number of one or more target nucleic acid molecules are associated with the surface without hybridizing with one or more surface-bound nucleic acid molecules. In some embodiments, when a fluorescent image is captured using a fluorescent imaging device under non-signal saturation conditions, the fluorescent image of the surface containing the amplified one or more target nucleic acid molecules exhibits a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the CNR is at least 50.

[0039] In some embodiments, one or more surface-bound nucleic acid molecules are hybridized with one or more target nucleic acid molecules for a period of no more than 25 minutes. In some embodiments, the method further includes removing the composition from the surface after a period of no more than 25 minutes. In some embodiments, one or more surface-bound nucleic acid molecules are hybridized with one or more target nucleic acid molecules for a period of 2-25 minutes. In some embodiments, one or more surface-bound nucleic acid molecules are hybridized with one or more target nucleic acid molecules for a period of 2-4 minutes. In some embodiments, one or more surface-bound nucleic acid molecules are hybridized with one or more target nucleic acid molecules for a period of 2 minutes. In some embodiments, at least one surface-bound nucleic acid is circular. In some embodiments, hybridization does not include cooling. In some embodiments, one or more surface-bound nucleic acid molecules are contacted with a hybridization composition comprising one or more target nucleic acid molecules to prevent one or more target nucleic acid molecules from hybridizing with non-complementary nucleic acid molecules under stringent conditions. In some embodiments, stringency is at least or about 70%, 80% or 90%. In some embodiments, stringency is at least 80%.

[0040] Incorporation by reference

[0041] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term in this document and a term in an incorporated reference, the term in this document controls. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0043] 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 obtained by reference to the following detailed description and its accompanying drawings, which set forth illustrative embodiments in which the principles of the disclosed compositions and methods are utilized, wherein:

[0044] Figure 1A-1B Non-limiting examples of image data are provided that demonstrate the improvements in hybridization stringency, speed, and efficiency that can be achieved by reformulating hybridization buffers for solid phase nucleic acid amplification as described herein. Figure 1A Examples of image data for two different hybridization buffer preparations and protocols are provided. Figure 1BExamples of corresponding image data obtained using standard hybridization buffers and protocols are provided.

[0045] Figure 2 A workflow for nucleic acid sequencing using the disclosed hybridization method on a low binding surface is shown, along with non-limiting examples of achievable processing times.

[0046] Figure 3 Shown are surface template hybridization images of samples corresponding to the compositions used for hybridization (NASA results at 100 pM).

[0047] Figure 4 Table showing the hybridization design with experimental spot counts.

[0048] Figure 5 Shown is a PCR image of the sample after nucleic acid surface amplification.

[0049] Figure 6 A workflow according to various embodiments disclosed herein is shown.

[0050] Figure 7 A workflow for sequential reactions according to various embodiments described herein is shown.

[0051] Figure 8 A sample nucleic acid hybridization workflow according to various embodiments described herein is shown.

[0052] Figure 9A-9B 1 shows how sample nucleic acids hybridized to nucleic acid molecules coupled to a low non-specific binding surface can be visualized according to various embodiments described herein. Fig.9A ) or amplified ( Fig. 9B ).

[0053] Fig.10 An example computer controlled system is schematically depicted.

[0054] Fig.11 Shown are workflows for purification and isolation of sample nucleic acid from a biological sample, library preparation, and hybridization according to various embodiments described herein. DETAILED DESCRIPTION

[0055] Disclosed herein are methods, compositions, systems and kits for carrying out nucleic acid hybridization with nucleic acid molecules coupled to surfaces. The methods, compositions, systems and kits described herein are particularly suitable for nucleic acid amplification, nucleic acid sequencing or a combination thereof. Compared with existing standard nucleic acid hybridization methods, the methods, compositions, systems and kits described herein can achieve excellent nucleic acid hybridization performance and can be performed at a fraction of the cost and time. This is achieved by utilizing optimized hybridization compositions (e.g., buffer, organic solvents) combined with hydrophilic low non-specific binding surfaces.

[0056] Existing standard nucleic acid hybridization methods are complicated, time-consuming, and lack the required specificity and efficiency for cost-effective high-throughput applications. In many cases, existing hybridization methods require high temperature (e.g., 90 degrees Celsius) incubation, long incubation time (e.g., 1-2 hours) and a large amount of input nucleic acid (e.g., 10 nanomoles). At least one reason why standard nucleic acid hybridization methods lack specificity and efficiency is that the surface used is prone to non-specific binding with proteins or nucleic acids, thereby causing an increase in background signal.

[0057] Compared to standard nucleic acid hybridization methods, the methods, compositions, systems and kits described herein provide excellent hybridization specificity and efficiency of target nucleic acid molecules and surface-bound nucleic acid molecules. This paper describes methods and systems that utilize low non-specific binding surfaces to reduce background signals. The low non-specific binding surfaces described herein are engineered so that proteins, nucleic acids and other biomolecules do not "stick" to the substrate on the surface. The low non-specific binding surfaces described herein are hydrophilic. In some cases, the low non-specific binding surfaces have a water contact angle of less than or equal to about 50 degrees.

[0058] In some embodiments, the method comprises hybridizing a target nucleic acid to a nucleic acid molecule coupled to a hydrophilic surface (e.g., a low non-specific binding surface) using a hybridization composition described herein. The methods described herein can be used for nucleic acid hybridization, amplification, sequencing, or a combination thereof. The methods described herein achieve excellent hybridization performance on a low non-specific binding surface. In addition, the methods described herein achieve less than about 0.25 molecules / μm 2 Nonspecific cyanine dye-3 (Cy3) dye adsorption.

[0059] The optimized hybridization compositions described herein, particularly when used with low non-specific binding surfaces, enable isothermal hybridization reactions to be performed in as little as 2 minutes at 60 degrees Celsius using input nucleic acids at concentrations as low as 50 picomolar. The methods described herein provide (i) excellent hybridization rates, (ii) excellent hybridization specificity, (iii) excellent hybridization stringency, (iv) excellent hybridization efficiency (or yield), (v) reduced demand for the amount of necessary starting material, (vi) reduced temperature requirements for isothermal or thermal gradient amplification protocols, (vii) increased annealing rates, and (viii) a lower percentage of the total number of target nucleic acid molecules (or amplified clusters of target nucleic acid molecules) that are associated with a surface and not hybridized to surface-bound nucleic acids than comparable hybridization reactions using standard hybridization protocols and reagents. The improved performance and reduced cost and time required to perform hybridization reactions make these methods, compositions, systems and kits ideally suited for high-throughput nucleic acid hybridization, amplification and sequencing applications.

[0060] When used with a standard hybridization protocol using a non-specific binding surface as described herein, standard hybridization preparations (e.g., saline sodium citrate buffer) achieve poor hybridization specificity or efficiency. The hybridization reaction or annealing interaction between the target nucleic acid molecule in the solution and the nucleic acid molecule coupled to the low non-specific binding surface may be affected by a variety of factors, including the availability of hydrogen bonding partners in the solution and the polarity of the solution. Typically, nucleic acids are preferentially present in bulk solution where possible, in order to utilize the additional entropy stabilization brought about by the ability to obtain dynamic states in three dimensions, rather than in two dimensions (e.g., available on a solid surface). Under equilibrium conditions, in a system comprising nucleic acids, solutions, and hydrophilic surfaces (e.g., low non-specific binding surfaces), when the solvent is aqueous, the nucleic acid molecules will be preferentially stabilized in solution, rather than in a surface-bound state.

[0061] Existing hybridizations utilize protic solvents (e.g., saline sodium citrate buffer), which is disadvantageous for nucleic acid hybridization reactions with low non-specific binding surfaces as described herein, because aprotic solvents provide a favorable environment for target nucleic acid molecules to remain in solution rather than bind to the low non-specific binding surface. This is because protic solvents can provide sufficient hydrogen bonding partners of sufficient size and distribution to allow hydrogen bonding interactions to occur between exposed hydrogen bond donors and acceptors along the nucleic acid backbone or any exposed side chain moieties.

[0062] In contrast, the hybridization compositions described herein drive target nucleic acid molecules to low nonspecific binding surfaces when in solution by utilizing aprotic organic solvents, such as formamide. The aprotic solvents described herein reduce the proportion of solvent molecules that can meet the hydrogen bonding requirements of nucleic acid chains, and make it possible to generate entropy penalties in the bulk solution, which will drive the system to stabilize by depositing nucleic acids on the surface (for example, the entropy penalty caused by adapting the bulk solution to the unbonded hydrogen bonding elements in the nucleic acids becomes greater than the entropy penalty caused by the loss of three-dimensional dynamic freedom when the polymer is adsorbed to the surface). In addition, introducing aprotic organic solvents into the solution can help reduce entropy, thereby providing a more favorable environment for nucleic acids to bind to hydrophilic surfaces. For example, adding the aprotic solvent acetonitrile helps drive nucleic acids in solution to a surface-bound state.

[0063] The hybridization compositions described herein further comprise a certain concentration of proton and aprotic organic solvents to prevent the target nucleic acid from precipitating from the solution, which may be caused by a high concentration of aprotic solvents in the solution. In this way, the hybridization compositions described herein selectively associate nucleic acids with hydrophilic surfaces (e.g., low nonspecific binding surfaces) while remaining substantially solvated.

[0064] Hybridization compositions as described herein optionally include a clustering agent that can regulate the interaction of nucleic acid with a bulk solution. In some cases, hybridization compositions include a relaxant, a divalent cation, or an intercalator that can regulate the dynamics of the polymer itself, and can also regulate the interaction of nucleic acid with a surface in the presence of a partially non-protonated bulk solvent. In some cases, such agents are provided in combination with a buffer containing a certain proportion of non-protonated or non-hydrogen-bonded components to better control the interaction of nucleic acid molecules with hydrophilic surfaces.

[0065] Various aspects of the disclosed nucleic acid hybridization methods can be applied to solution phase or solid phase nucleic acid hybridization, and can also be applied to any other type of nucleic acid amplification, or analytical applications (e.g., nucleic acid sequencing), or any combination thereof. It should be understood that different aspects of the disclosed methods, devices, and systems can be interpreted individually, collectively, or in combination with each other.

[0066] The methods, compositions, systems and kits described herein can be used in a wide range of applications in addition to those involving nucleic acid-surface interactions, because the same thermodynamic parameters optimized by the methods and compositions described herein can control many interactions between polymers and biomolecules, as well as polymer and surface interactions and biomolecule and surface interactions. Therefore, the methods, compositions, systems and kits described herein can be used to adjust the polarity of the solvent, or the hydrogen bonding potential, or a combination thereof, in other systems involving these interactions.

[0067] Solution-based hybridization is the basis of many solution-based molecular biology and solution-phase DNA manipulation applications, most notably the 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 functional duplex forms, which can be kinetically analyzed as second-order kinetic reactions, whereby the forward reaction of duplex formation is a second-order reaction and the reverse reaction involving the dissociation of the duplex structure to form two single-stranded complementary chains (chains A and B) is a first-order reaction (Han, C., Improvement of the Speed ​​and Sensitivity of DNA Hybridization Using Isotachophoresis, Stanford Thesis. 2015). These reactions can be written as:

[0068]

[0069] There are a number of methods that have been used to not only increase the speed of hybridization reactions, but also to improve the specificity of reactions in the presence of promiscuous non-complementary DNA fragments. Such methods include, but are not limited to, adding MgCl 2and higher salt concentrations, and lowering the temperature to accelerate the reaction (H. Kuhn, VV Demidov, JM Coull, MJ Fiandaca, BD Gildea and MD 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 trade-off for accelerating the reaction rate is usually reaction specificity (J. M. S. Bartlett and D. Stirling, PCR protocols, Humana Press, 2003; W. Rychlik, W. J. Spencer and R. E. Rhoads, Nucleic Acids Res., 1990, 18). Sometimes another approach is adopted, namely by using size exclusion or molecular clustering techniques or a combination thereof (which use inert polymers as hybridization buffer additives) to produce potential improvements in reaction specificity (R. Wieder and JG Wetmur, Biopolymers, 1981, 20, 1537-1547; JG Wetmur, Biopolymers, 1975, 14, 2517-2524). In addition, organic solvents have been used as additives to accelerate hybridization kinetics and maintain reaction specificity (N. Dave and J. Liu, J. Phys. Chem. B, 2010, 114, 15694-15699).

[0070] Although improvements in hybridization in solution can be translated to surface-based hybridization techniques, the requirement for surface-based hybridization has profound implications for many key biological assays, such as gene expression analysis (DT Ross, U. Scherf, M. Beisen, C. M. Perou, C. Rees, P. Spellman, V. Iyer, S. S. Jeffrey, M. Van de Rijn, M. Waltham, A. Pergamenschikov, J. C. Lee, D. Lashkari, D. Shalon, T. G. Myers, J. N. Weinstein, D. Botstein, and P. O. Brown, Nat. Genet., 2000, 24, 227). –235; A. Adomas, G. Heller, A. Olson, J. Osborne, M. Karlsson, J. Nahalkova, L. Van Zyl, R. Sederoff, J. Stenlid, R. Finlay and FOAsiegbu, Tree Physiol., 2008, 28, 885 – 897; M.Schena, D.Shalon, RWDavis and POBrown, 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, R Mayer, B. Sun, L. Hsie, CM Robbins, LC Brody, D. Wang, ES Lander, R. Lipshutz, SPFodor and FS Collins, Nat. Genet., 1999, 22, 164 – 167), rapid pathogen screening based on pathogen nucleic acid, next generation sequencing (NGS) and many other genomics-based applications (MJ Heller, Annu. Rev. Biomed. Eng., 2002, 4, 129 – 53). The common necessity of all these reactions is high reaction specificity in a highly multiplexed solution of target sequences (which may range from thousands to billions of different sequences) in order to rapidly tether the targets to a solid surface for subsequent detection or amplification or a combination thereof to enable DNA (or other nucleic acid) interrogation for applications such as sequencing or array-based analysis. The efficiency of surface-based hybridization reactions has been found to be much lower than that of solution reactions, for example, by about an order of magnitude. A great deal of work has been done in the past to attempt to create a hybridization method for solid surfaces that provides high specificity and accelerated hybridization reaction rates (D. Y. Zhang, S. X. Chen and P. Yin, Nat. Chem., 2012, 4, 208 – 14).

[0071] Disclosed herein is an innovative combination of methods gleaned from the surface-based and solution-based hybridization studies outlined above and from other research areas including DNA hydration and quadruplex studies, which results in substantial improvements in hybridization kinetics and specificity. When used with low nonspecific binding surfaces for applications such as next-generation sequencing (NGS) and other bioassays requiring highly specific nucleic acid hybridization in multiplexed pools consisting of a large number of target sequences, the disclosed hybridization compositions provide highly specific (e.g., >2 orders of magnitude improvement over traditional methods) and accelerated hybridization (e.g., >1-2 orders of magnitude improvement over traditional methods).

[0072] Hybridization method

[0073] Provided herein are methods for nucleic acid hybridization between sample nucleic acid molecules and capture nucleic acid molecules. Fig.11 , sample nucleic acid molecules are isolated and purified from a biological sample obtained from a subject 1110. A library of isolated and purified sample nucleic acid molecules is prepared 1111. In the presence of a hybridization composition, the library of sample nucleic acid molecules is hybridized 1112 with nucleic acid molecules coupled to a low non-specific binding surface as described herein.

[0074] Biological sample.Biological samples disclosed herein include nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, fats or viruses.In one example, a biological sample is a nucleic acid sample including one or more nucleic acid molecules.Exemplary biological samples may include polynucleotides, nucleic acids, oligonucleotides, cell-free nucleic acids (e.g., cell-free DNA (cfDNA)), circulating cell-free nucleic acids, circulating tumor nucleic acids (e.g., circulating tumor DNA (ctDNA)), circulating tumor cells (CTC) nucleic acids, nucleic acid fragments, nucleotides, DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosome 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.

[0075] Any material comprising nucleic acid can be the source of biological sample.Material can be fluid, for example biological fluid.Fluid material can include but is not limited to, blood, cord blood, saliva, urine, sweat, serum, semen, vaginal fluid, gastric juice and digestive fluid, cerebrospinal fluid, placental fluid, cavity fluid, eye fluid, serum, breast milk, lymph or its combination.The material can be solid, for example biological tissue.The material can include normal healthy tissue, diseased tissue or the mixture of healthy tissue and diseased tissue.

[0076] Biological samples as described herein are obtained from various subjects. The subject may be a living subject or a dead subject. Examples of subjects may include, but are not limited to, humans, mammals, non-human mammals, rodents, amphibians, reptiles, canines, felines, bovines, equines, goats, sheep, hens, birds, mice, rabbits, insects, slugs, microorganisms, bacteria, parasites, or fish. In some cases, the subject is a patient suffering from, suspected of suffering from, or at risk of developing a disease or condition. 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 who is at risk of having a baby with a certain congenital defect.

[0077] Samples can be obtained from subjects by various methods. For example, samples can be obtained from subjects by accessing the circulatory system (e.g., intravenously or intra-arterially via a syringe or other device), collecting secreted biological samples (e.g., saliva, sputum, urine, feces), surgery (e.g., biopsy), obtaining biological samples (e.g., intraoperative samples, postoperative samples), swabs (e.g., cheek swabs, oropharyngeal swabs), or pipetting.

[0078] Biological sample processing. In some cases, biological samples described herein are processed. Processing includes filtering the sample, combining sample components containing the analyte, combining the analyte, stabilizing the analyte, purifying the analyte, or a combination thereof. Non-limiting examples of sample components are cells, viral particles, bacterial particles, exosomes, and nucleosomes. In some cases, plasma or serum is separated from a whole blood sample. In some cases, whole blood is obtained from venous blood or capillary blood of a subject described herein.

[0079] Library preparation of sample nucleic acid. In some cases, the sample nucleic acid described herein is converted into a library by labeling the sample nucleic acid with a marker, a barcode or a tag. In some embodiments, the sample nucleic acid library is amplified, 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), nickase amplification reaction (NEAR), recombinase polymerase amplification (RPA) and branched amplification method (RAM).

[0080] In some cases, isothermal amplification is used. In some cases, except the initial heating step before the isothermal amplification begins, amplification is isothermal. Many isothermal amplification methods with different considerations and different advantages are known in the art and have been discussed in the literature, for example, 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, the entire contents of each are incorporated herein by reference.

[0081] In some cases, the amplification method is rolling circle amplification (RCA). RCA is an isothermal nucleic acid amplification method that allows the probe DNA sequence to be amplified by more than 10 at a single temperature (usually about 30°C). 9 times. Multiple rounds of isothermal enzymatic synthesis are performed by a DNA polymerase which extends the circular hybridization primer by successively advancing around the circular DNA probe. In some cases, the amplification reaction is performed using RCA at about 28° C. to about 32° C. A suitable method for RCA is described in US 6,558,928.

[0082] In some cases, amplification includes targeted amplification. In some cases, amplifying nucleic acid includes contacting nucleic acid with at least one primer having a sequence corresponding to a target chromosome sequence. Amplification can be multiple, including contacting nucleic acid with multiple sets of primers, wherein each of the first pair in the first group and each pair in the second group are different.

[0083] Hybridization. The methods described herein include contacting a sample nucleic acid molecule with a capture nucleic acid molecule optionally coupled to a low non-specific binding surface in the presence of a hybridization composition as described herein. In some cases, the capture nucleic acid molecule is coupled to a low non-specific binding surface and hybridization occurs on the surface. In some cases, the capture nucleic acid molecule is not coupled to a low non-specific binding surface, and hybridization occurs in solution. The methods provided herein further include hybridizing the sample nucleic acid molecule with the capture nucleic acid molecule.

[0084] The method comprises hybridizing at least a portion of a sample nucleic acid molecule comprising a nucleic acid sequence that is substantially complementary to a portion of a capture nucleic acid molecule. The capture nucleic acid molecule and the portion of the sample nucleic acid molecule can be at least or equal to about 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 capture nucleic acid molecule and the portion of the sample nucleic acid molecule may be 4 to 50, 5 to 49, 6 to 48, 7 to 47, 8 to 46, 9 to 45, 10 to 44, 11 to 43, 12 to 42, 13 to 41, 14 to 40, 15 to 39, 16 to 38, 17 to 37, 18 to 36, 19 to 35, 20 to 34, 21 to 33, 22 to 32, 23 to 31, 24 to 30, 25 to 29, 26 to 28 nucleotides. The capture nucleic acid molecule and the portion of the sample nucleic acid molecule may be 8 to 20 nucleotides. In some cases, at least 90% of the nucleic acids in the portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule are completely hybridized. In some cases, at least 95% of the nucleic acids in the portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule are completely hybridized. In some cases, 95-100% of the nucleic acids in the portion of the sample nucleic acid molecule and the portion of the capture nucleic acid molecule are completely hybridized.

[0085] Figure 8 The non-limiting example provided in shows one or more sample nucleic acid molecules 801, which are circularized 802 using a connection (e.g., a splint connection) 802, and introduced to one or more nucleic acid molecules 808 coupled to a hydrophilic substrate 807 of a low non-specific binding surface 806 in the presence of a hybridization composition 805. In this example, the low non-specific binding surface is immersed in the hybridization composition. In an alternative embodiment, one or more sample nucleic acid molecules are introduced to the hybridization composition prior to introduction to the one or more nucleic acid molecules 808 coupled to a hydrophilic substrate 807 of a low non-specific binding surface 806. Hybridization occurs between the sample nucleic acid molecules and the surface-coupled nucleic acid molecules 809.

[0086] Sample nucleic acid.One or more sample nucleic acid molecules described herein are derived from biological samples described herein.Sample nucleic acid molecules are deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules.In some cases, 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 (CTC) nucleic acid, nucleic acid fragments, nucleotides, DNA, complementary DNA (cDNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA (gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial DNA).In some cases, RNA is selected from ribosomal RNA, cell-free DNA, cell-free embryonic DNA (cffDNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, viral RNA, etc.

[0087] Capture nucleic acid is coupled to the surface. Nucleic acid molecules (e.g., capture molecules) coupled to the surface can be coupled to the surface by a variety of suitable options. In some cases, the nucleic acid molecules are coupled to the surface by covalent bonds. In some cases, the nucleic acid molecules are coupled to the surface by non-covalent bonds. In some cases, the nucleic acid molecules are attached to the surface by biological interactions. Non-limiting examples of biological interaction surface chemistry include biotin-streptavidin interactions (or variants thereof), polyhistidine (his) tags. – Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and silanes.

[0088] Composition

[0089] Hybridization compositions are provided herein. The hybridization compositions of the present disclosure include at least one organic solvent, which is polar and aprotic in some cases (e.g., having a dielectric constant of less than or equal to about 115 measured at 68 degrees Fahrenheit). The hybridization compositions include a pH buffer. Optionally, the hybridization compositions include one or more molecular clustering agents / volume exclusion agents, one or more additives affecting the DNA melting temperature, one or more additives affecting DNA hydration, or any combination thereof. Hybridization compositions described herein for sequencing, genotyping, or sequencing-related techniques with low nonspecific binding surfaces (e.g., silica coated with low binding polymers such as polyethylene glycol (PEG)) can be obtained using any one or combination of the following hybridization composition components.

[0090] Organic solvent: An organic solvent is a solvent or solvent system that contains carbon-based or carbon-containing substances capable of dissolving or dispersing other substances. Organic solvents may be miscible or immiscible with water.

[0091] Polar solvent: The polar solvents included in the hybridization compositions described herein are solvents or solvent systems that contain one or more molecules characterized by the presence of a permanent dipole moment (e.g., molecules with unevenly distributed charge density in space). Polar solvents are characterized by a dielectric constant of 20, 25, 30, 35, 40, 45, 50, 55, 60 or higher, or are characterized by containing a value or range of values including any of the above values. For example, a polar solvent may have a dielectric constant 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 about 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 about -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 °F. In some cases, the dielectric constant is measured at a temperature of 68 °F. In some cases, the dielectric constant is measured at a temperature of 20 °F.

[0092] The polar solvents as described herein may include polar aprotic solvents. The polar aprotic solvents as described herein may also not contain ionizable hydrogen in the molecule. Further, in the context of the compositions of the present disclosure, the polar solvents or polar aprotic solvents may preferably be substituted with strongly polar functional groups such as nitrile, carbonyl, thiol, lactone, sulfone, sulfite and carbonate groups such that the following solvent molecules have a dipole moment. The polar solvents and polar aprotic solvents may exist in aliphatic and aromatic or cyclic forms. In some embodiments, the polar solvent is acetonitrile.

[0093] The organic solvents described herein may have a dielectric constant the same as or close to that of acetonitrile. The dielectric constant of the organic solvent may be in the range of 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 below 30, 40, 45, 50, 55 or 60. The dielectric constant of the organic solvent may be about 35, 36, 37, 38 or 39.

[0094] 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 can specifically include, but are not limited to, acetonitrile, diethylene glycol, N,N-dimethylacetamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, formamide, hexamethylphosphoramide, glycerol, methanol, N-methyl-2-pyrrolidone, nitrobenzene, or nitromethane.

[0095] The organic solvents 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 be in the range of about 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 about 5.5, 5.6, 5.7, or 5.8.

[0096] The Snyder polarity index can be calculated according to the method disclosed in Snyder, LR, Journal of Chromatography A, 92 (2): 223-30 (1974), the entire contents of which are incorporated herein by reference. Representative polar aprotic solvents having a Snyder polarity index between 6.2 and 7.3 can be used. Such solvents may specifically include, but are not limited to, acetonitrile, dimethylacetamide, dimethylformamide, N-methylpyrrolidone, N,N-dimethylsulfoxide, methanol or formamide.

[0097] Relative polarity can be determined according to Reichardt, C., Solvents and Solvent Effects in Organic Chemistry, the 3rd edition, the method given in 2003, the full content of the document is incorporated herein by reference, especially about its disclosure of the method for polarity to polarity and determining or assessing the polarity of solvent and solvent molecules. Polar aprotic solvents with relative polarity between 0.44 and 0.82 can be used. Such solvents can especially include but are not limited to dimethyl sulfoxide, acetonitrile, 3-pentanol, 2-pentanol, 2-butanol, cyclohexanol, 1-octanol, 2-propyl alcohol, 1-heptanol, isobutyl alcohol, 1-hexanol, 1-pentanol, acetylacetone, ethyl acetoacetate, 1-butanol, benzyl alcohol, 1-propyl alcohol, 2-aminoethanol, ethanol, diethylene glycol, methanol, ethylene glycol, glycerine or formamide.

[0098] Solvent polarity (E T(30)) can be calculated according to the method disclosed in Reichardt, C., Molecular Interactions, Volume 3, Ratajczak, H. and Orville, WJ, Eds (1982), the entire contents of which are incorporated herein by reference.

[0099] 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 (ε), chloromaleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonyl lactone, 5-cyano-2-thiouracil, cyclopropanenitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyltetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone (dipheynyl sulfone), 1,2-dinitrobenzene, 2,4-dinitrotoluene, diphenyl alkynyl sulfone, ε-caprolactam, ethanesulfonyl chloride, ethyl ethyl phosphinate, N-ethyltetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, ethylene glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxybenzonitrile, l-methoxy-2-nitrobenzene, methyl alpha-bromoacetoacetic acid lactone, 1-methylimidazole, N-methylimidazole, 3-methylisoxazole, N-methylmorpholine-N-oxide, methylphenyl sulfone, N-methylpyrrolidone, methyl cyclopentane, methyl 4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, l-nitroso-2-pyrrolidone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenylsydney ketone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfonamide, cyclopentane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiopyran oxide, tetramethylene sulfone (cyclopentane), thiazole, 2-thiouracil, 3,3,3-trichloropropene, 1,1,2-trichloropropene, 1,2,3-trichloropropene, sulfided cyclopropane dioxide, and trimethylene sulfite.

[0100] Polar aprotic solvents having a solvent polarity between 44 and 60 can be used. Such solvents may specifically include, but are not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol, triethyl phosphite, 3-pentanol, acetonitrile, nitromethane, cyclohexanol, 2-pentanol, 4-methyl-1,3, dioxolane-2-one, propylene carbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol, 2,6, dimethylphenol, 2,6-dimethylphenol, 1-decanol, cyclopentanol, dimethyl sulfone, 1-octanol, diethylene glycol mono-n-butyl ether, butyl diglycol. , 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, isopentanol, 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, ethylenediaminetetraacetic acid, 1-hexanol, 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, isopentanol, 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, alcohol monobutyl ether, 1-butanol, 2-hydroxymethyl-tetrahydrofuran, tetrahydrofurfuryl alcohol, 2-hydroxymethylfuran, furfuryl alcohol, 1-propanol, 2,4-dimethylphenol, 2,4-dimethylphenol, benzyl alcohol, 2-ethoxyphenol, 2-ethoxyethanol, 1,5-pentanediol, 1-bromo-2-propanol, 2-methyl-5-isopropylphenol, carvacrol, 2-aminoethanol, ethanol, n-methylacetamide, 3-chloropropionitrile, 2-propene-1-ol, allyl alcohol, 2-methoxyethanol, 2-methylphenol , o-cresol, 1,3-butanediol, 2-propyn-1-ol, propargyl alcohol, 3-methylphenol, m-cresol, triethylene glycol, diethylene glycol, n-methylformamide, 1,2-propylene glycol, 1,3-propylene glycol, 2-chlorophenol, methanol, 1,2-ethylene glycol, ethylene glycol, formamide, 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, or p-cresol.

[0101] Polar aprotic solvents having a dielectric constant in the range of about 30-115 can be used. Such solvents can specifically include, but are not limited to, dimethyl sulfoxide, 2-methoxycarbonylphenol, triethyl phosphite, 3-pentanol, acetonitrile, nitromethane, cyclohexanol, 2-pentanol, 4-methyl-1,3, dioxolane-2-one, propylene carbonate, acrylonitrile, 1-phenylethanol, 1-dodecanol, 2-butanol, 2-methylcyclohexanol, 2,6, dimethylphenol, 2,6-dimethylphenol, 1-decanol, cyclopentanol, dimethyl sulfone, 1-octanol, diethylene glycol mono-n-butyl ether, butyl diglycol. , 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, isopentanol, 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, ethylenediaminetetraacetic acid, 1-hexanol, 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, isopentanol, 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, alcohol monobutyl ether, 1-butanol, 2-hydroxymethyl-tetrahydrofuran, tetrahydrofurfuryl alcohol, 2-hydroxymethylfuran, furfuryl alcohol, 1-propanol, 2,4-dimethylphenol, 2,4-dimethylphenol, benzyl alcohol, 2-ethoxyphenol, 2-ethoxyethanol, 1,5-pentanediol, 1-bromo-2-propanol, 2-methyl-5-isopropylphenol, carvacrol, 2-aminoethanol, ethanol, n-methylacetamide, 3-chloropropionitrile, 2-propene-1-ol, allyl alcohol, 2-methoxyethanol, 2-methylphenol , o-cresol, 1,3-butanediol, 2-propyn-1-ol, propargyl alcohol, 3-methylphenol, m-cresol, triethylene glycol, diethylene glycol, n-methylformamide, 1,2-propylene glycol, 1,3-propylene glycol, 2-chlorophenol, methanol, 1,2-ethylene glycol, ethylene glycol, formamide, 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, or p-cresol.

[0102] Organic solvent addition: In some cases, the disclosed hybridization buffer formulations may include the addition of an organic solvent. Examples of suitable solvents include, but are not limited to, acetonitrile, ethanol, DMF and methanol, or any combination of different percentages thereof (e.g., >5%). In some cases, the percentage (by volume) of the organic solvent contained in the hybridization buffer may be in the range of about 1% to about 20%. In some cases, the volume percentage of the 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 volume percentage of the organic solvent may be at most 20%, 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 lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, the volume percentage of the organic solvent can be in the range of about 4% to about 15%. The volume percentage of the organic solvent can have any value within the range, for example, about 7.5%.

[0103] When the organic solvent includes a polar aprotic solvent, the amount of the polar aprotic solvent is present in an amount that effectively denatures the double-stranded nucleic acid. In some embodiments, based on the total volume of the preparation, the amount of the polar aprotic solvent is greater than or equal to about 10 volume percents. Based on the total volume of the preparation, the amount of the polar aprotic solvent is about or greater than about 5 volume percents, 10 volume percents, 15 volume percents, 20 volume percents, 25 volume percents, 30 volume percents, 35 volume percents, 40 volume percents, 50 volume percents, 60 volume percents, 70 volume percents, 80 volume percents, 90 volume percents or higher. Based on the total volume of the preparation, the amount of the polar aprotic solvent is less than about 15 volume percents, 20 volume percents, 25 volume percents, 30 volume percents, 35 volume percents, 40 volume percents, 50 volume percents, 60 volume percents, 70 volume percents, 80 volume percents, 90 volume percents or higher. In some embodiments, based on the total volume of the preparation, the amount of the polar aprotic solvent is in the range of about 10 volume percents to 90 volume percents. In some embodiments, the amount of polar aprotic solvent is in the range of about 25 volume percent to 75 volume percent based on the total volume of the formulation. In some embodiments, the amount of polar aprotic solvent is in the range of about 10 volume percent to 95 volume percent, 10 volume percent to 85 volume percent, 20 volume percent to 90 volume percent, 20 volume percent to 80 volume percent, 20 volume percent to 75 volume percent, or 30 volume percent to 60 volume percent based on the total volume of the formulation. In some embodiments, the polar aprotic solvent is formamide.

[0104] When the organic solvent includes a polar aprotic solvent, the amount of the aprotic solvent is present in an amount that effectively denatures the double-stranded nucleic acid. In some embodiments, based on the total volume of the preparation, the amount of the aprotic solvent is greater than or equal to about 10 volume percents. Based on the total volume of the preparation, the amount of the aprotic solvent is about or greater than about 5 volume percents, 10 volume percents, 15 volume percents, 20 volume percents, 25 volume percents, 30 volume percents, 35 volume percents, 40 volume percents, 50 volume percents, 60 volume percents, 70 volume percents, 80 volume percents, 90 volume percents or higher. Based on the total volume of the preparation, the amount of the aprotic solvent is less than about 15 volume percents, 20 volume percents, 25 volume percents, 30 volume percents, 35 volume percents, 40 volume percents, 50 volume percents, 60 volume percents, 70 volume percents, 80 volume percents, 90 volume percents or higher. In some embodiments, based on the total volume of the preparation, the amount of the aprotic solvent is in the range of about 10 volume percents to 90 volume percents. In some embodiments, the amount of aprotic solvent is in the range of about 25 volume percent to 75 volume percent based on the total volume of the formulation. In some embodiments, the amount of aprotic solvent is in the range of about 10 volume percent to 95 volume percent, 10 volume percent to 85 volume percent, 20 volume percent to 90 volume percent, 20 volume percent to 80 volume percent, 20 volume percent to 75 volume percent, or 30 volume percent to 60 volume percent based on the total volume of the formulation.

[0105] Addition of Molecular Clustering Agents / Size Exclusion Agents: The compositions described herein may include one or more clustering agents that enhance molecular clustering. The clustering agent may be selected from polyethylene glycol (PEG), dextran, hydroxypropyl methylcellulose (HPMC), hydroxyethyl methylcellulose (HEMC), hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose, and hydroxymethylcellulose, and combinations thereof. Exemplary clustering agents may include one or more of polyethylene glycol (PEG), dextran, a protein (e.g., ovalbumin or hemoglobin), or Ficoll.

[0106] The appropriate amount of clustering agent in the composition allows, enhances or promotes molecular clustering. Based on the total volume of the preparation, the amount of the clustering agent is about or greater than about 1 volume percent, 2 volume percent, 3 volume percent, 5 volume percent, 10 volume percent, 15 volume percent, 20 volume percent, 25 volume percent, 30 volume percent, 35 volume percent, 40 volume percent, 50 volume percent, 60 volume percent or higher. In some cases, based on the total volume of the preparation, the amount of the molecular clustering agent is greater than or equal to about 5 volume percent. Based on the total volume of the preparation, the amount of the clustering agent is less than about 3 volume percent, 5 volume percent, 10 volume percent, 12.5 volume percent, 15 volume percent, 20 volume percent, 25 volume percent, 30 volume percent, 35 volume percent, 40 volume percent, 50 volume percent, 60 volume percent, 70 volume percent, 80 volume percent, 90 volume percent or higher. In some cases, based on the total volume of the preparation, the amount of the molecular clustering agent can be less than or equal to about 30 volume percent. In some embodiments, the amount of organic solvent is in the range of about 25 volume percent to 75 volume percent based on the total volume of the preparation. In some embodiments, the amount of organic solvent is in the range of about 1 volume percent to 40 volume percent, 1 volume percent to 35 volume percent, 2 volume percent to 50 volume percent, 2 volume percent to 40 volume percent, 2 volume percent to 35 volume percent, 2 volume percent to 30 volume percent, 2 volume percent to 25 volume percent, 2 volume percent to 20 volume percent, 2 volume percent to 10 volume percent, 5 volume percent to 50 volume percent, 5 volume percent to 40 volume percent, 5 volume percent to 35 volume percent, 5 volume percent to 30 volume percent, 5 volume percent to 25 volume percent, 5 volume percent to 20 volume percent. In some cases, the amount of molecular clustering agent can be in the range of about 5 volume percent to about 20 volume percent based on the total volume of the preparation. In some embodiments, the amount of clustering agent is in the range of about 1 volume percent to 30 volume percent based on the total volume of the preparation.

[0107] An example of a clustering agent in the composition is polyethylene glycol (PEG). In some embodiments, the PEG used may have a molecular weight sufficient to enhance or promote molecular clustering. In some embodiments, the PEG used in the composition has a molecular weight in the range of about 5k-50k Da. In some embodiments, the PEG used in the composition has a molecular weight in the range of about 10k-40kDa. In some embodiments, the PEG used in the composition has a molecular weight in the range of about 10k-30k Da. In some embodiments, the PEG used in the composition has a molecular weight in the range of about 20k Da.

[0108] In some cases, the disclosed hybridization buffer formulations may include the addition of molecular clustering agents or size exclusion agents. Molecular clustering agents or size exclusion agents are, for example, macromolecules (e.g., proteins) that, when added to a solution at high concentrations, can change the properties of other molecules in the solution by reducing the volume of solvent available to other molecules. In some cases, the volume percentage of molecular clustering agents or size exclusion agents included in the hybridization buffer formulation may be in the range of about 1% to about 50%. In some cases, the volume percentage of molecular clustering agents or size exclusion agents 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 clustering agent or size 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 lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, the volume percentage of the molecular clustering agent or size exclusion agent can be in the range of about 5% to about 35%. The volume percentage of the molecular clustering agent or size exclusion agent can have any value within this range, for example, about 12.5%.

[0109] pH buffer system: The compositions described herein include a pH buffer system that maintains the pH of the composition within a range suitable for hybridization processes. The pH buffer system can include one or more buffers selected from Tris, HEPES, TAPS, Tricine, Bicine, Bis-Tris, NaOH, KOH, TES, EPPS, MES, and MOPS. The pH buffer system can further include a solvent. Exemplary pH buffer systems include MOPS, MES, TAPS, phosphate buffer in combination with methanol, acetonitrile, ethanol, isopropanol, butanol, tert-butanol, DMF, DMSO, or any combination thereof.

[0110] The amount of the pH buffer system effectively maintains the pH of the preparation 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 lower and upper limits 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 within the range of about 4 to about 8. The pH of the hybridization buffer may have any value within this range, for example, about pH 7.8. In some cases, the pH range is about 3 to about 10. In some cases, the disclosed hybridization buffer formulation may include adjusting the pH within the range of about pH 3 to pH 10, with a narrower buffer range of 5-9.

[0111] Additives that affect the melting temperature of DNA: The compositions described herein may include one or more additives to allow for better control of the melting temperature of nucleic acids and to enhance stringency control of hybridization reactions. Hybridization reactions are typically performed under stringent conditions to achieve hybridization specificity. In some cases, the additive that controls the melting temperature of nucleic acids is formamide.

[0112] The amount of the additive for controlling the melting temperature of nucleic acid can change according to other medicaments used in the compositions.The cumulative volume based on preparation, the amount of the additive for controlling the melting temperature of nucleic acid is about or greater than about 1 volume percent, 2 volume percents, 3 volume percents, 5 volume percents, 10 volume percents, 15 volume percents, 20 volume percents, 25 volume percents, 30 volume percents, 35 volume percents, 40 volume percents, 50 volume percents, 60 volume percents or higher.In some cases, the cumulative volume based on preparation, the amount of the additive for controlling the melting temperature of nucleic acid is greater than or equal to about 2 volume percents.In some cases, the cumulative volume based on preparation, the amount of the additive for controlling the melting temperature of nucleic acid is greater than or equal to about 5 volume percents. In some cases, the amount of the additive used to control the melting temperature of the nucleic acid is less than about 3 volume percent, 5 volume percent, 10 volume percent, 12.5 volume percent, 15 volume percent, 20 volume percent, 25 volume percent, 30 volume percent, 35 volume percent, 40 volume percent, 50 volume percent, 60 volume percent, 70 volume percent, 80 volume percent, 90 volume percent or more based on the total volume of the formulation. In some embodiments, based on the total volume of the preparation, the amount of the additive for controlling the melting temperature of the nucleic acid is in the range of about 1 volume percent to 40 volume percent, 1 volume percent to 35 volume percent, 2 volume percent to 50 volume percent, 2 volume percent to 40 volume percent, 2 volume percent to 35 volume percent, 2 volume percent to 30 volume percent, 2 volume percent to 25 volume percent, 2 volume percent to 20 volume percent, 2 volume percent to 10 volume percent, 5 volume percent to 50 volume percent, 5 volume percent to 40 volume percent, 5 volume percent to 35 volume percent, 5 volume percent to 30 volume percent, 5 volume percent to 25 volume percent, 5 volume percent to 20 volume percent. In some embodiments, based on the total volume of the preparation, the amount of the additive for controlling the melting temperature of the nucleic acid is in the range of about 2 volume percent to 20 volume percent. In some cases, based on the total volume of the preparation, the amount of the additive for controlling the melting temperature of the nucleic acid is in the range of about 5 volume percent to 10 volume percent.

[0113] In some cases, the disclosed hybridization buffer formulations may include additives that add changes in the melting temperature of the nucleic acid duplex. Examples of suitable additives that can be used to change the melting temperature of nucleic acids include, but are not limited to, formamide. In some cases, the volume percentage of the melting temperature additive included in the hybridization buffer formulation may be in the range of about 1% to about 50%. In some cases, the volume percentage of the 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 the 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 lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, the volume percentage of the melting temperature additive can be in the range of about 10% to about 25%. The volume percentage of the melting temperature additive can have any value within the range, for example, about 22.5%.

[0114] Additives that affect DNA hydration: In some cases, the disclosed hybridization buffer formulations may include the addition of additives that affect nucleic acid hydration. Examples include, but are not limited to, betaine, urea, glycine betaine, or any combination thereof. In some cases, the volume percentage of the hydration additive included in the hybridization buffer formulation may be in the range of about 1% to about 50%. In some cases, the volume percentage of the 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 the 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 lower and upper values ​​described in this paragraph can be combined to form a range included in the present disclosure, for example, the volume percentage of the hydration additive can be in the range of about 1% to about 30%. The volume percentage of the melting temperature additive can have any value within the range, for example, about 6.5%.

[0115] system

[0116] Provided herein is a system comprising a hybridization composition as described herein and a low non-specific binding surface. In some cases, the system as described herein includes a flow cell device. In some cases, the system further includes an imaging system (e.g., a camera and an inverted fluorescence microscope). The system may further include one or more computer control systems to perform a computer-implemented nucleic acid analysis method.

[0117] Low non-specific binding surfaces: The present disclosure includes low non-specific binding surfaces that can improve nucleic acid hybridization and amplification performance. In general, the disclosed surfaces can include one or more covalently or non-covalently attached low binding chemical modification layers (e.g., silane layers, polymer films) and one or more covalently or non-covalently attached primer sequences that can be used to tether single-stranded template oligonucleotides 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 to the surface or to each other or to a combination thereof, and the total number of layers can be changed so that the non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the surface is minimized or reduced relative to a comparable monolayer. In general, the formulation of the surface can be changed so that the non-specific hybridization on the surface is minimized or reduced relative to a comparable monolayer. The formulation of the surface can be changed so that the non-specific amplification on the surface is minimized or reduced relative to a comparable monolayer. The formulation of the surface can be changed so that the specific amplification rate or yield or a combination thereof on the surface is maximized. In some cases disclosed herein, amplification levels suitable for detection are achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 30 cycles of amplification.

[0118] Non-limiting examples of low non-specific binding surfaces are provided in co-pending U.S. Patent Application No. 16 / 739,007, the entire contents of which are incorporated herein by reference. The terms "low non-specific binding surface" and "low binding surface" are used interchangeably and refer to a hydrophilic surface that exhibits a lower amount of non-specific binding of proteins or nucleic acids than a non-hydrophilic surface. In some cases, the low non-specific binding surface is passivated, meaning that it is coated with a hydrophilic substrate.

[0119] Examples of materials that can be used to make the substrate or support structure include, but are not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of glass and plastic substrates are contemplated.

[0120] Substrate or support structure can be presented in any one of multiple geometric shapes and sizes, and substrate or support structure can comprise any one of multiple materials.For example, in some cases, substrate or support structure can be locally flat (for example, including microscope slide, or the surface of microscope slide).In general, substrate or support structure can be cylindrical (for example, including the inner surface of capillary or capillary), spherical (for example, including the outer surface of non-porous bead) or irregular (for example, including the outer surface of irregular shape, non-porous bead or particle).In some cases, the surface of substrate or support structure for nucleic acid hybridization and amplification can be solid, non-porous surface.In some cases, the surface of substrate or support structure for nucleic acid hybridization and amplification can be porous, so that coating as described herein penetrates porous surface, and nucleic acid hybridization and amplification reaction carried out thereon can occur in hole.

[0121] The substrate or support structure including one or more chemically modified layers (e.g., a layer of low non-specific binding polymer) can be independent or integrated into another structure or assembly. For example, in some cases, the substrate or support structure can include one or more surfaces in an integrated or assembled microfluidic flow cell. The substrate or support structure can include one or more surfaces in a microplate format, such as the bottom surface of a hole in a microplate. As described above, in some embodiments, the substrate or support structure includes the inner surface (e.g., lumen surface) of a capillary. In an alternative embodiment, the substrate or support structure includes the inner surface (e.g., lumen surface) of a capillary etched into a planar chip.

[0122] The chemical modification layer can be applied uniformly on the surface of substrate or support structure. Alternatively, the surface of substrate or support structure can be unevenly distributed or patterned so that the chemical modification layer is confined in one or more discrete regions of substrate. For example, photolithography can be used to pattern substrate surface to form an ordered array or random pattern of chemically modified regions on the surface. Contact printing or inkjet printing technology or its combination can be used to pattern substrate surface. In some cases, the ordered array or random pattern of chemically modified discrete regions may include 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 intermediate number within the scope of this article.

[0123] To achieve a low nonspecific binding surface (also referred to herein as a "low binding" or "passivated" surface), a hydrophilic polymer can be nonspecifically adsorbed or covalently grafted to the substrate or support surface. For example, passivation can be performed using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA), polyglutamic acid (P GA), polylysine, polyglucosides, streptavidin, dextran or other hydrophilic polymers with different molecular weights and end groups are attached to the surface using, for example, silane chemistry. The end groups away from the surface can include, but are not limited to, biotin, methoxy ethers, carboxylates, amines, NHS esters, maleimides, and disilazane. In some cases, two or more layers of hydrophilic polymers, such as linear polymers, branched polymers, or multi-branched polymers, can be deposited on the surface. In some cases, the two or more layers can be covalently coupled to each other or internally cross-linked to improve the resulting surface. stability. In some cases, oligonucleotide primers (or other biomolecules, such as enzymes or antibodies) with different base sequences and base modifications can be bound to the resulting surface layer at various surface densities. In some cases, for example, the surface functional group density and oligonucleotide concentration can be changed to target a certain primer density range. In addition, the primer density can be controlled by diluting the oligonucleotide with other molecules with the same functional group. For example, in the reaction with the surface coated with NHS-ester, the amine-labeled oligonucleotide can be diluted with amine-labeled polyethylene glycol to reduce the final primer density. Primers with different lengths of joints between the hybridization region and the surface attachment functional group can also be used to control the surface density. Examples of suitable joints include polythymidylic acid (poly-T) chains and polyadenylic acid (poly-A) chains (e.g., 0 to 20 bases), PEG joints (e.g., 3 to 20 monomer units) and carbon chains (e.g., C6, C12, C18, etc.) at the 5' end of the primer. In order to measure the primer density, fluorescently labeled primers can be bound to the surface, and then the fluorescence readings are compared with the fluorescence readings of dye solutions of known concentrations.

[0124] Due to the surface passivation technology disclosed herein, proteins, nucleic acids and other biomolecules do not "stick" to the substrate, that is, they exhibit low nonspecific binding (nonspecific binding). Examples of standard monolayer surface preparation methods using different glass preparation conditions are shown below. Hydrophilic surfaces that have been passivated to achieve ultra-low nonspecific binding of proteins and nucleic acids require novel reaction conditions to improve primer deposition reaction efficiency, hybridization performance and induce effective amplification. All of these methods require oligonucleotide attachment to low binding surfaces and subsequent protein binding and delivery. As described below, the results produced by the combination of a new primer surface conjugate preparation (Cy3 oligonucleotide grafting titration) with the resulting ultra-low nonspecific background (nonspecific binding functional testing using red and green fluorescent dyes) demonstrate the feasibility of the disclosed method. Some surfaces disclosed herein exhibit specific binding (e.g., hybridization with bound primers or probes) of fluorophores (e.g., Cy3) and nonspecific binding (e.g., B inter ) is 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 any intermediate value within the scope herein. Some surfaces disclosed herein exhibit a specific fluorescent signal of a fluorophore (e.g., Cy3) and a nonspecific fluorescent signal (e.g., a specifically hybridized labeled oligonucleotide and a nonspecifically bound labeled oligonucleotide, or a specifically amplified labeled oligonucleotide and a nonspecifically bound (B inter ) labeled oligonucleotides or nonspecific amplification (B intra ) labeled oligonucleotides or combinations thereof (B inter +B intra 1, 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 any intermediate value within the scope herein.

[0125] In order to scale the primer surface density and add extra dimensions to hydrophilic or amphiphilic surfaces, substrates containing multilayer coatings of PEG and other hydrophilic polymers have been developed. By using hydrophilic and amphiphilic surface layering methods, including but not limited to polymer / copolymer materials described below, the primer loading density on the surface can be significantly increased. Traditional PEG coating methods use monolayer primer deposition, which has been generally reported for single molecule applications, but does not produce high copy numbers in nucleic acid amplification applications. As described herein, "layering" can be completed using any compatible polymer or monomer subunit using traditional cross-linking methods, so that a surface containing two or more highly cross-linked layers can be sequentially constructed. Examples of suitable polymers include but are not limited to streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some cases, different layers can be attached to each other by various conjugation reactions, including but not limited to biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers. In some cases, materials with high primer density can be constructed in solution and then layered onto a surface in multiple operations.

[0126] The attachment chemistry used to graft the first chemically modified layer to the support surface generally depends on the material and chemical properties of the layer used to make the support. In some cases, the first layer can be covalently attached to the support surface. In some cases, the first layer can be, for example, adsorbed on the surface by 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 rather than covalently attached. In either case, the substrate surface can be treated before attaching or depositing the first layer. Any of a variety of surface preparation techniques can be used to clean or treat the support surface. For example, glass or silicon surfaces can be treated with piranha solution (sulfuric acid (H2O)). 2 SO 4 ) and hydrogen peroxide (H 2 O 2 ) a mixture of) acid pickling, or cleaning using an oxygen plasma treatment method, or a combination thereof.

[0127] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, Cl2, C18 hydrocarbons or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to generate any of the disclosed low-binding support surfaces include, but are not limited to, any of (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), various PEG-silanes (e.g., including molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (e.g., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0128] Any of a variety of molecules, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof, can be used to create one or more chemically modified layers on the support surface, wherein the selection of components used can be varied to alter one or more properties of the support surface, such as the surface density of functional groups or tethered oligonucleotide primers, or combinations thereof; the hydrophilicity / hydrophobicity of the support surface, or the three-dimensional properties of the support surface (i.e., "thickness"). Examples of polymers that can be used to create one or more layers of low nonspecific binding material in any disclosed support surface include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., polymer layers) to the surface of a support or to cross-link layers to each other, or a combination thereof, include, but are not limited to, biotin-streptavidin interaction (or variants thereof), his tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxides, azides, hydrazides, alkynes, isocyanates, and silanes.

[0129] One or more layers of the multilayer surface may comprise a branched polymer or may be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (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 polyglucosides, and dextran.

[0130] In some cases, the branched polymers used to create one or more layers of any multilayer surface disclosed herein may include 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, or at least 40 branches. The molecules typically exhibit a "power of 2" number of branches, such as 2, 4, 8, 16, 32, 64, or 128 branches.

[0131] PEG multilayers including PEG (8,16,8) on PEG-amine-APTES exposed to two layers of 7uM pre-loaded primers exhibited concentrations of 2,000,000 to 10,000,000 on the surface. Similar concentrations were observed for 3 layers of multi-arm PEG (8,16,8) and (8,64,8) on PEG-amine-APTES exposed to 8uM primers, and for 3 layers of multi-arm PEG (8,8,8) with star-shaped PEG-amines replacing dumbbell-shaped 16mers and 64mers. PEG multilayers with comparable first, second, and third PEG levels were also considered.

[0132] The molecular weight of the linear, branched or multi-branched polymer used to produce one or more layers of any multi-layer surface disclosed herein can be 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.

[0133] In some cases, for example, where at least one layer of the multilayer surface includes a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the underlying layer can be in the range of about one covalent bond per molecule and about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the underlying layer 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, 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 covalent bonds per molecule.

[0134] Any reactive functional groups remaining after the material layer is coupled to the support surface can optionally be blocked by coupling a small, inert molecule using a high-yield coupling chemistry. For example, where an amine coupling chemistry is used to attach a new material layer to an underlying layer, any remaining amine groups can then be acetylated or inactivated by coupling with a small amino acid (e.g., glycine).

[0135] The number of layers of low non-specific binding materials, such as hydrophilic polymer materials, deposited on the surface of the disclosed low binding support can be in the range of 1 to about 10. In some cases, the number of layers is 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 layers. 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 layer. Any of the lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the number of layers can be in the range of about 2 to about 4. In some cases, all layers can include the same material. In some cases, each layer can include different materials. In some cases, multiple layers can include multiple materials. In some cases, at least one layer can include a branched polymer. In some cases, all layers can include a branched polymer.

[0136] In some cases, one or more layers of low non-specific binding materials can be deposited on the substrate surface or conjugated to the substrate surface, or a combination thereof, using a polar protic solvent, a polar aprotic solvent, a non-polar solvent, or any combination thereof. In some cases, the solvent used for layer deposition or coupling, or a combination thereof, can include an alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, an aqueous buffer solution (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino) propanesulfonic acid (MOPS), etc.), or any combination thereof. In some cases, the organic component of the solvent mixture used may account for 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 within the scope of this article or close to it, the balance is made up by water or aqueous buffer solution. In some cases, the aqueous component of the solvent mixture used may account for 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 within the scope of this article or close to it, the balance is made up by organic solvent. The pH of the solvent mixture used can be less than or equal to about 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or any value within or near the ranges described herein. The pH of the solvent mixture can be greater than or equal to about 10.

[0137] In some cases, one or more layers of low non-specific binding material can be deposited on a substrate surface, or conjugated to a substrate surface, or a combination thereof, using a mixture of organic solvents, wherein at least one component has a dielectric constant of less than 40 and constitutes at least 50% of the total mixture volume. In some cases, the dielectric constant of at least one component can be less than 10, less than 20, less than 30, less than 40. In some cases, at least one component constitutes 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 total mixture volume.

[0138] As noted, the low non-specific binding supports of the present disclosure exhibit reduced non-specific binding of proteins, nucleic acids, and other components of hybridization or amplification formulations for solid-phase nucleic acid amplification, or combinations thereof. The degree of non-specific binding exhibited by a given support surface can be evaluated qualitatively or quantitatively. For example, in some cases, the exposure of a surface to a fluorescent dye (e.g., Cy3, Cy5, etc.), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, or a fluorescently labeled protein (e.g., polymerase), or combinations thereof, under a set of standardized conditions, followed by a specified wash procedure and fluorescence imaging, can be used as a qualitative tool for comparing non-specific binding on supports containing different surface formulations. In some cases, the exposure of a surface to a fluorescent dye, a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, or a fluorescently labeled protein (e.g., polymerase), or combinations thereof, under a set of standardized conditions, followed by a specified wash procedure and fluorescence imaging, can be used as a quantitative tool for comparing non-specific binding on supports containing different surface formulations —— Provided that care has been taken to ensure that fluorescence imaging is performed under conditions where the fluorescence signal is linearly related (or related in a predictable manner) to the number of fluorophores on the support surface (e.g., conditions where signal saturation or fluorophore self-quenching or combinations thereof are not a problem) and appropriate calibration standards are used. In some cases, other techniques such as radioisotope labeling and counting methods can be used to quantitatively evaluate the degree of non-specific binding exhibited by different support surface formulations of the present disclosure.

[0139] Some surfaces disclosed herein exhibit a ratio of specific binding to non-specific binding 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 greater than 100, or any intermediate value within the scope herein. Some surfaces disclosed herein exhibit a ratio of specific fluorescence to non-specific 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 greater than 100, or any intermediate value within the scope herein.

[0140] As noted, in some cases, the extent of non-specific binding exhibited by the disclosed low binding supports can be assessed using a standardized protocol to contact the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc. under a set of standard incubation and washing conditions, followed by detecting the amount of label retained on the surface, and comparing the resulting signal with an appropriate calibration standard. In some cases, the label may include a fluorescent label. In some cases, the label may contain a radioactive isotope. In some cases, the label may include any other detectable label. In some cases, the extent of non-specific binding exhibited by a given support surface preparation can thus be assessed based on the number of protein molecules (or other molecules) non-specifically bound per unit area. In some cases, the low binding supports of the present disclosure may exhibit less than or equal to about 0.001 molecules / μm 2 , less than or equal to about 0.01 molecules / μm 2 , less than or equal to about 0.1 molecule / μm 2 , less than or equal to about 0.25 molecules / μm 2 , less than or equal to about 0.5 molecules / μm 2 , less than or equal to about 1 molecule / μm 2 , less than or equal to about 10 molecules / μm 2 , less than or equal to about 100 molecules / μm 2 or less than or equal to about 1,000 molecules / μm 2 A given support surface of the present disclosure may exhibit nonspecific binding falling anywhere within this range, e.g., less than 86 molecules / μm 2 .

[0141] In some cases, the surfaces disclosed herein exhibit a ratio of specific binding to non-specific 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 intermediate value within the scope herein. In some cases, the surfaces disclosed herein exhibit a ratio of specific binding to non-specific binding of a fluorophore, such as Cy3, of greater than or equal to about 100. In some cases, the surfaces disclosed herein exhibit a ratio of specific fluorescence signal to non-specific fluorescence signal 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 any intermediate value within the scope herein. In some cases, a surface disclosed herein exhibits a ratio of a specific fluorescent signal to a nonspecific fluorescent signal of a fluorophore, such as Cy3, of greater than or equal to about 100.

[0142] Low background surfaces consistent with the disclosure herein can exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) 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 greater than 50 specifically attached dye molecules per non-specifically adsorbed molecule. Similarly, low background surfaces consistent with the disclosure herein to which fluorophores (e.g., Cy3) have been attached can exhibit a ratio of specific fluorescent signal (e.g., derived from Cy3-labeled oligonucleotides attached to the surface) to non-specifically adsorbed dye fluorescent signal 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 when subjected to excitation energy.

[0143] In some cases, the hydrophilicity degree (or "wettability" with aqueous solution) of the disclosed support surface can be evaluated, for example, by measuring the water contact angle (wherein a small water droplet 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 or receding contact angle can be determined. In some cases, the water contact angle of the hydrophilic, low-binding support surface disclosed herein can be in the range of about 0 degree to about 30 degrees. In some cases, the water contact angle of the hydrophilic, low-binding 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 hydrophilic, low-binding support surface can exhibit a water contact angle with any numerical value within this range.

[0144] In some cases, the hydrophilic surfaces disclosed herein help reduce wash times for bioassays, typically due to reduced nonspecific binding of biomolecules to low binding surfaces. In some cases, a sufficient wash can be performed in less than or equal to about 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, in some cases, a sufficient wash can be performed in less than 30 seconds.

[0145] Some low binding surfaces of the present disclosure exhibit significant improvements in stability or durability to long-term exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be detected by fluorescently labeling functional groups on the surface or bound biomolecules (e.g., oligonucleotide primers) on the surface, and monitoring the fluorescent signal before, during, and after long-term 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 assess surface quality can be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured within these time periods) within 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 a solvent or elevated temperature, or a combination thereof. In some cases, the degree of change in fluorescence used to assess surface quality can be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% (or any combination of these percentages measured over this cycle range) within 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 of repeated exposure to solvent changes or temperature changes, or a combination thereof.

[0146] In some cases, surfaces disclosed herein can show a high ratio of specific signal to non-specific signal or other backgrounds. For example, when used for nucleic acid amplification, the amplified signal shown on some surfaces can be at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times or more than 100 times larger than the signal in the adjacent non-dense region of the surface. Similarly, the amplified signal shown on some surfaces can be at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times or more than 100 times larger than the signal in the adjacent amplified nucleic acid population region of the surface.

[0147] Fluorescence excitation energy varies between specific fluorophores and protocols, and can be within a range of excitation wavelengths, consistent with the fluorophore selection or other use parameters of the surface disclosed herein. In some cases, the wavelength is less than or equal to about 400 nanometers (nm). In some cases, the wavelength is greater than or equal to about 800 nm. In some cases, the wavelength is between 400 nm and 800 nm.

[0148] Thus, the low background surfaces disclosed herein exhibit low background fluorescence signals or high contrast-to-noise (CNR) ratios. For example, in some cases, the surface background fluorescence at a location that is spatially different or distant from a labeled feature (e.g., a surface labeled spot, cluster, discrete region, sub-portion, or subgroup) on a surface comprising a hybridized nucleic acid molecule cluster or comprising a clonal amplified nucleic acid molecule cluster generated by 20 cycles of nucleic acid amplification via thermal cycling can be no more than 20 times, 10 times, 5 times, 2 times, 1 times, 0.5 times, 0.1 times, or less than 0.1 times greater than the background fluorescence measured at the same location before performing the hybridization or the 20 cycles of nucleic acid amplification.

[0149] In some cases, fluorescent images of the disclosed low background surfaces exhibit 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 greater than 250 when used in nucleic acid hybridization or amplification applications to produce hybridized or clonally amplified nucleic acid molecule clusters (e.g., that have been directly or indirectly labeled with a fluorophore).

[0150] The surface comprising one or more chemically modified layers (e.g., low non-specific binding polymer layer) can be independent or integrated into another structure or assembly. The chemically modified layer can be uniformly applied on the entire surface. Alternatively, the surface can be patterned so that the chemically modified layer is confined to one or more discrete regions of the substrate. For example, photolithography patterning can be used to pattern the surface to produce an ordered array or random pattern of chemically modified regions on the surface. For example, contact printing or inkjet printing technology or a combination thereof can be used to pattern the substrate surface. In some cases, the ordered array or random pattern of chemically modified regions may include 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.

[0151] In order to achieve low non-specific binding surface (also referred to as "low binding" or "passivation" surface in this article), hydrophilic polymers can be non-specifically adsorbed or covalently grafted to the surface. For example, passivation can be carried out using poly (ethylene glycol) (PEG, also referred to as polyethylene oxide (PEO) or polyoxyethylene) or other hydrophilic polymers with different molecular weights and end groups, which are connected to the surface using, for example, silane chemistry. The end groups away from the surface can include, but are not limited to, biotin, methoxy ethers, carboxylates, amines, NHS esters, maleimides and disilanes. In some cases, two or more layers of hydrophilic polymers, such as linear polymers, branched polymers or multi-branched polymers, can be deposited on the surface. In some cases, two or more layers can be covalently coupled to each other or internally cross-linked to improve the stability of the resulting surface. In some cases, oligonucleotide primers (or other biomolecules, such as enzymes or antibodies) with different base sequences and base modifications can be bound to the resulting surface layer with various surface densities. In some cases, for example, the surface functional group density and oligonucleotide concentration can be changed to target a certain primer density range. In addition, the primer density can be controlled by diluting the oligonucleotide with other molecules with the same functional group. For example, in the reaction with the surface coated with NHS-ester, the amine-labeled oligonucleotide can be diluted with amine-labeled polyethylene glycol to reduce the final primer density. Primers with joints of different lengths between the hybridization region and the surface attachment functional group can also be used to control the surface density. Examples of suitable joints include polythymidylic acid chains and polyadenylic acid chains (e.g., 0 to 20 bases), PEG joints (e.g., 3 to 20 monomer units) and carbon chains (e.g., C6, C12, C18, etc.) at the 5' end of the primer. In order to measure the primer density, fluorescently labeled primers can be bound on the surface, and then the fluorescence readings are compared with the fluorescence readings of the dye solution of known concentration.

[0152] In order to scale the primer surface density and add extra dimensions to the hydrophilic or amphiphilic surface, a surface of a multilayer coating containing PEG and other hydrophilic polymers has been developed. The primer loading density on the surface can be significantly increased by using a hydrophilic and amphiphilic surface layering method including but not limited to the following polymer / copolymer materials. The traditional PEG coating method uses a monolayer primer deposition, which has been generally reported for single molecule applications, but does not produce a high copy number for nucleic acid amplification applications. As described herein, "layering" can be achieved using traditional cross-linking methods with any compatible polymer or monomer subunit, so that a surface containing two or more highly cross-linked layers can be constructed in sequence. Examples of suitable polymers include but are not limited to streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some cases, different layers can be attached to each other by any of a variety of conjugation reactions, including but not limited to, biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers. In some cases, high primer density materials can be constructed in solution and then layered onto a surface.

[0153] The attachment chemistry used to graft the first chemically modified layer to the surface generally depends on the material and chemical properties of the layer from which the surface is made. In some cases, the first layer can be covalently attached to the surface. In some cases, the first layer can be non-covalently attached, such as adsorbed to the surface by non-covalent interactions, such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the molecular components of the surface and the first layer. In either case, the substrate surface can be treated prior to attachment or deposition of the first layer. Any of a variety of surface preparation techniques can be used to clean or treat the surface. For example, glass or silicon surfaces can be treated using piranha solution (sulfuric acid (H2O)). 2 SO 4 ) and hydrogen peroxide (H 2 O 2 ) mixture), acid washing, alkaline treatment in KOH and NaOH, or cleaning using an oxygen plasma treatment method, or a combination thereof.

[0154] Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine or carboxyl groups), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths, such as C6, C12, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to generate any of the disclosed low binding surfaces include, but are not limited to, any of (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), various PEG-silanes (e.g., including molecular weights of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (e.g., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0155] Any of a variety of molecules, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof, can be used to create one or more chemically modified layers on a surface, wherein the selection of components used can be varied to alter one or more properties of the surface, such as the surface density of functional groups or tethered oligonucleotide primers or combinations thereof; the hydrophilicity / hydrophobicity of the surface, or the three-dimensional properties of the surface (e.g., "thickness"). Examples of polymers that can be used to create one or more layers of low nonspecific binding material in any disclosed surface include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., a polymer layer) to a surface, or to cross-link layers to each other, or combinations thereof include, but are not limited to, biotin-streptavidin interaction (or variants thereof), his tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and silanes.

[0156] One or more layers of the multilayer surface may comprise a branched polymer or may be linear. Examples of suitable branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (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 polyglucosides, and dextran.

[0157] In some cases, the branched polymer used to create one or more layers of any multi-layer surface disclosed herein may contain 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, or at least 40 branches.

[0158] The linear, branched or multi-branched polymers used to produce one or more layers of any multi-layer surface disclosed herein can 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.

[0159] In some cases, for example, where at least one layer of the multilayer surface includes a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the underlying layer can be in the range of about one covalent bond per molecule and about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the underlying layer 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, 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.

[0160] Any reactive functional groups remaining after the material layer is coupled to the surface can optionally be blocked by coupling a small inert molecule using a high yield coupling chemistry. For example, where an amine coupling chemistry is used to attach a new material layer to an underlying material layer, any remaining amine groups can then be acetylated or passivated by coupling with a small amino acid such as glycine.

[0161] The number of layers of low non-specific binding materials, such as hydrophilic polymer materials, deposited on the surface can be in the range of 1 to about 10. In some cases, the number of layers is 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 lower and upper limits described in this paragraph can be combined to form a range included in the present disclosure, for example, in some cases, the number of layers can be in the range of about 2 to about 4. In some cases, all layers can contain the same material. In some cases, each layer can include different materials. In some cases, multiple layers can include multiple materials. In some cases, at least one layer can include a branched polymer. In some cases, all layers can include a branched polymer.

[0162] In some cases, one or more layers of low non-specific binding materials can be deposited on the substrate surface or conjugated to the substrate surface or a combination thereof using a polar protic solvent, a polar aprotic solvent, a non-polar solvent, or any combination thereof. In some cases, the solvent used for layer deposition or coupling or a combination thereof may include an alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, an aqueous buffer solution (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino) propanesulfonic acid (MOPS), etc.), or any combination thereof. In some cases, the organic component of the solvent mixture used may account for 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 supplemented by water or an aqueous buffer solution. In some cases, the aqueous component of the solvent mixture used may account for 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 balance being made up by organic solvents. The pH value 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.

[0163] As mentioned above, low non-specific binding surface shows nucleic acid and the non-specific binding of the reduction of other components of hybridization or amplification preparation or its combination for solid phase nucleic acid amplification.The non-specific binding degree that can be qualitatively or quantitatively assessed given surface shows.For example, in some cases, the surface can be exposed to fluorescent dye (for example, Cy3, Cy5, etc.), fluorescently labeled nucleotide, fluorescently labeled oligonucleotide or fluorescently labeled protein (for example, polymerase) or its combination under a set of standardized conditions and the subsequent specified flushing scheme and fluorescence imaging as a qualitative tool, for comparing the non-specific binding surface comprising different surface preparations.In some cases, the surface can be exposed to fluorescent dye, fluorescently labeled nucleotide, fluorescently labeled oligonucleotide or fluorescently labeled protein (for example, polymerase) or its combination under a set of standardized conditions and the subsequent specified flushing scheme and fluorescence imaging can be used as a quantitative tool, for comparing the non-specific binding on the surface comprising different surface preparations-prerequisite is to ensure that the number of fluorophores on the surface is linearly correlated (or correlated in a predictable manner) under the condition of having noted to ensure (for example, under the condition that signal saturation or fluorophore self-quenching or its combination is not a problem) to carry out fluorescence imaging and use suitable calibration standards. In some cases, other techniques, such as radioisotope labeling and counting methods, can be used to quantitatively assess the extent of non-specific binding exhibited by various surface formulations of the present disclosure.

[0164] As described above, in some cases, the degree of nonspecific binding exhibited by the disclosed low binding surfaces can be assessed using a standardized protocol that contacts the surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., under a set of standardized incubation and wash conditions, and then detects the amount of the label retained on the surface and compares the signal generated therefrom with an appropriate calibration standard. In some cases, the label may include a fluorescent label. In some cases, the label may contain a radioactive isotope. In some cases, the label may include any other detectable label. In some cases, the degree of nonspecific binding exhibited by a given surface preparation can therefore be assessed based on the number of nonspecifically bound protein molecules (or other molecules) per unit area. In some cases, the low binding surface of the present disclosure may exhibit less than or equal to about 0.001 molecules / μm 2 , less than or equal to about 0.01 molecules / μm 2 , less than or equal to about 0.1 molecule / μm 2 , less than or equal to about 0.25 molecules / μm 2 , less than or equal to about 0.5 molecules / μm2 , less than or equal to about 1 molecule / μm 2 , less than or equal to about 10 molecules / μm 2 , less than or equal to about 100 molecules / μm 2 , or less than or equal to about 1,000 molecules / μm 2 A given surface of the present disclosure may exhibit nonspecific binding falling anywhere within this range, for example, less than or equal to about 86 molecules / μm 2 For example, some modified surfaces disclosed herein exhibit less than or equal to about 0.5 molecules / μm after contact with a 1 μM solution of bovine serum albumin (BSA) in phosphate buffered saline (PBS) buffer for 30 minutes followed by a 10 minute PBS rinse. 2 In another example, some modified surfaces disclosed herein exhibited less than or equal to about 0.5 molecules / μm after contact with a 1 μM solution of cyanine 3 dye-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes and then rinsed three times with deionized water. 2 Some modified surfaces disclosed herein exhibit less than or equal to about 0.25 molecules / μm 2 Non-specific binding of Cy3 dye molecules.

[0165] Low background surfaces consistent with the disclosure herein can exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) 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 greater than 50 specifically attached dye molecules per non-specifically adsorbed molecule. Similarly, low background surfaces consistent with the disclosure herein to which fluorophores (e.g., Cy3) have been attached can exhibit a ratio of specific fluorescent signals (e.g., derived from Cy3-labeled oligonucleotides attached to the surface) to non-specifically 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 when subjected to excitation energy.

[0166] In some cases, the degree of hydrophilicity (or "wettability" with aqueous solutions) of the disclosed surface can be assessed, for example, by measuring the water contact angle, wherein a small water droplet 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 or receding contact angle can be determined. In some cases, the water contact angle of the hydrophilic low binding surface disclosed herein can be in the range of about 0 degrees to about 30 degrees. In some cases, the water contact angle of the hydrophilic low binding 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 hydrophilic low binding surface of the present disclosure can exhibit a water contact angle with any value within this range.

[0167] In some cases, the low binding surfaces of the present disclosure can show significant improvements in stability or durability to long-term exposure to solvents and elevated temperatures or repeated cyclic exposure to solvents or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups on the surface or biomolecules (e.g., oligonucleotide primers) bound to the surface and monitoring the fluorescent signal before, during, and after long-term exposure to solvents and elevated temperatures or repeated exposure to solvents or temperature changes. In some cases, the degree of change in fluorescence used to assess surface quality can be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured within these time periods) within 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 a solvent, or elevated temperature, or a combination thereof. In some cases, the degree of change in fluorescence used to assess surface quality can be less than or equal to about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured over this cycle range) within 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 of repeated exposure to solvent changes, or temperature changes, or a combination thereof.

[0168] In some cases, surfaces disclosed herein can show a high ratio of specific signal to nonspecific signal or other background. For example, when used for nucleic acid amplification, the amplification signal shown on some surfaces can be at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 or more than 100 times greater than the signal of the adjacent non-dense area on the surface. Similarly, the amplification signal shown on some surfaces is at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 or more than 100 times greater than the signal of the adjacent amplified nucleic acid population area on the surface.

[0169] Thus, the low background surfaces disclosed herein exhibit low background fluorescence signals or high contrast-to-noise (CNR) ratios.

[0170] Flow cell device : In some aspects, the low nonspecific binding surface is a surface of a flow device described herein. The flow device described herein may include a first reservoir containing a first solution and having an inlet end and an outlet end, wherein the first agent flows from the inlet end to the outlet end in the first reservoir; a second reservoir containing a second solution and having an inlet end and an outlet end, wherein the second agent flows from the inlet end to the outlet end in the second reservoir; and a central region, whose inlet end is fluidically coupled to the outlet end of the first reservoir and the outlet end of the second reservoir through at least one valve. In the 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.

[0171] The reservoirs described in the device can be used to hold different reagents. In some aspects, the first solution contained in the first reservoir is different from the second solution contained in the second reservoir. The second solution includes at least one reagent common to multiple reactions occurring in the central region. In some aspects, the second solution includes at least one reagent selected from a solvent, a polymerase, and dNTPs. In some aspects, the second solution includes a low-cost reagent. In some aspects, the first reservoir is coupled to the central region fluid through a first valve, and the second reservoir is coupled to the central region fluid through a second valve. The valve can be a diaphragm valve or other suitable valve.

[0172] The central region may include a capillary or microfluidic chip with one or more microfluidic channels. In some embodiments, the capillary is a readily available product. The capillary or microfluidic chip may also be removed from the device. In some embodiments, the capillary or microfluidic channel comprises an oligonucleotide group for sequencing a eukaryotic genome. In some embodiments, the capillary or microfluidic channel in the central region may be removable.

[0173] A single capillary flow cell device is disclosed herein, comprising a single capillary and one or two fluid adapters fixed to one or both ends of the capillary, wherein the capillary provides a fluid flow channel of a specific cross-sectional area and length, wherein the fluid adapter is configured to cooperate with standard tubing to provide a convenient, interchangeable fluid connection with an external fluid flow control system. Typically, the capillary used in the disclosed flow cell device (and the flow cell cartridge described below) will have at least one internal, axially aligned fluid flow channel (or "inner cavity") that runs through the entire length of the capillary. In some aspects, the capillary may have two, three, four, five, or more than five internal, axially aligned fluid flow channels (or "inner cavities").

[0174] Many specified cross-sectional geometries of a single capillary (or its lumen) are consistent with the disclosure herein, including but not limited to circular, elliptical, square, rectangular, triangular, rounded square, rounded rectangular or rounded triangle cross-sectional geometries. In some aspects, a single capillary (or its lumen) may have any specified cross-sectional dimensions or size groups. For example, in some aspects, the maximum cross-sectional dimension of the capillary lumen (e.g., the diameter when the shape of the lumen is circular, or the diagonal when the shape of the lumen is square or rectangular) may be in the range of about 10 μm to about 10 mm. The length of one or more capillaries for making the disclosed single capillary flow cell device or flow cell cartridge may be in the range of about 5 mm to about 5 cm or more. In some cases, the gap height of the capillary is about or exactly 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400 or 500 um, or any value falling within the range of this definition.

[0175] The present disclosure also includes a flow cell device comprising one or more microfluidic chips and one or two fluid adapters secured to one or both ends of the microfluidic chip, wherein the microfluidic chip provides one or more fluid flow channels having a specific cross-sectional area and length, wherein the fluid adapter is configured to cooperate with the microfluidic chip to provide a convenient, interchangeable fluid connection with an external fluid flow control system.

[0176] The microfluidic chip described herein includes one or more microfluidic channels etched on the chip surface. A microfluidic channel is defined as a fluid conduit having at least one minimum dimension from <1 nm to 1000 μm. A microfluidic channel system fabricated on a glass or silicon substrate has a channel height and width of about <1 nm to 1000 μm. The channel length can be in the micrometer range.

[0177] The capillaries or microfluidic chips used to construct the disclosed flow cell devices can be made of 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), polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) as a more chemically inert alternative. PEI is somewhat between polycarbonate and PEEK in terms of cost and compatibility. FFKM is also known as Kalrez or any combination thereof.

[0178] A flow cell device (eg, a microfluidic chip or a capillary flow cell) can be operably coupled to the imaging system described herein to capture or detect signals of DNA bases for applications such as nucleic acid sequencing, analyte capture and detection.

[0179] Oligonucleotide Primers and Adapter Sequences: Typically, at least one of the one or more layers of low non-specific binding material may contain functional groups for covalently or non-covalently attaching oligonucleotide adapter or primer sequences, or at least one layer may already contain covalently or non-covalently attached oligonucleotide adapter or primer sequences when it is deposited on the support surface. In some cases, the oligonucleotides tethered to the polymer molecules of at least one third layer may be distributed at multiple depths throughout the layer.

[0180] One or more types of oligonucleotide primers can be attached or tethered to the support surface. In some cases, one or more types of oligonucleotide adapters or primers can include a spacer sequence, an adapter sequence for hybridizing to a template library nucleic acid sequence connected to the adapter, a forward amplification primer, a reverse amplification primer, a sequencing primer, or a molecular barcode sequence, or any combination thereof. In some cases, one primer or adapter sequence can be tethered 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 primers or adapter sequences can be tethered to at least one layer of the surface.

[0181] In some cases, the length of the bound oligonucleotide adapter or primer sequence or its combination can be in the range of about 10 nucleotides to about 100 nucleotides. In some cases, the length of the bound oligonucleotide adapter or primer sequence or its combination can be 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 length of the bound oligonucleotide adapter or primer sequence or its combination can be 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. Any lower and upper limits 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 bound oligonucleotide adapter or primer sequence or its combination can be in the range of about 20 nucleotides to about 80 nucleotides. The length of a tethered oligonucleotide adaptor or primer sequence, or a combination thereof, can have any value within this range, such as about 24 nucleotides.

[0182] In some cases, the tethered primer sequence may include modifications designed to promote specificity and efficiency of nucleic acid amplification on low binding supports. For example, in some cases, the primer may include a polymerase stop point so that the primer sequence segment between the surface conjugation point and the modification site is always in single-stranded form and serves as a loading site for the 5' to 3' helicase in some isothermal amplification methods that rely on helicases. Other examples of primer modifications that can be used to generate polymerase stop points include, but are not limited to, inserting a PEG chain between two nucleotides toward the 5' end of the primer backbone, inserting abasic nucleotides (e.g., nucleotides that have neither purine nor pyrimidine bases) or lesions that can be bypassed by helicases.

[0183] As will be discussed further in the embodiments hereinafter, the surface density of the primer that is bound on the support surface or the spacing (for example, by changing the length of the adapter molecule that is used to bind the primer to the surface) or its combination that is bound away from the support surface can be changed, so that when using a given amplification method, "adjusting" the support obtains optimal performance. As described below, the surface density of the primer that adjusts the constraint may affect the level of specific or non-specific amplification or its combination observed on the support, and its mode can be different according to the selected amplification method. In some cases, the surface density of the oligonucleotide primer that is bound can be changed by adjusting the ratio of the molecular components that are used to produce the support surface. For example, in the case of using oligonucleotide primer-PEG conjugates to produce the final layer of low binding support, the ratio of oligonucleotide primer-PEG conjugates and non-conjugated PEG molecules can be changed. Any one of a variety of techniques can be used to assess or measure the surface density of the bound primer molecule then. Examples include, but are not limited to, the use of radioisotope labeling and counting methods, covalent coupling of cleavable molecules comprising optically detectable labels (e.g., fluorescent labels) that can be cleaved from a defined area of ​​the support surface, collected in a fixed volume of an appropriate solvent, and then by comparing the fluorescence signal to that of a calibration solution of known optical label concentration, or using fluorescence imaging techniques (as long as care is taken with the labeling reaction conditions and image acquisition settings) to ensure that the fluorescence signal is linearly related to the number of fluorophores on the surface (e.g., there is no significant self-quenching of the fluorophores on the surface).

[0184] In some cases, the resulting surface density of oligonucleotide primers on the surface of a low binding support of the present disclosure can be about 1,000 primer molecules / μm 2 To about 1,000,000 primer molecules / μm 2 In some cases, the surface density of the oligonucleotide primers may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm 2 In some cases, the surface density of the oligonucleotide primers may be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules / μm 2 Any of the lower and upper limits described in this paragraph may be combined to form a range encompassed by the present disclosure. For example, in some cases, the surface density of primers may be about 10,000 molecules / μm. 2 to about 100,000 molecules / μm 2 The surface density of primer molecules can have any value within this range, for example, about 455,000 molecules / μm 2In some cases, the surface density of template library nucleic acid sequences that initially hybridize to adapter or primer sequences on the support surface can be less than or equal to the density indicated by the surface density of the tethered oligonucleotide primers. In some cases, the surface density of clonally amplified template library nucleic acid sequences that hybridize to adapter or primer sequences on the support surface can span the same range of densities as indicated by the surface density of the tethered oligonucleotide primers.

[0185] The local densities listed above do not exclude variations in density across the surface, such that a surface may contain, for example, 500,000 / um 2 The invention further comprises a region of oligonucleotide density having a substantially different local density.

[0186] Imaging system. The imaging system described herein is used to detect hybridization between one or more sample nucleic acid molecules and the capture nucleic acid molecules coupled to the low non-specific binding surface. In some cases, the imaging system includes a camera. In some cases, the imaging system includes a microscope, such as a fluorescence microscope. The combination of an inverted fluorescence microscope and a camera can be used to capture the image of the low non-specific binding surface and visualize the hybridization between one or more sample nucleic acid molecules and the capture nucleic acid molecules. Non-limiting examples of imaging systems described herein are Olympus IX83 microscopes (Olympus Corp., Center Valley, PA) with a total internal reflection fluorescence (TIRF) objective lens (100X, 1.5NA, Olympus), CCD cameras (e.g., Olympus EM-CCD black and white camera, Olympus XM-10 black and white camera or Olympus DP80 color and black and white camera), illumination sources (e.g., Olympus 100W Hg lamp, Olympus 75W Xe lamp or Olympus U-HGLGPS fluorescent light source), and excitation wavelengths of 532nm or 635nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), for example, 405, 488, 532, or 633 nm dichroic mirrors / beamsplitters, and the bandpass filters were selected as 532LP or 645LP, which corresponded to the appropriate excitation wavelength.

[0187] Computer Control System. The present disclosure provides computer systems that are programmed or otherwise configured to implement the methods provided herein, such as, for example, methods for nucleic acid sequencing, storing reference nucleic acid sequences, performing sequence analysis, and / or comparing sample and reference nucleic acid sequences, as described herein. An example of such a computer system is shown in Fig.10 In. Fig.10As shown, computer system 1001 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1005, which can be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 1001 also includes a memory or memory location 1010 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1015 (e.g., a hard disk), a communication interface 1020 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 1025, such as cache, other memory, data storage, and / or an electronic display adapter. Memory 1010, storage unit 1015, interface 1020, and peripherals 1025 communicate with CPU 1005 via a communication bus (solid line) such as a motherboard. Storage unit 1015 can be a data storage unit (or data repository) for storing data. Computer system 1001 can be operably coupled to a computer network ("network") 1030 by means of communication interface 1020. The network 1030 may be the Internet, the Internet and / or an extranet, or an intranet and / or an 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, which may enable distributed computing, such as cloud computing. The network 1030 may, in some cases, implement a peer-to-peer network with the aid of the computer system 1001, which may enable devices coupled to the computer system 1001 to act as clients or servers.

[0188] The CPU 1005 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location such as the memory 1010. Examples of operations performed by the CPU 1005 may include fetching, decoding, executing, and writing back.

[0189] Storage unit 1015 can store files, such as drivers, libraries, and saved programs. Storage unit 1015 can store user data, such as user preferences and user programs. In some cases, computer system 1001 may include one or more other data storage units external to computer system 1001, such as located on a remote server that communicates with computer system 1001 via an intranet or the Internet.

[0190] Computer system 1001 may communicate with one or more remote computer systems via network 1030. For example, computer system 1001 may communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), tablets or tablet computers (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or a personal digital assistant. A user can access the computer system 1001 through the network 1030.

[0191] The methods described herein may be implemented by means of machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 1001 (e.g., on the memory 1010 or electronic storage unit 1015). The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 1005. In some cases, the code may be retrieved from the storage unit 1015 and stored on the memory 1010 for access by the processor 1005 at any time. In some cases, the electronic storage unit 1015 may be excluded and the machine executable instructions may be stored in the memory 1010.

[0192] The code may be precompiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language, which may be selected to enable the code to be executed in a precompiled or compile-time manner.

[0193] Various aspects of the systems and methods (e.g., computer system 1001) provided herein can be embodied in programming. Various aspects of the technology can be considered as "products" or "articles" in the form of machine (or processor) executable code and / or associated data, which are usually carried or embodied in the type of machine-readable media. Machine executable code can be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of a computer, processor, etc., or its related modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage at any time for software programming. All or part of the software can sometimes communicate through the Internet or other various telecommunications networks. For example, such communication can enable software to be loaded from one computer or processor to another computer or processor, such as from a management server or host to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as those used on physical interfaces between local devices through wired and optical landline networks and through various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as media that carry the software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0194] Therefore, machine-readable media such as computer executable code can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any one or more computers, such as can be used to implement the database shown in the accompanying drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wires and optical fibers, including wires that contain buses within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Therefore, common forms of computer-readable media include, for example: floppy disks, foldable disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tapes, any other physical storage media with hole patterns, RAM, ROMs, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or boxes, carriers for transmitting data or instructions, cables or links for transmitting such carriers, or any other media from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0195] The computer system 1001 may include or communicate with an electronic display 1035, which includes a user interface (UI) for providing an output or readout of a nucleic acid sequencing instrument, such as a nucleic acid sequencing instrument coupled to the computer system 1001. This readout may include a nucleic acid sequencing readout, such as a sequence of nucleic acid bases constituting a given nucleic acid sample. The UI may also be used to display the results of the analysis using this readout. Examples of UIs include, but are not limited to, a graphical user interface (GUI) and a web-based user interface. The electronic display 1035 may be a computer monitor, or a capacitive or resistive touch screen.

[0196] Composition and system performance

[0197] Improvement of hybridization rate: In some cases, the use of the buffer formulations disclosed herein (optionally, in combination with a low non-specific binding surface) produces a relative hybridization rate that is about 2 to about 20 times faster than a standard hybridization protocol. In some cases, the relative hybridization rate can be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times that of a standard hybridization protocol.

[0198] The methods and compositions described herein can contribute to shortening the time required for completing hybridization. In some embodiments, the hybridization time can be in the range of about 1 second (s) to 2 hours (h), about 5s to 1.5h, about 15s to 1h or about 15s to 0.5h. In some embodiments, the hybridization time can be in the range of about 15s to 1h. In some embodiments, the hybridization time can be shorter than 15s, 30s, 1 minute (min), 1.5min, 2min, 2.5min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 25min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min or 120min. In some embodiments, the hybridization time can be longer than 1s, 5s, 10s, 15s, 30s, 1min, 1.5min, 2min, 2.5min, 3min, 4min or 5min.

[0199] The annealing methods described herein can significantly shorten the annealing time. In some embodiments, at least 90% of the target nucleic acids are annealed to the surface-bound nucleic acids in less than or equal to about 15 seconds, 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes or 120 minutes. In some embodiments, at least 80% of the target nucleic acids anneal to the surface-bound nucleic acids in less than or equal to about 15 seconds, 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, or 120 minutes. In some embodiments, at least 90% of the target nucleic acids anneal to the surface-bound nucleic acids in greater than or equal to about 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes, or 5 minutes. In some embodiments, at least 90% of the target nucleic acids anneal to the surface-bound nucleic acids in the range of about 10 seconds to about 1 hour, about 30 seconds to about 50 minutes, about 1 minute to about 50 minutes, or about 1 minute to about 30 minutes. In some embodiments, at least 90% of the target nucleic acids anneal to the surface-bound nucleic acids within 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 minutes.

[0200] Improvement in hybridization efficiency: As used herein, hybridization efficiency (or yield) is a measure of the percentage of total available bound adapter sequences, primer sequences, or oligonucleotide sequences that hybridize to complementary sequences on a solid surface. In some cases, use of the optimized buffer formulations disclosed herein (optionally, used in combination with a low non-specific binding surface) results in improved hybridization efficiency compared to standard hybridization protocols. In some cases, the hybridization efficiency achievable in any of the hybridization reaction times specified above is better than 80%, 85%, 90%, 95%, 98% or 99%.

[0201] The methods and compositions described herein can be used for isothermal annealing conditions. In some embodiments, the methods described herein can eliminate the cooling required for most hybridizations. In some embodiments, the annealing 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 less than about 40°C, 50°C, 60°C, 70°C, 80°C, or 90°C.

[0202] Improvement of hybridization specificity: Compared with the comparable hybridization reaction carried out with 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 carried out on the low non-specific binding surface described herein at 90 degrees Celsius in a buffer comprising saline-sodium citrate for 5 minutes, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius. In some cases, the hybridization specificity that can be achieved is better than 1 base mismatch in 10 hybridization events, 1 base mismatch in 100 hybridization events, 1 base mismatch in 1,000 hybridization events, or 1 base mismatch in 10,000 hybridization events. Hybridization specificity can be measured using the technology described herein.

[0203] In some cases, at least or about 70%, 80%, or 90% of the sample nucleic acid molecules correctly hybridize with the capture nucleic acid molecules (e.g., adapter sequences, primer sequences, or oligonucleotide sequences) having complementary sequences. In some cases, greater than 90% of the sample nucleic acid molecules correctly hybridize with the capture nucleic acid molecules. In some cases, 90%-99% of the sample nucleic acid molecules correctly hybridize with the capture nucleic acid molecules. In some cases, 100% of the sample nucleic acid molecules correctly hybridize with the capture nucleic acid molecules.

[0204] Can be by (for example, Cy3) complementary oligonucleotide of mark and be fixed to the surface binding nucleic acid molecule hybridization on the surface, dehybridize and collect the oligonucleotide of hybridization, under suitable excitation and emission wavelength (for example, 532, peak 570 / 30), use fluorescence microplate reader to measure the fluorescent signal from the oligonucleotide of collection, measure hybridization specificity.The result is used to draw standard curve, and measure accurate concentration.Can repeat this mensuration with the oligonucleotide that demonstrates complementarity in different degree and each specificity.

[0205] The hybridization specificity measured on the surface can be measured by dividing the nonspecific background counts (e.g., calculated using the method provided in Example 3) by the nonspecific probe hybridization-nonspecific background counts (also calculated using the method in Example 3). A calibration curve can be established, and experiments with oligonucleotides having different degrees of complementarity can be added to more accurately calculate the specificity of each.

[0206] The specificity p of a given nucleic acid probe can be quantified by the relative sensitivity when the p spot is exposed to a perfectly matched target t or a mismatch m,

[0207] The proportion of mis-hybridized probes P m To quantify the specificity of the assay. In this case, y=x(c m / c t )(K m / K t ).

[0208] Improvement of hybridization sensitivity. "Hybridization sensitivity" refers to the concentration range in which sample (or target) nucleic acid molecules hybridize with target hybridization specificity. In some cases, target hybridization specificity is 90% or higher. In some cases, the methods, systems, compositions and kits described herein utilize sample nucleic acid molecules less than 10 nanomolar concentrations to hybridize sample nucleic acid molecules with high specificity to capture nucleic acid molecules. In some cases, sample nucleic acid molecules with a concentration of 10 nanomolar to 50 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 9 nanomolar to 100 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 9 nanomolar to 150 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 7 nanomolar to 200 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 6 nanomolar to 250 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 5 nanomolar to 250 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 4 nanomolar to 300 picomolar are used. In some cases, sample nucleic acid molecules with a concentration of 3 nanomolar to 350 picomolar are used. In some cases, 2 nanomoles to 400 picomoles of sample nucleic acid molecules are used. In some cases, 1 nanomoles to 500 picomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 1 nanomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 250 picomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 200 picomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 150 picomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 100 picomoles of sample nucleic acid molecules are used. In some cases, less than or equal to about 50 picomoles of sample nucleic acid molecules are used.

[0209] In some cases, if the hybridization sensitivity calculated by the International Union of Pure and Applied Chemistry (IUPAC) is used, the sensitivity S e The calibration curve describes the response of the target concentration c t The measured response R, R(c t ), and S e =dR / dc t Then, the quantitative resolution of the determination is Δc t By Δc t =∈ r (c t ) / S e (c t ) specifies, where ∈ r is the measurement error given by its standard deviation. Detection limit, the lowest detectable c t , by Δc t (c t =0) is determined because when the concentration c t Below Δct (c t =0), the error is greater than the signal; and R(ct) is assumed to be proportional to the equilibrium hybridization fraction x of the surface; that is, R(ct) = κx + const, where κ is a constant. This assumption is reasonable when the following conditions are met: (1) nonspecific adsorption is negligible and R is determined only by hybridization at the surface; (2) the experimental time is long enough for hybridization to reach equilibrium; and (3) the measured signal is linearly related to the amount of oligonucleotide at the surface.

[0210] Nucleic Acid Sequencing Applications

[0211] Nucleic acid sequencing is one of many applications in which the methods, compositions, systems and kits described herein can be used. Figure 2 In some embodiments, the methods disclosed herein include preparing a sample nucleic acid molecule library for sequencing, hybridizing the sample nucleic acid library with a nucleic acid molecule coupled to a low non-specific binding surface in the presence of a hybridization composition described herein, amplifying the sample nucleic acid library in situ, optionally linearizing the amplified sample nucleic acid in situ, dehybridizing the linearized and amplified sample nucleic acid with a nucleic acid molecule coupled to a low non-specific binding surface, hybridizing a primer sequence with the sample nucleic acid, and sequencing the sample nucleic acid.

[0212] refer to Figure 6 , preparing a sample nucleic acid molecule library 601, for example by a split-ligation scheme, hybridizing the sample nucleic acid molecule library with a nucleic acid molecule coupled to a low non-specific binding surface in the presence of a hybridization composition described herein 602, hybridizing the sample nucleic acid molecule with a nucleic acid molecule coupled to a low non-specific binding surface 603, hybridizing a sequencing primer with a complementary primer binding sequence on the sample nucleic acid 604, and sequencing the sample nucleic acid 605.

[0213] Figure 7 An exemplary sequencing workflow is provided in which labeled deoxynucleoside triphosphates (dNTPs) are bound to sample nucleic acid molecules to determine the identity of complementary nucleotides in the nucleic acid sequence of the sample nucleic acid molecules 701. In some cases, the dNTPs are directly labeled with a fluorophore (e.g., Cy3) or are labeled by interacting with a labeled detection reagent. The surface is optionally washed to remove unbound labeled dNTPs. The surface is imaged to detect the presence of labeled dNTPs 702. The labeled dNTPs are unbound from the sample nucleic acid molecules, and the blocked unlabeled dNTPs are incorporated into the sample nucleic acid molecules 703. The blocked unlabeled nucleotides are cleaved 704. Steps 701-704, 705 are repeated for the next nucleotide in the sample nucleic acid molecule.

[0214] The methods, compositions, systems and kits described herein provide at least the following advantages, particularly in nucleic acid sequencing processes: (i) reduced fluid wash times (due to reduced non-specific binding, thereby accelerating sequencing cycle times), (ii) reduced imaging time (thereby accelerating turnaround time for assay readout and sequencing cycles), (iii) reduced overall workflow time requirements (due to reduced cycle times), (iv) reduced detection instrumentation costs (due to improved contrast-to-noise ratios), (v) improved read (base calling) accuracy (due to improved contrast-to-noise ratios), (vi) improved reagent stability and reduced reagent usage requirements (thereby reducing reagent costs), and (vii) fewer runtime failures due to nucleic acid amplification failures.

[0215] definition

[0216] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0217] 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.

[0218] Any reference herein to "or" is intended to include "and / or" unless stated otherwise.

[0219] As used herein, the term "about" means plus or minus 10% of the number. When the term "about" is used in a range, it means the range minus 10% of its minimum and 10% of its maximum.

[0220] As used herein, unless otherwise indicated, the terms "DNA hybridization" and "nucleic acid hybridization" are used interchangeably and are intended to cover any type of nucleic acid hybridization, eg, DNA hybridization, RNA hybridization.

[0221] As used herein, the term "isothermal" refers to conditions in which the temperature remains substantially constant. A "substantially constant" temperature may deviate (e.g., increase or decrease) by no more than 0.25, 0.50, 0.75, or 1.0 degrees over a period of time.

[0222] The terms "annealing" or "hybridization" are used interchangeably herein and refer to the ability of two nucleic acid molecules to bind together. In some cases, "combination" refers to Watson-Crick base pairing between bases in each of the two nucleic acid molecules.

[0223] As used herein, "hybridization specificity" refers to a measure of the ability of a nucleic acid molecule (e.g., an adaptor sequence, primer sequence, or oligonucleotide sequence) to hybridize correctly to a region of a target nucleic acid molecule having a nucleic acid sequence that is completely complementary to that nucleic acid molecule.

[0224] As used herein, "hybridization sensitivity" refers to the concentration range of sample (or target) nucleic acid molecules in which hybridization occurs with high specificity. In some cases, the sample nucleic acid molecules in which high specific hybridization is achieved using the methods, compositions, systems and kits described herein are as low as 50 picomolar concentrations. In some cases, the range is between about 1 nanomolar to about 50 picomolar concentrations of sample nucleic acid molecules.

[0225] As used herein, "hybridization efficiency" refers to a measure of the percentage of total available nucleic acid molecules (e.g., adaptor sequences, primer sequences, or oligonucleotide sequences) that hybridize to a region of a target nucleic acid molecule having a nucleic acid sequence that is completely complementary to that nucleic acid molecule.

[0226] As used herein, the term "hybridization stringency" refers to the percentage of nucleotide bases that are complementary by standard Watson-Crick base pairing within at least a portion of a nucleic acid sequence that undergoes a hybridization (e.g., hybridization region) reaction. In a non-limiting example, 80% hybridization stringency means that a stable duplex can be formed, wherein 80% of the hybridization region undergoes Watson-Crick base pairing. Higher hybridization stringency means that a higher degree of Watson-Crick base pairing is required in a given hybridization reaction to form a stable duplex.

[0227] As used herein, unless otherwise indicated, the terms "isolate" and "purify" are used interchangeably herein.

[0228] abbreviation

[0229] Dimethyl sulfoxide (DMSO)

[0230] Dimethylformamide (DMF)

[0231] 3-(N-Morpholino)propanesulfonic acid (MOPS)

[0232] Acetonitrile (ACN)

[0233] 2-(N-Morpholino)ethanesulfonic acid (MES)

[0234] Saline-sodium citrate (SSC)

[0235] Formamide(Form.)

[0236] Tris(hydroxymethyl)aminomethane (Tris)

[0237] The present invention provides embodiments including but not limited to the following:

[0238] 1. A method for hybridizing a target nucleic acid molecule to a nucleic acid molecule coupled to a hydrophilic polymer surface, the method comprising:

[0239] (a) providing at least one nucleic acid molecule coupled to a hydrophilic polymer surface; and

[0240] (b) contacting the at least one nucleic acid molecule coupled to the polymer surface with a hybridization composition comprising the target nucleic acid molecule at a concentration of 1 nanomolar or less under conditions sufficient to allow the target nucleic acid molecule to hybridize to the at least one nucleic acid molecule coupled to the polymer surface within 30 minutes or less.

[0241] 2. The method of embodiment 1, wherein the hydrophilic polymer surface has a water contact angle of less than 45 degrees.

[0242] 3. The method of embodiment 1 or 2, wherein the conditions are maintained at a substantially constant temperature.

[0243] 4. The method of embodiment 3, wherein the target nucleic acid molecule is present in the hybridization composition at a concentration of 0.50 nanomolar or less.

[0244] 5. The method of embodiment 4, wherein the target nucleic acid molecule is present in the hybridization composition at a concentration of 250 picomolar or less.

[0245] 6. The method of embodiment 5, wherein the target nucleic acid molecule is present in the hybridization composition at a concentration of 100 picomolar or less.

[0246] 7. The method according to any one of embodiments 1-4, wherein 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.

[0247] 8. A method according to embodiment 7, wherein the time period is less than 20 minutes.

[0248] 9. The method of embodiment 8, wherein the time period is less than 15 minutes.

[0249] 10. The method of embodiment 9, wherein the time period is less than 10 minutes.

[0250] 11. The method of embodiment 10, wherein the time period is less than 5 minutes.

[0251] 12. The method of any one of embodiments 1-11, further comprising hybridizing the target nucleic acid molecule with the at least one nucleic acid molecule coupled to the polymer surface with an increased hybridization efficiency compared to a comparable hybridization reaction, wherein the comparable hybridization reaction is performed in a buffer comprising saline-sodium citrate for 120 minutes, at 90 degrees Celsius for 5 minutes, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius.

[0252] 13. The method of any one of embodiments 1-12, wherein the temperature is about 30 degrees Celsius to 70 degrees Celsius.

[0253] 14. The method of embodiment 13, wherein the temperature is about 50 degrees Celsius.

[0254] 15. The method according to any one of embodiments 1-14, further comprising hybridizing the target nucleic acid molecule to the at least one nucleic acid molecule with a hybridization stringency of at least 80%.

[0255] 16. The method of any one of embodiments 1-15, wherein the hydrophilic polymer surface exhibits a non-specific cyanine 3 dye adsorption level of less than about 0.25 molecules / micrometer squared.

[0256] 17. The method according to any one of embodiments 1 to 16, wherein the hybridization composition further comprises:

[0257] (a) at least one organic solvent having a dielectric constant of no greater than about 115 when measured at 68 degrees Fahrenheit; and

[0258] (b) pH buffer.

[0259] 18. The method according to any one of embodiments 1 to 16, wherein the hybridization composition further comprises:

[0260] (a) at least one organic solvent that is polar and aprotic; and

[0261] (b) pH buffer.

[0262] 19. The method according to embodiment 17 or 18, wherein the at least one organic solvent comprises at least one functional group selected from the group consisting of hydroxyl, nitrile, lactone, sulfone, sulfite, and carbonate.

[0263] 20. The method of embodiment 19, wherein the at least one organic solvent comprises formamide.

[0264] 21. The method according to embodiment 17 or 18, wherein the at least one organic solvent is miscible with water.

[0265] 22. The method of embodiment 17 or 18, wherein the at least one organic solvent is at least about 5 volume percent based on the total volume of the hybridization composition.

[0266] 23. The method of embodiment 22, wherein the at least one organic solvent is up to about 95 volume percent based on the total volume of the hybridization composition.

[0267] 24. The method of embodiment 17 or 18, wherein the pH buffer is at most about 90 volume percent of the total volume of the hybridization composition.

[0268] 25. The method of embodiment 17 or 18, wherein the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or a combination thereof.

[0269] 26. The method of embodiment 17 or 18, wherein the pH buffer further comprises a second organic solvent.

[0270] 27. A method according to embodiment 17 or 18, wherein the pH buffer is present in the hybridization composition in an amount effective to maintain the pH of the hybridization composition in the range of about 3 to about 10.

[0271] 28. A method according to any one of embodiments 1-27, wherein the hybridization composition further comprises a molecular clustering agent.

[0272] 29. The method according to embodiment 28, wherein the molecular clustering agent is selected from polyethylene glycol, dextran, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethyl cellulose and any combination thereof.

[0273] 30. The method of embodiment 29, wherein the molecular clustering agent is polyethylene glycol.

[0274] 31. The method of any one of embodiments 28-30, wherein the molecular clustering agent has a molecular weight in the range of about 5,000 to 40,000 Daltons.

[0275] 32. The method of any one of embodiments 28-31, wherein the amount of the molecular clustering agent is at least about 5 volume percent based on the total volume of the hybridization composition.

[0276] 33. The method according to any one of embodiments 28-32, wherein the amount of the molecular clustering agent is at most about 50 volume percent based on the total volume of the hybridization composition.

[0277] 34. The method of any one of embodiments 1-33, wherein the at least one nucleic acid molecule coupled to the polymer surface is coupled to the polymer surface by covalent bonding.

[0278] 35. The method of any one of embodiments 1-33, wherein the hydrophilic polymer surface comprises one or more hydrophilic polymer layers, and wherein the at least one nucleic acid molecule is coupled to the one or more hydrophilic polymer layers.

[0279] 36. A method according to embodiment 35, wherein the one or more hydrophilic polymer layers comprise molecules selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin and dextran.

[0280] 37. The method according to any one of embodiments 35-36, wherein the one or more hydrophilic polymer layers comprise at least one dendritic polymer.

[0281] 38. A method 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 the capture probe coupled thereto under conditions sufficient to allow the target nucleic acid molecule to be captured by the capture probe in a period of time less than 30 minutes.

[0282] 39. The method of embodiment 38, wherein the mixture comprises a polar aprotic solvent.

[0283] 40. The method of any one of embodiments 38-39, wherein the polar aprotic solvent comprises formamide.

[0284] 41. The method of any one of embodiments 38-40, wherein the capture probe is a nucleic acid molecule.

[0285] 42. The method of any one of embodiments 38-41, wherein the concentration is 0.50 nanomolar or less.

[0286] 43. The method of embodiment 42, wherein the concentration is 250 picomolar or less.

[0287] 44. The method of embodiment 43, wherein the concentration is 100 picomolar or less.

[0288] 45. A method according to any one of embodiments 38-44, wherein the time period is less than or equal to 20 minutes.

[0289] 46. ​​A method according to embodiment 45, wherein the time period is less than or equal to 15 minutes.

[0290] 47. A method according to embodiment 46, wherein the time period is less than or equal to 10 minutes.

[0291] 48. A method according to embodiment 47, wherein the time period is less than or equal to 5 minutes.

[0292] 49. The method of any one of embodiments 38-48, wherein the hydrophilic surface is maintained at a temperature of about 30 degrees Celsius to about 70 degrees Celsius.

[0293] 50. The method of any one of embodiments 38-49, wherein the hydrophilic surface is maintained at a substantially constant temperature.

[0294] 51. The method according to any one of embodiments 38-50 further comprises hybridizing the target nucleic acid molecule to the capture probe with an increased hybridization efficiency compared to a comparable hybridization reaction, wherein the comparable hybridization reaction is carried out in a buffer composition comprising saline-sodium citrate for 120 minutes, at 90 degrees Celsius for 5 minutes, and then cooled for 120 minutes to reach a final temperature of 37 degrees Celsius.

[0295] 52. The method according to any one of embodiments 38-51 further comprises hybridizing the target nucleic acid molecule to the capture probe with a hybridization stringency of at least 80%.

[0296] 53. The method of any one of embodiments 38-52, wherein the hydrophilic surface exhibits a non-specific cyanine 3 dye adsorption level of less than about 0.25 molecules / micrometer squared.

[0297] 54. A method according to any one of embodiments 38-53, wherein the mixture further comprises a pH buffer, and the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol or a combination thereof.

[0298] 55. A method according to any one of embodiments 38-54, wherein the mixture further comprises a clustering agent, and the clustering agent is selected from polyethylene glycol, dextran, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, hydroxybutyl methylcellulose, hydroxypropyl cellulose, methylcellulose and hydroxymethylcellulose, and any combination thereof.

[0299] 56. A method according to any one of embodiments 38-55, wherein the hydrophilic surface comprises one or more hydrophilic polymer layers.

[0300] 57. A method according to embodiment 56, wherein the one or more hydrophilic polymer layers comprise molecules selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin and dextran.

[0301] 58. The method according to embodiment 56, wherein the one or more hydrophilic polymer layers comprise at least one dendritic polymer.

[0302] Example

[0303] These examples are provided for illustrative purposes only, and not to limit the scope of the claims provided herein.

[0304] Example 1 - DNA hybridization on low non-specific binding surfaces

[0305] Figure 1A-1B Examples of optimized hybridization achieved on low binding surfaces using the disclosed hybridization methods are provided ( Figure 1A ), the concentration of hybridization reporter probes was reduced and the hybridization time was shortened compared to the results obtained using traditional hybridization protocols on the same low-binding surface ( Figure 1B ).

[0306] Figure 1A Hybridization reactions on low binding surfaces according to embodiments described herein are shown. These rows provide two tested hybridization conditions, hybridization condition 1 ("Hyb 1") and hybridization condition 2 ("Hyb 2"). Hyb 1 refers to hybridization buffer composition C10 in Table 1. Hyb 2 refers to hybridization buffer composition D18 in Table 1. Figure 1AHybridization reporter probes (complementary oligonucleotide sequences labeled with CyTM3 fluorophore at the 5' end) at the concentrations reported in the assay (10 nM, 1 nM, 250 pM, 100 pM and 50 pM) were hybridized in a buffer composition at 60 degrees Celsius for 2 minutes.

[0307] Figure 1B Hybridization reactions on low binding surfaces according to a standard hybridization protocol with standard hybridization conditions ("Standard Hyb Conditions") are shown. Standard hybridization buffers of 2X-5X saline-sodium citrate (SSC) and the same concentrations as above (eg Figure 1A The standard hybridization reaction was performed at 90 degrees Celsius with a slow cooling process (2 hours) to reach 37 degrees Celsius.

[0308] for Figure 1A and Figure 1B In each hybridization reaction provided in FIG, the top row of each hybridization reaction is the test ("T"), which is the complementary oligonucleotide (e.g., CY3 TM -5'-ACCCTGAAAGTACGTGCATTACATG-3'), and the bottom row of each hybridization reaction is a control ("C"), which is non-complementary (e.g., CY3 TM -5'-ATGTCTATTACGTCACACTATTATG-3').

[0309] The surface for all test conditions was a surface having a relative density corresponding to less than or equal to about 0.25 molecules / μm 2 Ultra-low non-specific binding surface with low non-specific Cy3 dye adsorption level. In this example, the low non-specific binding surface used was a glass substrate functionalized with Silane-PEG-5K-COOH (Nanocs Inc.).

[0310] After the hybridization reaction was completed, the wells were washed with 50 mM Tris (pH 8.0); 50 mM NaCl.

[0311] While immersing the sample in buffer (25 mM A CES, pH 7.4 buffer), an inverted microscope (Olympus IX83) equipped with a 100X TIRF objective (NA=1.4) (Olympus), a dichroic mirror optimized for 532 nm light (Semrock, Di03-R532-t1-25x36), a bandpass filter optimized for Cy3 emission (Semrock, FF01-562 / 40-25), and a camera (sCMOS, Andor Zyla) was used for 1 sec under non-signal saturating conditions (Laser Quantum, Gem 532, <1 W / cm at the sample). 2) to obtain fluorescence images. Images were collected as described above and the results are displayed Figure 1A (optimization) and Figure 1B (Standard) Medium.

[0312] Compared to the negative control, significant signals were observed from reactions with 250 picomolar (pM) in both Hyb 1 and Hyb 2 hybridization reactions ( Figure 1A ). In contrast, no signal was observed from the reaction with 250 pM under standard Hyb conditions compared to the negative control. The same results were observed for hybridization reporter probes at lower input concentrations (e.g., 100 pM, 50 pM). Figure 1A The results show that the input DNA (labeled oligonucleotides) required for specific DNA capture on the tested low non-specific binding surfaces was reduced by more than 200-fold, the hybridization time was reduced by 50-fold, and the hybridization temperature was reduced by half ( 1.5 fold) compared to using standard hybridization methods and reagents on the same low non-specific binding substrate. Figure 1B ). The buffer compositions and methods described herein have improved hybridization specificity, reduced workflow time, and increased hybridization sensitivity.

[0313] Example 2

[0314] Buffer compositions according to various embodiments described herein are optimized to promote hybridization of single template oligonucleotide fragments to low non-specific binding surfaces described herein.

[0315] Prepare low non-specific binding surface. Glass substrate (175um 22x 60mm 2 , Corning Glass) was cleaned with KOH and ethanol. Low binding glass surfaces were prepared by incubating Silane-PEG5K-NHS (Nanocs) in ethanol at 65 degrees for 30 minutes. In a mixture of 1 micromolar (uM), 5.1 uM and 46 uM oligonucleotides in methanol / phosphate buffer, NH with 5' modification was added. 2 Oligonucleotides were grafted onto these surfaces for 20 minutes to form immobilized oligonucleotides coupled to the glass substrate.

[0316] Circularization of single template oligonucleotide fragments into the library. Single template oligonucleotide fragments (about 100 base pairs in length) were circularized using a splint ligation protocol containing fragments complementary to the surface grafted primers.

[0317] The circularized library was hybridized to the immobilized oligonucleotide. After library circularization, the circularized library fragments were added to the various test hybridization test mixtures shown in rows BF at a concentration of 100 picomolar (pM). Separate buffer / library hybridization mixtures were added to a 384-well plate where the functionalized surface was fixed at 50 degrees Celsius for 4 minutes.

[0318] Hybridization was visualized using the assay buffer composition. An intercalating DNA dye was added to the buffer / library hybridization mixture after the hybridization reaction to visualize hybridization of the circularized library. The 384-well plate was imaged using a fluorescence microscope and 488 nanometer (nm) excitation with a 60x water immersion objective (1.2NA, Olympus) (see Figure 3 A number of buffer compositions were tested for hybridization of target nucleic acids (e.g., circularized libraries) to surface-bound nucleic acids (e.g., immobilized oligonucleotides). Table 1 provides Figure 3 The buffer composition and fixed oligonucleotide concentration for each reaction are shown in Table 1. Columns 10-21 correspond to Figure 3 , and row BF corresponds to columns 10-21 of Figure 3 F10 and F11 are negative controls using standard hybridization conditions, where no background signal was detected, demonstrating the effectiveness of the negative controls and the low nonspecific binding properties of the test surface.

[0319] Table 1. Buffer compositions tested for hybridization of target nucleic acids to surface-bound nucleic acids

[0320]

[0321]

[0322] The "grafted" concentration refers to the concentration of surface-bound oligonucleotides. Spot counts for each hybridization condition were tabulated, whereby higher counts indicated a more efficient hybridization buffer formulation, e.g. Figure 4 Table 1 provides Figure 4 The buffer composition and fixed oligonucleotide concentration for each reaction are shown in Table 1. Columns 10-21 correspond to Figure 4 , and row BF corresponds to columns 10-21 of Figure 4 The BFth row of .

[0323] The hybridized target nucleic acid was amplified using surface-bound nucleic acid. After hybridization, the target nucleic acid was amplified to quantify the hybridization effectiveness. The hybridization was performed according to the manufacturer's instructions (New England Biolabs). ), using an amplification mixture containing Bst for rolling circle amplification (RCA). These amplified target nucleic acid colonies are further amplified using an RCA / PCR amplification strategy, whereby PCR cycles are performed on RCA polymer nanospheres to increase the detection sensitivity of the assay and more stringently quantify the hybridization library.

[0324] The resulting surface amplification products were stained again with an embedded DNA dye and imaged to verify hybridization specificity and effectiveness (see Figure 5 Table 1 provides Figure 5The buffer composition and fixed oligonucleotide concentration for each reaction are shown in Table 1. Columns 10-21 correspond to Figure 5 , and row BF corresponds to columns 10-21 of Figure 5 The BFth row of .

[0325] Analysis of hybridization buffers and conditions. Hybridization conditions were based on Figure 3 , Figure 4 and Figure 5 The correlation with the maximum spot count was evaluated. Figure 4 Hybridization buffers C10, D18, and E21 showed the highest spot counts compared to the negative controls provided in F10 and F11, in which water was used instead of hybridization buffer. Figure 5 Verified in.

[0326] Example 3

[0327] In this example, nonspecific binding of cyanine 3 dye (Cy3)-labeled molecules was measured on the low nonspecific binding surfaces disclosed herein. In a separate nonspecific binding assay, 1 uM of labeled Cy3 dCTP (GE Amersham), 1 uM of Cy5 dGTP dye (Jena Biosciences), 10 uM of aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 uM of aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 uM of aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 uM of cCTP-Cy3.5 (GE Amersham) and 10 uM of 7-propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated separately on the low nonspecific binding surface described in Example 2 (glass substrate treated with silane-PEG5K, Nanocs) in a 384-well plate format at 37°C for 15 minutes. Each well was rinsed 2-3 times with 50ul of RNase / DNase-free deionized water and 2-3 times with 25mM ACES buffer (pH7.4). The 384-well plate was imaged at single-molecule resolution on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with a TIRF objective (100X, 1.4NA, Olympus), a sCMOS camera (Zyla4.2, Andor), and an illumination source with an excitation wavelength of 532nm or 635nm. The dichroic mirror was purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), such as a 405, 488, 532 or 633nm dichroic mirror / beam splitter, and the bandpass filter was selected as 532LP or 645LP, which is consistent with the appropriate excitation wavelength. 5.

[0328] The imaging device enables visualization of individual dye molecules bound to the substrate. Individual fluorescent spots are counted and the total number of spots is divided by the corresponding ROI area. For example, using a 100x objective and an Andor sCMOS camera with a pixel size of 6.5 microns, the area of ​​the region of interest (ROI) can be calculated.

[0329] Low non-specific binding of dye molecules less than or equal to about 0.50 molecules / μm2 or more was observed. Some non-specific binding of dye molecules less than or equal to 0.25 molecules / μm2 was observed.

[0330] Example 4

[0331] The hybridization compositions and methods disclosed in Examples 1 and 2 were used on the surfaces used in Examples 1-3. Figure 2 The nucleic acid sequencing reaction was performed according to the workflow provided in . In this non-limiting example, the processing time achieved is also Figure 2 Available in.

[0332] Although preferred embodiments of the compositions and methods disclosed herein have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present disclosure, those skilled in the art will now appreciate many variations, changes, and substitutions. It should be understood that, when practicing the methods and compositions of the present disclosure, various alternatives to the embodiments of the methods and compositions described herein may be employed in any combination.

Claims

1. A method for hybridizing a target nucleic acid molecule with a nucleic acid molecule coupled to a hydrophilic polymer surface, the method comprising: include: (a) providing at least one nucleic acid molecule coupled to a hydrophilic polymer surface; and (b) contacting the at least one nucleic acid molecule coupled to the polymer surface with a hybridization composition comprising the target nucleic acid molecule at a concentration of 1 nanomolar or less under conditions sufficient to allow the target nucleic acid molecule to hybridize to the at least one nucleic acid molecule coupled to the polymer surface within 30 minutes or less.

2. The method of claim 1, wherein the hydrophilic polymer surface has a water contact angle of less than 45 degrees.

3. The method of claim 1 or 2, wherein the conditions are maintained at a substantially constant temperature. The method of claim 3 , wherein the target nucleic acid molecule is present in the hybridization composition at a concentration of 0.50 nanomolar or less.

5. The method of claim 4, wherein the target nucleic acid molecule is present in the hybridization composition at a concentration of 250 picomolar or less. The method of claim 5 , wherein the target 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 to 4, wherein 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. The method of claim 7 , wherein the period of time is less than 20 minutes.

9. The method of claim 8, wherein the time period is less than 15 minutes.

10. A method for attaching a target nucleic acid molecule to a surface, the method comprising: include: A mixture comprising said target nucleic acid molecules at a concentration of 1 nanomolar or less is contacted with a hydrophilic surface comprising said capture probes coupled thereto under conditions sufficient to allow said target nucleic acid molecules to be captured by the capture probes in a period of time less than 30 minutes.

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