KR20210144929A - Multivalent Binding Compositions for Nucleic Acid Analysis - Google Patents

Abstract

Multivalent binding compositions comprising a particle-nucleotide conjugate having a plurality of copies of nucleotides attached to the particle are described. The multivalent binding composition enables localization of a detectable signal to an active region of a biochemical interaction, e.g., a site of a protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and a polymerase reaction. It can be used to identify sites of base incorporation in nucleic acid chains that extend during nucleic acid chains and to provide improved base identification for sequencing and array-based applications.

Description

Multivalent Binding Compositions for Nucleic Acid Analysis

cross reference

This application is a continuation-in-part of U.S. Patent Application No. 16/579,794, filed September 23, 2019, U.S. Provisional Patent Application No. 62/897,172, filed September 6, 2019, and U.S. Provisional Patent Application No. 62/852,876, each of which is incorporated herein by reference in its entirety.

field of invention

The present invention relates generally to multivalent binding compositions and their use for use in the analysis of nucleic acid molecules. In particular, the present invention relates to multivalent binding compositions having multiple copies of nucleotides attached to a particle or polymer core that effectively increase the local concentration of nucleotides and enhance the binding signal. The multivalent binding composition can be applied in, for example, sequencing and biosensor microarray fields.

Nucleic acid sequencing can be used to obtain information in a variety of biomedical contexts, including diagnostic, prognostic, biotechnology and forensic biology. Maxam-Gilbert sequencing and chain-termination methods, or de novo sequencing methods including shotgun sequencing and bridge PCR, or polony sequencing, 454 pyrosequencing (454 pyrosequencing), Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single molecule sequencing, SMRT® sequencing, and others. A variety of sequencing methods have been developed, including next-generation methods, including Despite advances in DNA sequencing, many challenges to cost-effective high-throughput sequencing remain unresolved. The present invention provides novel solutions and approaches that address many of the shortcomings of the prior art.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method comprising the steps of: a. providing a composition comprising: i. two or more copies of the target nucleic acid sequence; ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and iii. two or more polymerase molecules; b. Under conditions sufficient to form a multivalent binding complex between the polymer nucleotide conjugate and the two or more copies of the target nucleic acid sequence of the composition of (a), the composition is contacting the polymer nucleotide conjugate, wherein the polymer-nucleotide conjugate comprises two or more copies of a nucleotide moiety and optionally one or more detectable labels; and c. detecting the multivalent binding complex to determine the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the step of detecting the multivalent binding complex is performed in the absence of an unbounded or solution-borne polymer nucleotide conjugate. In some embodiments, the target nucleic acid sequence has been replicated or amplified or has been generated by replication or amplification. In some embodiments, the at least one detectable label is a fluorescent label. In some embodiments, detecting the multivalent complex comprises fluorescence measurement. In some embodiments, the contacting step comprises the use of one type of polymer-nucleotide conjugate. In some embodiments, the contacting step comprises the use of two or more types of polymer-nucleotide conjugates. In some embodiments, each type of two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the contacting step comprises the use of three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotides are 3'-O-azidomethyl nucleotides, 3'-O-methyl (3-O-Methyl) nucleotides, or 3'-0-alkyl hydrogen It is a 3'-O-alkyl hydroxylamine nucleotide. In some embodiments, said contacting occurs in the presence of ions that stabilize said multivalent binding complex. In some embodiments, the contacting step is in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule has been made catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule has been rendered catalytically inactive by the absence of a required ion or cofactor. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. In some embodiments, the multivalent binding complex has a duration greater than 2 seconds. In some embodiments, the method may be performed at a temperature within the range of 25 °C to 62 °C. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, wherein the two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to the surface, wherein the fluorescent image of the multivalent binding complex on the surface is detected The contrast to noise ratio is greater than 20 in the step. In some embodiments, the composition of step (a) is deposited on a surface using a buffer incorporating a polar aprotic solvent. In some embodiments, the contacting step stabilizes the multivalent binding complex if the nucleotide moiety is complementary to the next base of the target nucleic acid sequence, wherein the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence. In this case, it is carried out under conditions that destabilize the multivalent binding complex. In some embodiments, said polymer-nucleotide conjugate comprises a polymer having a plurality of branches, wherein said two or more nucleotide moieties are attached to said branches. In some embodiments, the polymer has a star, comb, cross-linked, bottle brush, or dendrimer morphology. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tag, and combinations thereof. In some embodiments, the method further comprises a dissociation step destabilizing the multivalent binding complex formed between the composition of step (a) and the polymer-nucleotide conjugate, wherein the dissociation step comprises removal of the polymer-nucleotide conjugate. make it possible In some embodiments, the method further comprises an extension step incorporating into said two or more primer nucleic acid molecules a nucleotide complementary to the next base of the target nucleic acid sequence. In some embodiments, the extending step occurs concurrently with or after the dissociating step.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method comprising the steps of: a. providing a composition comprising: i. two or more copies of the target nucleic acid sequence; ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and iii. two or more polymerase molecules; b. contacting said composition with said polymeric nucleotide conjugate under conditions sufficient to allow a multivalent binding complex to form between said polymer-nucleotide conjugate and said two or more copies of said target nucleic acid sequence of said composition of (a); , wherein the polymer-nucleotide conjugate comprises at least two copies of a reversibly terminated nucleotide moiety and optionally at least one cleavable and detectable label; and c. detecting the multivalent complex to determine the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the method comprises contacting the composition of step (a) with a polymer-nucleotide conjugate comprising a reversibly terminated nucleotide or two or more copies of a reversibly terminated nucleotide after detection of said multivalent binding complex. additionally include In some embodiments, the target nucleic acid sequence has been replicated or amplified or has been generated by replication or amplification. In some embodiments, the at least one detectable label is a fluorescent label. In some embodiments, detecting the multivalent complex comprises fluorescence measurement. In some embodiments, the contacting step comprises the use of one type of polymer-nucleotide conjugate. In some embodiments, the contacting step comprises the use of two or more types of polymer-nucleotide conjugates. In some embodiments, each type of two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the contacting step comprises the use of three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is 3'-0-azidomethyl, 3'-0-methyl, or 3'-0-alkyl hydroxylamine. In some embodiments, said contacting occurs in the presence of ions that stabilize said multivalent binding complex. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule has been made catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. In some embodiments, the method may be performed at a temperature within the range of 25 °C to 80 °C. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels, wherein the two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to the surface, wherein the fluorescent image of the multivalent binding complex on the surface is detected The contrast-to-noise ratio in the step is greater than 20.

Also disclosed herein is a system comprising: a) one or more computer processors individually or collectively programmed to implement a method comprising the steps of: i) tethered to the surface of a substrate contacting a substrate comprising multiple copies of a target nucleic acid sequence with a reagent comprising a polymerase and one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence to form a primed target nucleic acid sequence; ii) contacting the substrate surface with a reagent comprising the polymer nucleotide conjugate under conditions sufficient to permit the formation of a multivalent binding complex between the polymer-nucleotide conjugate and two or more copies of the primed target nucleic acid sequence; , wherein the polymer-nucleotide conjugate comprises two or more copies of a known nucleotide moiety and a detectable label; iii) acquiring and processing an image of the substrate surface to detect the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence. In some embodiments, the system further comprises a fluid module configured to deliver a series of reagents to the substrate surface in a designated order and for a designated time interval. In some embodiments, the system further comprises an imaging module configured to acquire an image of the substrate surface. In some embodiments, steps (ii) and (iii) are repeated two or more times to determine the identity of a series of two or more nucleotides in the target nucleic acid sequence. In some embodiments, the sequence of steps further comprises a dissociation step destabilizing the multivalent binding complex, wherein the dissociation step enables removal of the polymer-nucleotide conjugate. In some embodiments, the sequence of steps further comprises an extension step incorporating into said two or more primer nucleic acid molecules a nucleotide complementary to the next base of the target nucleic acid sequence. In some embodiments, the extending step occurs concurrently with or after the dissociating step. In some embodiments, the detectable label comprises a fluorophore and the image comprises a fluorescence image. In some embodiments, the fluorescence image of the multivalent conjugate complex on the substrate surface is cyanine dye 3 (Cy3), wherein the fluorophore is cyanine dye 3 (Cy3), 20X objective, NA = 0.75, dichroic mirror optimized for 532 nm light, cyanine dye- 3 Using an inverted fluorescence microscope equipped with a bandpass filter optimized for emission, and a camera, the contrast-to-noise ratio was is greater than 20. In some embodiments, the series of steps is completed in less than 60 minutes. In some embodiments, the series of steps is completed in less than 30 minutes. In some embodiments, the series of steps are completed in less than 10 minutes. In some embodiments, the accuracy of the base determination is characterized by a Q-score greater than 25 for at least 80% of the determined nucleotide entities. In some embodiments, the accuracy of the base determination is characterized by a Q-score greater than 30 for at least 80% of the determined nucleotide entities. In some embodiments, the accuracy of the base determination is characterized by a Q-score greater than 40 for at least 80% of the determined nucleotide entities.

Disclosed herein is a composition comprising: a) a polymeric core; and b) two or more nucleotides, nucleotide analogues, nucleosides, or nucleoside analogue moieties attached to the polymer core; wherein the length of the linker depends on the nucleotide, nucleotide analogue, nucleoside, or nucleoside analogue moiety attached to the polymer core. Also disclosed herein is a composition comprising: a) a mixture of polymer-nucleotide conjugates, wherein each polymer-nucleotide conjugate comprises: i) a polymer core; and ii) at least two nucleotide, nucleotide analog, nucleoside, or nucleoside analog moieties attached to the polymer core, wherein the length of the linker is the nucleotide, nucleotide analog, nucleoside analog moiety attached to the polymer core. depending on the cleoside, or nucleoside analog moiety; wherein the mixture comprises a polymer-nucleotide conjugate having at least two different types of attached nucleotides, nucleotide analogues, nucleosides, or nucleoside analogue moieties. In some embodiments, the polymeric core comprises a polymer having a plurality of branches, wherein two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties are attached to the branches. In some embodiments, the polymer has a star, comb, crosslinked, bottle brush, or dendrimer shape. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the polymeric core comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotides are 3'-0-azidomethyl nucleotides, 3'-0-methyl nucleotides, or 3'-0-alkyl hydroxylamine nucleotides. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels.

In some embodiments, the present specification provides a method of determining the identity of a nucleotide in a target nucleic acid, the method comprising, regardless of any particular sequence of operations: 1) providing a composition comprising: Step: a target nucleic acid comprising two or more repeats of the same sequence; two or more primer nucleic acids complementary to one or more regions of the target nucleic acid; and two or more polymerase molecules; 2) contacting said composition with a multivalent binding or incorporation composition comprising a polymer-nucleotide conjugate under conditions sufficient to allow formation of a bound or incorporated complex between said polymer-nucleotide conjugate and the composition of step (a); wherein the polymer-nucleotide conjugate comprises at least two copies of the nucleotide and optionally at least one detectable label; and 3) detecting the bound or incorporated complex, thereby establishing the identity of the nucleotide in the target nucleic acid polymer. In some further embodiments, the methods provided herein include, wherein the target nucleic acid is DNA, and/or wherein the target nucleic acid is, for example, rolling circle amplification, bridge amplification, helicase dependent amplification ( Any commonly practiced DNA replication or amplification method, such as helicase dependent amplification, isothermal bridge amplification, rolling circle multiple displacement amplification (RCA/MDA), and/or recombinase (recombinase)-based replication or amplification method. In some further embodiments, the methods provided herein are wherein the detectable label is a fluorescent label and/or the step of detecting the complex comprises fluorescence measurement. In some further embodiments, the methods provided herein include, wherein the multivalent binding composition comprises one type of polymer-nucleotide conjugate and the multivalent binding composition comprises two or more types of polymer-nucleotide conjugate; , and/or each type of two or more types of polymer-nucleotide conjugates is one comprising a different type of nucleotide. In some embodiments, the methods provided herein further comprise a blocked nucleotide, wherein the binding complex or the incorporated complex, in particular, the blocked nucleotide is 3'-O-azidomethyl nucleotide, 3'-O- alkyl hydroxylamino nucleotides, or 3'-O-methyl nucleotides. In some further embodiments, the methods provided herein are wherein the contacting step is in the presence of strontium ions, barium, magnesium ions, and/or calcium ions. In some embodiments, the methods provided herein include wherein the polymerase molecule is catalytically inactive, such as the polymerase molecule is catalytically inactive, by mutation, by chemical modification, or due to the absence of a required ion or cofactor. has become inactive. In some embodiments, the method also provided herein is wherein the polymerase molecule is catalytically active and/or the binding complex does not comprise blocked nucleotides. In some embodiments, the methods provided herein include, wherein the binding complex has a duration of greater than 2 seconds, and/or the method comprises at least 15 °C, at least 20 °C, at least 25 °C, at least 35 °C, at least 37 °C, and/or , 42 °C or higher, 55 °C or higher, 60 °C or higher, or 72 °C or higher, or at a temperature within the range defined by any one of the foregoing. In some embodiments, the methods provided herein are wherein the binding complex is deposited, adhered, or hybridized to a surface that exhibits a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, the methods provided herein are wherein the composition is deposited under buffer conditions incorporating a polar aprotic solvent. In some embodiments, the method provided herein stabilizes the binding complex when the nucleotide is complementary to the next base of the target nucleic acid, and when the nucleotide is not complementary to the next base of the target nucleic acid The contacting step is performed under conditions that destabilize the binding complex. In some embodiments, the method provided herein, wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches, and wherein the plurality of copies of the first nucleotide are attached to the branches, and in particular, the 1 The polymer has a star, comb, crosslinked, bottle brush, or dendrimer shape. In some embodiments, the methods provided herein are such that the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the method provided herein further comprises a dissociation step of destabilizing the binding complex formed between the composition of step (a) and the polymer-nucleotide conjugate, thereby removing the polymer-nucleotide conjugate. will include In some embodiments, the method provided herein further comprises an extension step of incorporating into said primer nucleic acid a nucleotide complementary to said next base of a target nucleic acid, optionally wherein said extending step is concurrent with said dissociation step or it will happen after that

In some embodiments, the present disclosure provides a composition comprising a branched polymer having two or more branches and two or more copies of a nucleotide, wherein said nucleotide is attached to a first plurality of said branches or arms; , optionally, one or more interacting moieties are attached to a second plurality of said branches or arms. In some embodiments, the composition may further comprise one or more labels on the polymer. In some embodiments, the compositions provided herein are those wherein the nucleosides have a surface density of at least 4 nucleotides per polymer. In some embodiments, the compositions provided herein comprise or incorporate modified nucleotides or nucleotide analogs to prevent incorporation into the extending nucleic acid chain during the polymerase reaction. In some embodiments, the composition may comprise or incorporate reversibly modified nucleotides or nucleotide analogs to prevent incorporation into the extending nucleic acid chain during the polymerase reaction. In some embodiments, the compositions provided herein are such that at least one label comprises a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more branches or arms, or 2, 4, 8, 16, 32, 64 or more, branches or arms. In some embodiments, a branch or arm may radiate from a central moiety. In some embodiments, the composition may comprise one or more interacting moieties, wherein the interacting moieties include avidin or streptavidin; biotin moieties; affinity tag; enzymes, antibodies, minibodies, receptors, or other proteins; non-protein tags; a metal affinity tag, or any combination thereof. In some embodiments, the compositions provided herein are those wherein the polymer comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid. In some embodiments, the compositions provided herein include: a nucleotide or nucleotide analog attached to a branch or arm via a linker; In particular, the linker comprises PEG, wherein the PEG linker moiety has an average molecular weight of about 1 K Da, about 2 K Da, about 3 K Da, about 4 K Da, about 5 K Da, about 10 K Da, about 15 K Da, about 20 K Da, about 50 K Da, about 100 K Da, about 150 K Da, or about 200 K Da, or greater than about 200 K Da. In some embodiments, the compositions provided herein, wherein the linker comprises PEG, wherein the PEG linker moiety has an average molecular weight of from about 5 K Da to about 20 K Da. In some embodiments, the compositions provided herein include, wherein at least one nucleotide or nucleotide analog comprises deoxyribonucleotides, ribonucleotides, deoxyribonucleosides, or ribonucleosides; and/or wherein the nucleotide or nucleotide analogue is conjugated to a linker via the 5' end of the nucleotide or nucleotide analogue. In some embodiments, the compositions provided herein comprise: deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5- methyl-uridine, and/or cytidine; Here, the length of the linker is 1 nm to 1,000 nm. In some embodiments, the composition provided herein comprises at least one nucleotide or nucleotide analogue modified to inhibit elongation during a polymerase reaction or a sequencing reaction, such as wherein at least one nucleotide or nucleotide analogue is 3' nucleotides that do not have a hydroxyl group; a nucleotide modified to include a blocking group at the 3' position; and/or 3'-O-azido group, 3'-O-azidomethyl group, 3'-O-alkyl hydroxylamino group, 3'-phosphorothioate group, 3'-O-malonyl group, or a nucleotide modified with a 3'-O-benzyl group. In some embodiments, in the compositions provided herein, at least one nucleotide or nucleotide analog that is not modified at the 3' position is a nucleotide.

In some embodiments, the present specification provides a method of determining the sequence of a nucleic acid molecule, the method comprising, regardless of any particular sequence of operations: 1) a template strand and at least partially complementary to the template strand providing a nucleic acid molecule comprising a complementary strand; 2) contacting the nucleic acid molecule with one or more nucleic acid binding compositions according to any of the embodiments disclosed herein; 3) detecting binding of the nucleic acid binding composition to the nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the present specification provides a method of determining the sequence of a nucleic acid molecule, the method comprising, regardless of any particular sequence of operations: 1) a template strand and at least partially complementary to the template strand providing a nucleic acid molecule comprising a complementary strand; 2) contacting the nucleic acid molecule with one or more nucleic acid binding compositions according to any of the embodiments disclosed herein; 3) detecting partial or complete incorporation of the nucleic acid binding composition to the nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule from the partial or complete incorporation of the embodiments described herein. decision step. In some embodiments, the methods provided herein include incorporation of the terminal nucleotide into the complementary strand, and repeating the contacting, detecting, and incorporation steps for one or more additional iterations, whereby the nucleic acid molecule It will further comprise the step of determining the sequence of the template strand. In some embodiments, the method provided herein comprises: the nucleic acid molecule is tethered to a solid support; In particular a solid support is one comprising a glass or polymer matrix, at least one hydrophilic polymer coating layer, and a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer. In some embodiments, the methods provided herein include: at least one hydrophilic polymer coating layer comprising PEG; and/or an embodiment wherein the at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least 8 branches. In some embodiments, the method provided herein comprises a method wherein the plurality of oligonucleotide molecules is at least 500 molecules/mm ² , at least 1,000 molecules/mm ² , at least 5,000 molecules/mm ² , at least 10,000 molecules/mm ² , at least 20,000 molecules/mm ² , at least 50,000 molecules/mm ² , at least 100,000 molecules/mm ² , or at least 500,000 molecules/mm ² . In some embodiments, in the method provided herein, the nucleic acid molecule is clonal amplified on a solid support. In some embodiments, the method provided herein comprises: clonal amplification comprises polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase dependent amplification, recombinase dependent amplification, single-strand binding (SSB) ) protein dependent amplification, or any combination thereof. In some embodiments, the methods provided herein include: one or more nucleic acid binding compositions are labeled with a fluorophore and the detecting step comprises the use of fluorescence imaging; In particular, fluorescence imaging is one that includes dual wavelength excitation/4 wavelength emission fluorescence imaging. In some embodiments, the methods provided herein include four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, wherein the composition is used to determine the identity of a terminal nucleotide, the four different nucleic acid binding compositions is labeled with distinct individual fluorophores, and the detection step comprises simultaneous excitation at a wavelength sufficient to excite all four fluorophores and imaging of the fluorescence emission at a wavelength sufficient to detect each individual fluorophore. will be. In some embodiments, the methods provided herein include four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, wherein the composition is used to determine the identity of a terminal nucleotide, the four different nucleic acid binding compositions were labeled with cyanine dye 3 (Cy3), cyanine dye 3.5 (Cy3.5), cyanine dye 5 (Cy5), and cyanine dye 5.5 (Cy5.5), respectively, and the detection steps were 532 nm, 568, respectively. simultaneous excitation at any two wavelengths of nm and 633 nm, and imaging of fluorescence emission at about 570 nm, 592 nm, 670 nm, and 702 nm; and/or fluorescence imaging is one comprising dual wavelength excitation/double wavelength emission fluorescence imaging. In some embodiments, the methods provided herein include four different nucleic acid binding compositions, each composition comprising a different nucleotide or nucleotide analogue is used to determine the identity of a terminal nucleotide, wherein 1, 2, 3 , or four different nucleic acid binding compositions are each labeled with a separate set of fluorophores or fluorophores, and the detection step is performed simultaneously at a wavelength sufficient to excite one, two, three, or four fluorophores or sets of fluorophores. excitation, and imaging of fluorescence emission at a wavelength sufficient to detect each individual fluorophore. In some embodiments, the methods provided herein are three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analog, wherein the composition is used to determine the identity of a terminal nucleotide, wherein 1, 2 , or three different nucleic acid binding or incorporation compositions are each labeled with a separate set of fluorophores or fluorophores, wherein the detection step is performed simultaneously at a wavelength sufficient to excite one, two, or three fluorophores or sets of fluorophores. excitation, and imaging of fluorescence emission at a wavelength sufficient to detect each individual fluorophore, wherein detection of the fourth nucleotide is determined with reference to a “dark” or unlabeled spot or target nucleotide, or it can be decided In some embodiments, the methods provided herein include, wherein the multivalent binding or incorporation composition may comprise three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates is a different type of nucleotides. In some embodiments, the methods provided herein are wherein detection of binding or incorporation complexes is performed in the absence of unbound or solution polymeric nucleotide conjugates.

In some embodiments, the methods provided herein comprise four different nucleic acid binding compositions, or three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analog to determine the identity of a terminal nucleotide. wherein one of the four or three different nucleic acid binding or incorporation compositions is labeled with a first fluorophore, one is labeled with a second fluorophore, and one is labeled with both the first and second fluorophores. and one is unlabeled or absent, wherein the detecting step includes simultaneous excitation at a first excitation wavelength and a second excitation wavelength and an image is acquired at the first fluorescence emission wavelength and the second fluorescence emission wavelength. In some embodiments, the methods provided herein include, wherein the first fluorophore is Cy3, the second fluorophore is Cy5, the first excitation wavelength is 532 nm or 568 nm, the second excitation wavelength is 633 nm, and , the first fluorescence emission wavelength is about 570 nm, and the second fluorescence emission wavelength is about 670 nm. In some embodiments, the methods provided herein are those wherein the detection label comprises one or more portions of a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications can be performed under a single excitation and imaging step. In some embodiments, the methods provided herein are such that a sequencing reaction cycle comprising contacting, detecting, and incorporating/extending steps is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the methods provided herein have an average Q-score for base determination accuracy over a sequencing run of at least 30, and/or at least 40. In some embodiments, the methods provided herein include: at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides have a Q-score greater than 30 and/or greater than or equal to 40 will have In some embodiments, the methods provided herein are wherein at least 95% of the identified terminal nucleotides have a Q-score greater than 30.

In some embodiments, provided herein are reagents comprising one or more nucleic acid binding compositions disclosed herein and a buffer. For example, in some embodiments, provided herein is a reagent, wherein the reagent comprises one, two, three, four or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition is of a single type. containing nucleotides. In some embodiments, the reagents herein comprise one, two, three, four or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, wherein said nucleotide or The nucleotide analogues may each correspond to: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), and at least one from the group consisting of deoxyadenosine monophosphate (dAMP); Thymidine triphosphate (TTP), thymidine diphosphate (TDP), thymidine monophosphate (TMP), deoxythymidine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate (dTMP), uridine triphosphate (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP), deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), and di at least one from the group consisting of oxyuridine monophosphate (dUMP); Cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), and deoxycytidine mono at least one from the group consisting of phosphate (dCMP); and guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), deoxyguanosine triphosphate (dGTP), deoxyguanosine diphosphate (dGDP), and deoxyguanosine at least one from the group consisting of monophosphate (dGMP). In some other examples or some additional examples, the disclosure provides reagents comprising, or further comprising, 1, 2, 3, 4 or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition is of a single type. a nucleotide or nucleotide analogue of, wherein the nucleotide or nucleotide analogue is ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, It may correspond to one or more from the group consisting of dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

[0062] The present specification includes a nucleic acid binding or incorporation composition of any embodiment disclosed herein and/or a reagent of any embodiment disclosed herein, and/or one or more buffers; and a kit comprising instructions for use thereof.

Disclosed herein is a system for performing a method of any of the embodiments disclosed herein, comprising a nucleic acid binding or incorporation composition of any embodiment disclosed herein, and/or of any of the embodiments disclosed herein. Includes reagents. In some embodiments, the system comprises sequential contacting of a tethered, primed nucleic acid molecule with said nucleic acid binding or incorporation composition and/or said reagent; and detection of binding or incorporation of the disclosed nucleic acid binding or incorporation composition to one or more primed nucleic acid molecules.

In some embodiments, provided herein is a composition comprising a particle (e.g., a nanoparticle or polymeric core), wherein the particle comprises a plurality of enzyme or protein binding or incorporation substrates, wherein the enzyme or protein binding or The incorporation substrate binds one or more enzymes or proteins to form one or more binding or incorporation complexes (e.g., multivalent binding or incorporation complexes), wherein the binding or incorporation is determined by the location, presence, and/or incorporation of the one or more binding or incorporation complexes; Or it can be monitored or identified by observing persistence. In some embodiments, the particles may comprise polymers, branched polymers, dendrimers, liposomes, micelles, nanoparticles, or quantum dots. In some embodiments, the substrate may comprise a nucleotide, a nucleoside, a nucleotide analog, or a nucleoside analog. In some embodiments, the enzyme or protein binding or incorporation substrate may comprise an agent capable of binding a polymerase. In some embodiments, the enzyme or protein may comprise a polymerase. In some embodiments, said observation of the location, presence, or persistence of one or more binding or incorporation complexes may comprise fluorescence detection. In some embodiments, the present specification provides a composition comprising a plurality of discrete particles as disclosed herein, wherein each particle comprises a single type of nucleoside or nucleoside analog, each such nucleoside A side or nucleoside analog is associated with a fluorescent label of detectably different emission or excitation wavelength. In some embodiments, the compositions provided herein further comprise one or more labels, eg, fluorescent labels, on the particle. In some embodiments, the compositions provided herein comprise at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 tethered compositions wherein the composition is tethered to a particle. nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues. In some embodiments, the composition provided by the present specification, the nucleoside is μm ² 0.001 per side or nucleoside analog to 1,000,000, μm ² per 0.01 to 1 million, 0.1 to 1 million, 1 to 1,000, 000 per μm ² per μm ² , μm ² any of the sugar from 10 to 1,000,000, μm ² 100 to 1,000,000, μm ² per 1,000 to 1,000,000, μm ² per 1,000 to 100,000 μm ² 10,000 to 100,000, or μm ² 50,000 to 100,000, or the above-mentioned values per per per It exists with a surface density within the range defined by the two values of . In some embodiments, the compositions provided herein are those in which a nucleoside or nucleoside analog is present in a nucleotide or nucleotide analog. In some embodiments, the compositions provided herein comprise or incorporate modified nucleotides or nucleotide analogs to prevent incorporation of the compositions into the extending nucleic acid chain during a polymerase reaction. In some embodiments, the compositions provided herein comprise or incorporate reversibly modified nucleotides or nucleotide analogs to prevent incorporation into the extending nucleic acid chain during the polymerase reaction. In some embodiments, the compositions provided herein are such that at least one label comprises a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the compositions provided herein are those in which a substrate (eg, a nucleotide, nucleotide analogue, nucleoside, or nucleoside analogue) is attached to a particle via a linker. In some embodiments, the compositions provided herein are nucleotides, such as, for example, that do not have a 3' hydroxyl group, wherein at least one nucleotide or nucleotide analog is modified to inhibit elongation during a polymerase reaction or a sequencing reaction nucleotides; a nucleotide modified to include a blocking group at the 3'position;3'-O-azido group, 3'-O-azidomethyl group, 3'-O-alkyl hydroxylamino group, 3'-phosphorothioate group, 3'-O-malonyl group, or nucleotides modified with a 3'-0-benzyl group; and/or unmodified nucleotides at the 3' position.

In some embodiments, the present specification provides a method of determining the sequence of a nucleic acid molecule, the method comprising, in any order, the following steps: 1) a template strand and a complementary strand that is at least partially complementary to the template strand providing a nucleic acid molecule comprising; 2) contacting the nucleic acid molecule with one or more nucleic acid binding or incorporation compositions according to any of the embodiments disclosed herein; 3) detecting binding or incorporation of the nucleic acid binding or incorporation composition to the nucleic acid molecule, and 4) determining the identity of the terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the method repeats the incorporation of the terminal nucleotide into the complementary strand, and the contacting, detecting, and incorporation steps for one or more additional iterations to obtain the sequence of the template strand of the nucleic acid molecule. It may further include the step of determining. In some embodiments, in the method provided herein, the nucleic acid molecule is clonal amplified on a solid support. In some embodiments, the method provided herein comprises, wherein the clonal amplification comprises polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase dependent amplification, recombinase dependent amplification, single-stranded binding (SSB) protein dependence amplification, or any combination thereof. In some embodiments, the methods provided herein are such that a sequencing reaction cycle comprising contacting, detecting, and incorporating steps is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the methods provided herein have an average Q-score for base determination accuracy over a sequencing run of at least 30, or at least 40. In some embodiments, the methods provided herein comprise at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the identified terminal nucleotides greater than 30; or having a Q-score greater than 40. In some embodiments, the methods provided herein are wherein at least 95% of the identified terminal nucleotides have a Q-score greater than 30.

In some embodiments, provided herein are reagents comprising one or more nucleic acid binding or incorporation compositions disclosed herein, and a buffer. In some embodiments, the reagents provided herein comprise one, two, three, four or more nucleic acid binding or incorporation compositions, wherein the nucleic acid binding or incorporation compositions each comprise a single type of nucleotide or nucleotide analog. and, wherein the nucleotide or nucleotide analogue includes nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues. In some embodiments, the methods provided herein include one, two, three, four or more nucleic acid binding or incorporation compositions, wherein the reagent comprises one, two, three, four or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog. wherein the nucleotide or nucleotide analogue is at least one from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP, respectively; one or more from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP; one or more from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP; And it may correspond to one or more from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, the methods provided herein include one, two, three, four or more nucleic acid binding or incorporation compositions, wherein the reagent comprises one, two, three, four or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog. Including, wherein the nucleotide or nucleotide analogue is ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, It may correspond to one or more from the group consisting of CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

In some embodiments, the present specification provides for any composition disclosed herein; and/or any reagent disclosed herein; one or more buffers; and instructions for use thereof.

In some embodiments, the present specification provides a system for performing any of the methods disclosed herein; wherein the method comprises any of the compositions disclosed herein; and/or any reagent disclosed herein; one or more buffers, and one or more nucleic acid molecules optionally tethered or attached to a solid support, wherein the system comprises sequential contacting of the nucleic acid molecule with the composition and/or the reagent; and repeatedly detecting binding or incorporation of the nucleic acid binding or incorporation composition to one or more nucleic acid molecules.

In some embodiments, provided herein is a composition disclosed herein for use in increasing the contrast-to-noise ratio (CNR) of a labeled nucleic acid complex, bound or associated with a surface.

In some embodiments, provided herein is a composition disclosed herein for use in establishing or maintaining control over the duration of a signal from a labeled nucleic acid complex, bound or associated with a surface.

In some embodiments, the specification is used to establish or maintain control over the duration of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex, bound or associated with a surface. A composition disclosed herein is provided for

In some embodiments, provided herein is a composition disclosed herein for use in increasing the specificity, accuracy, or read length of a nucleic acid sequencing and/or genotyping application.

In some embodiments, the present specification provides a composition disclosed herein for use in increasing specificity, accuracy, or read length in a sequencing-by-binding or incorporation-by-synthetic sequencing, single molecule sequencing, or ensemble sequencing method. .

In some embodiments, provided herein is a reagent disclosed herein for use in increasing the contrast-to-noise ratio (CNR) of a labeled nucleic acid complex, bound or associated with a surface.

In some embodiments, provided herein are reagents disclosed herein for use in establishing or maintaining control over the duration of a signal from a labeled nucleic acid complex, bound or associated with a surface.

In some embodiments, the specification is used to establish or maintain control over the duration of a fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signal from a labeled nucleic acid complex, bound or associated with a surface. Reagents disclosed herein are provided for:

In some embodiments, provided herein are reagents disclosed herein for use in increasing the specificity, accuracy, or read length of nucleic acid sequencing and/or genotyping applications.

In some embodiments, provided herein are reagents disclosed herein for use in increasing specificity, accuracy, or read length in methods of sequencing by binding or incorporation, sequencing by synthesis, single molecule sequencing, or ensemble sequencing. .

INCLUDING BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication 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 specification and a term in an incorporated reference, the term in this specification shall control.

A patent or application file contains at least one drawing in color. Copies of this patent or patent application published together with the color drawing(s) will be made available to the Patent Office upon request and payment of the necessary fee.
The novel features of the inventive concept disclosed herein are particularly set forth in the appended claims. A better understanding of the features and advantages of the disclosed constructions, methods and systems will be obtained by reference to the following detailed description and accompanying drawings, which set forth exemplary embodiments in which the principles of the inventive concept are utilized.
1A-1H depict steps utilizing a non-limiting example of a multivalent binding composition for sequencing a target nucleic acid: FIG. 1A depicts a non-limiting example 4 of attaching a target nucleic acid to a surface; 1B clones a target nucleic acid to form a cluster of amplified target nucleic acid molecules; 1C depicts a non-limiting example of priming a target nucleic acid to generate a primed target nucleic acid; 1D depicts a non-limiting example of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; 1E shows a non-limiting example of an image of a binding complex captured on a surface; 1F depicts a non-limiting example of extending a primer strand by one nucleotide; 1G depicts a non-limiting illustration of another cycle of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; 1H depicts a non-limiting example of an image of a binding complex captured on a surface in a subsequent sequencing cycle.
2 shows a flow diagram schematically outlining the steps of sequencing a target nucleic acid and extending a primer strand via single base addition.
3 depicts a flow diagram schematically outlining the steps of sequencing a target nucleic acid and extending a primer strand by incorporating nucleotides into a particle-nucleotide conjugate.
4A-4B illustrate non-limiting examples of target nucleic acid detection using polymer-nucleotide conjugates. 4A depicts the step of contacting a polymerase and a polymer-nucleotide conjugate to some nucleic acid molecule; 4B depicts the binding complex formed between a polymerase, a polymer-nucleotide conjugate, and a target nucleic acid molecule.
Figures 5A-5C schematically depict non-limiting examples of manifold arrangements of polymer-nucleotide conjugates: Figure 5A depicts polymer-nucleotide conjugates having various multi-arm structures; 5B depicts a polymer-nucleotide conjugate with polymer branches radiating from the center; 5C depicts a polymer-nucleotide conjugate with a binding moiety biotin.
6 depicts, in generalized graphical representations, the increase in signal intensity observed during binding, persistence and washing and removal of multivalent substrates.
7A-7J depict fluorescence images of steps in a sequencing reaction using a polyvalent PEG-substrate composition. 7A : DNA RCA template (G and A first base) in exposure buffer containing ^{-20 nM Klenow polymerase and 2.5 mM Sr +2} for 500 nM base labeled nucleotides (A-Cy3 and G-Cy5) Red and green fluorescence images after exposure. Images were collected after washing with an imaging buffer of the same composition as the exposure buffer but without nucleotides or polymerase. Contrast was adjusted to maximize visualization of the faintest signal, but the signal did not persist after washing with imaging buffer ( Figure 7A , inset). 7B-7E : Multivalent PEG-nucleotide (base-labeled) ligands PB1 ( FIG. 7B ), PB2 ( FIG. 7C ), PB3 ( FIG. 7D ), and at 500 nM after mixing in exposure buffer and imaging in the same imaging buffer as above. Fluorescence image showing PB5 ( Figure 7E). Figure 7F : Fluorescence image showing the multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uM after mixing in exposure buffer and imaging in the same imaging buffer as above. Figures 7G-7I : Inactive mutants of Klenow polymerase ( Figure 7G : D882H; Figure 7H : D882E; Figure 7I : D882A), vs. wild-type Klenow (control) enzyme ( Figure 7J ) Additional base discrimination by multivalent ligand exposure Fluorescence images shown.
8A-8B depict the efficacy of a multivalent reporter composition in determining the sequencing of a DNA sequence over five sequencing cycles: FIG. 8A depicts images and predicted sequences for templates taken after each sequencing cycle; Fig. 8B shows the sorted sequencing results using the images taken in Fig. 8A.
9A-9J show polyvalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates having an effective nucleotide concentration of 500 nM and various PEG branch lengths, containing 20 nM Klenow polymerase and 2.5 mM Sr ⁺² . Fluorescence images are shown after contacting the support surface (containing G or A as the first base; prepared using rolling circle amplification (RCA)) containing the DNA template in an exposure buffer that Images were acquired after washing with an imaging buffer of the same composition as the exposure buffer but without nucleotides and polymerase. The panels show images obtained using multivalent PEG-nucleotide ligands having the following arm lengths. Figure 9A : 1 K PEG. Figure 9B : 2K PEG. Figure 9C : 3K PEG. Figure 9D : 5K PEG. Figure 9E : 10 K PEG. Figure 9F : 20 K PEG. 9G depicts images obtained using an inactive klenow polymerase comprising 10 K PEG and mutation D882H. 9H depicts images obtained using an inactive klenow polymerase comprising 10 K PEG and the mutation D882E. Figure 9I depicts images obtained using an inactive klenow polymerase comprising 10 K PEG and the mutation D882A. 9J depicts images obtained using 10 K PEG and active wild-type klenow polymerase.
Figure 10 shows a quantitative representation of the fluorescence intensity of the images shown in Figures 9A-9F , with orange trace corresponding to the red label (Cy3 label; A base) and the green label corresponding to the green label (Cy5 label; G base). The blue traces are distinguished by color values.
11 depicts normalized fluorescence from multivalent substrates bound to DNA clusters as described in FIGS. 7A-7J , substrate complexes formed in the presence (Condition B) and absence (Condition A) of Triton-X100 (0.016%). do.
12A-12B show plots of normalized fluorescence intensities measured for multivalent polymer-nucleotide conjugates and free nucleotides. Figure 12A : After 1 min, two copies of a multivalent polymer-nucleotide conjugate bound to a DNA cluster (conditions A and B) versus a binding complex using labeled free nucleotides (condition C); 12B : Time course of fluorescence from the multivalent substrate complex over the course of 60 minutes.

details

I. Definition

As used herein, "nucleic acid" (also referred to as "polynucleotide", "oligonucleotide", ribonucleic acid (RNA), or deoxyribonucleic acid (DNA)) is two or more linked by a covalent internucleoside linkage. a linear polymer of nucleotides, or a variant or functional fragment thereof. In a naturally occurring example of a nucleic acid, the internucleoside linkage is a phosphodiester bond. However, other examples may optionally include other internucleoside linkages, such as phosphorothiolate linkages, or no phosphate groups. Nucleic acids include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids (PNA), hybrids between PNA and DNA or RNA, and other types of nucleic acids Variants may also be included.

As used herein, “nucleotide” refers to a nucleotide, a nucleoside, or an analog thereof. Nucleotide refers to both naturally occurring and chemically modified nucleotides and may include, but is not limited to, nucleosides, ribonucleotides, deoxyribonucleotides, protein-nucleic acid residues, or derivatives. Examples of nucleotides include adenine, thymine, uracil, cytosine, guanine, or residues thereof; deoxyadenine, deoxythymine, deoxyuracil, deoxycytosine, deoxyguanine, or residues thereof; Adenine PNA, thymine PNA, uracil PNA, cytosine PNA, guanine PNA, or residues or equivalents thereof, N- or C-glycosides of purine or pyrimidine bases (e.g., 2-deoxy-D-ribose deoxyribonucleosides comprising D-ribose or ribonucleosides comprising D-ribose).

As used herein, "complementary" or refers to the topological compatibility or matching together of the interacting surfaces of a ligand molecule and its receptor. Accordingly, the receptor and its ligand can be described as complementary, and furthermore, the tangential surface properties are complementary to each other.

As used herein, "branched polymer" refers to a polymer having a plurality of functional groups that help to conjugate a biologically active molecule, such as a nucleotide, the functional group being on the side chain of the polymer, or the central core or central backbone of the polymer. can be attached directly to Branched polymers may have a linear backbone with one or more functional groups emerging from the backbone for conjugation. A branched polymer may also be a polymer having one or more side chains, which side chains have suitable sites for conjugation. Examples of functional groups include hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanoic acid, acid halide, Isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate including, but not limited to.

As used herein, "polymerase" refers to an enzyme comprising a nucleotide binding moiety and aids in the formation of a binding complex between a target nucleic acid and a complementary nucleotide. A polymerase may have one or more activities, including, but not limited to, base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding or incorporation, strand displacement activity, and nucleotide binding or incorporation and recognition. does not Polymerases may include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing nucleotide binding or incorporation moieties.

As used herein, "duration" is the amount of time that a binding complex formed between a target nucleic acid, polymerase, conjugated or unconjugated nucleotide remains in a stable state without dissociation of any binding components from the binding complex. refers to the length of Duration indicates the stability of the binding complex and the strength of the binding interaction. Duration can be measured by observing the onset and/or duration of the binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in the binding complex, so that a signal from the label can be detected for the duration of the binding complex. One non-limiting example of a label is a fluorescent label.

II. Methods for analyzing target nucleic acids

Disclosed herein are multivalent binding or incorporation compositions and their use in the analysis of nucleic acid molecules, including sequencing or other bioassay applications. Increased binding or incorporation of nucleotides to an enzyme (eg, polymerase) or enzyme complex may be affected by increasing the effective concentration of nucleotides. This increase can be achieved by increasing the concentration of nucleotides in the free solution, or by increasing the amount of nucleotides near the relevant binding or incorporation site. This increase can also be achieved by locally increasing the concentration by physically limiting the number of nucleotides to a limited volume, so that such structures are unconjugated, untethered, or otherwise It can bind or be incorporated into a binding or incorporation site with a higher apparent avidity than that observed for unrestricted individual nucleotides. One non-limiting means of accomplishing this limitation is to provide multivalent binding or incorporation compositions in which multiple nucleotides are bound to particles, such particles as polymers, branched polymers, dendrimers, micelles, liposomes, microparticles, nanoparticles. , quantum dots, or other suitable particles known in the art.

The multivalent binding or incorporation compositions disclosed herein may comprise at least one particle-nucleotide conjugate, wherein the particle-nucleotide conjugate has multiple copies of the same nucleotide attached to the particle. When the nucleotide is complementary to the target nucleic acid, the particle-nucleotide conjugate forms a binding or incorporation complex with the polymerase and the target nucleic acid, and the binding or incorporation complex forms a bond formed using a single unconjugated or untethered nucleotide. or increased stability, and longer duration than the incorporation complex. Each of the nucleotide moieties of the multivalent binding composition binds to complementary N+1 nucleotides of the primed target nucleic acid molecule, such that two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., For example, a multivalent binding complex comprising a polymer-nucleotide conjugate) can be formed. Each of the nucleotide moieties of the multivalent binding composition binds to a complementary N nucleotide of the primed target nucleic acid molecule, such that two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a multivalent binding composition (e.g., , polymer-nucleotide conjugates). Nucleotides from the bound complex can be screened for complementary bases prior to incorporating modified reversibly blocked nucleotides that extend the replicating strand by one base. It is also conceivable to probe the N nucleotides into the bound complex, advance to the reversibly terminated nucleotide, and then probe the N+1 bases before and after deprotection. In this way, the overall accuracy of the base determination can be improved by performing error checking and reading the subject twice. An important distinguishing factor from traditional methods is that binding is used to search for matching bases, whereas stepping or incorporation steps are only used to move forward in the extending strand.

Multivalent binding or incorporation compositions can be used to localize a detectable signal to an active region of an interaction, such as a site of an enzymatic reaction such as a protein-nucleic acid interaction, a nucleic acid hybridization reaction, or a polymerase reaction. For example, the multivalent binding or incorporation compositions described herein can be used to identify base incorporation sites that extend nucleic acid chains during a polymerase reaction and to provide base discrimination for sequencing and array based applications. When the nucleotide is complementary to the target nucleic acid, increased binding or incorporation between the target nucleic acid and the nucleotide in the multivalent binding or incorporation composition enhances the signal, greatly improving the sequencing accuracy and shortening the imaging time.

In addition, the use of multivalent binding compositions allows for sequencing signals from a given sequence to occur within cluster regions comprising multiple copies of the target sequence. Sequencing methods that incorporate multiple copies of a target sequence offer the advantage that the signal is amplified due to the presence of multiple simultaneous sequencing reactions within a defined region, each providing a unique signal. The presence of multiple signals within the defined region also reduces the effect of any single skipped cycle due to the fact that the signal of a large number of correct base confirmations can overwhelm the signal of a small number of skips or false confirmations, resulting in paging errors ( A method for reducing phasing error and/or improving read length in a sequencing reaction is provided.

The multivalent binding compositions and uses thereof disclosed herein result in one or more of the following: (i) a stronger signal for better sequencing accuracy as compared to conventional nucleic acid amplification and sequencing methodologies; ii) allowing greater differentiation of sequence-specific signals from background signals; (iii) reduced requirements for the amount of starting material required, (iv) increased sequencing speed and reduced sequencing time; (v) reduced paging error, and (vi) improved read length in sequencing reactions.

In some embodiments, a target nucleic acid may refer to a target nucleic acid sample having one or more nucleic acid molecules. In some embodiments, a target nucleic acid may comprise a plurality of nucleic acid molecules. In some embodiments, a target nucleic acid may comprise two or more nucleic acid molecules. In some embodiments, a target nucleic acid may comprise two or more nucleic acid molecules having the same sequence.

A. Target Nucleic Acid Sequencing

1A-1H illustrate exemplary methods in which a multivalent binding composition is used to sequence a target nucleic acid. As shown in FIG. 1A , the target nucleic acid 102 may be tethered to the solid support surface 101 . The target nucleic acid may be directly or indirectly attached to the surface. Although not shown in FIG. 1A , the target nucleic acid 102 can hybridize to an adapter that attaches to a surface via covalent or non-covalent bonds. Where more than one adapter is used to attach a target nucleic acid to a surface, the target surface may include fragments that are complementary to the adapter and thus hybridize to the adapter. In some cases, one adapter sequence may be tethered to the surface. In some cases, a plurality of adapter sequences may be tethered to the surface. In some cases, the target nucleic acid 102 may also be attached directly to the solid-support surface without the use of an adapter. The solid support may be a low non-specific binding surface.

In FIG. 1B , after an initial step of attaching the target nucleic acid to the surface of a solid support surface (eg, via hybridization to an adapter), the target nucleic acid is clonal amplified to form clusters of amplified nucleic acids. When the target nucleic acid is attached to the surface via the adapter, the surface density of the cloned amplified nucleic acid sequence hybridized to the adapter on the surface of the support may span the same range as the surface density of the tethered adapter (or primer). Clonal amplification can include polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase dependent amplification, recombinase dependent amplification, single-stranded binding (SSB) protein dependent amplification, or any combination thereof. .

1C illustrates a non-limiting step in which a primer 103 anneals to a target nucleic acid 102 to form a primed target nucleic acid 104 . Although FIG. 1B shows that only one primer is used in the annealing step, more than one primer may be used depending on the type of target nucleic acid. In some cases, the adapter used to attach the target nucleic acid to the surface has the same sequence as the primer used to prepare the primed target nucleic acid. The primers may include forward amplification primers, reverse amplification primers, sequencing primers, and/or molecular barcoding sequences, or any combination thereof. In some cases, one primer sequence may be used in the hybridization step. In some cases, a plurality of different primer sequences may be used in the hybridization step.

1D , the primed target nucleic acid 104 is combined with a multivalent binding or incorporation composition and a polymerase 106 to form a binding or incorporation complex. Non-limiting examples of multivalent binding or incorporation compositions of FIG. 1D include the four particle-nucleotide conjugates (105a), (105b), (105c), and (105d). Each particle-nucleotide conjugate has multiple copies of a nucleotide attached to the particle, and the four particle-nucleotide conjugates cover each of the four types of nucleotides. A particle-nucleotide conjugate having a nucleotide complementary to the next base of the primed target nucleic acid will form a binding or incorporation complex with the polymerase and the target nucleic acid. In some cases, the multivalent binding or incorporation composition may comprise 1, 2 or 3 particle-nucleotide conjugates. In some embodiments, each different type of particle-nucleotide conjugate may be labeled with a separate label. In some embodiments, three of the four types of nucleotide conjugates may be labeled and the fourth type is conjugated to or to an unlabeled or undetectable label. In some embodiments, 1, 2, 3, or 4 particle-nucleotide conjugates may be labeled with the same label, or each with a label corresponding to the entity of its conjugated nucleotide, each, 3, 2, 1 The canine particle-nucleotide conjugate, or any conjugate, may remain unlabeled, or conjugated to or to an undetectable label. In some embodiments, detection of a polymerase complex incorporating a particle-nucleotide conjugate may be performed using four-color detection, so that a conjugate corresponding to all four nucleotides is present in the sample, each conjugate comprising: It has a distinct label corresponding to the nucleotide conjugated thereto. In some embodiments, the four particle-nucleotide conjugates may be simultaneously exposed or contacted to the target nucleic acid; In some other embodiments, the four particle-nucleotide conjugates may be exposed or contacted to the target nucleic acid sequentially, individually, or in groups of two or three. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates may be performed using tri-color detection, so that conjugates corresponding to 3 out of 4 nucleotides are present in the sample, and 3 The conjugates have distinct labels corresponding to the nucleotides conjugated thereto and one conjugate is conjugated to an unlabeled or undetectable label. In some embodiments, only three types of conjugates are provided such that conjugates corresponding to three of the four nucleotides are present in the sample, and the three conjugates have distinct labels corresponding to the nucleotides conjugated thereto. and one conjugate is absent. In some embodiments, the identity of a nucleotide corresponding to an unlabeled or nucleotide-free conjugate can be determined with respect to identification of known target nucleic acid positions and/or "dark" spots or positions that do not exhibit a fluorescent signal. In some embodiments, the methods provided herein are wherein detection of binding or incorporation complexes is performed in the absence of unbound or solution polymeric nucleotide conjugates.

In some embodiments in which three of the four particle-nucleotide conjugates are labeled, or only three of the four particle-nucleotide conjugates are present, the identity of the nucleotide corresponding to the unlabeled or absent conjugate is in the absence of a signal. or by monitoring the presence of unlabeled complexes, such as by identification of "dark" spots or unlabeled regions in a sequencing reaction. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using two-color detection, so that conjugates corresponding to 2 out of 4 nucleotides are present in the sample, and 2 The conjugates have distinct labels corresponding to the nucleotides conjugated thereto and the two conjugates are conjugated to unlabeled or undetectable labels. In some embodiments, only two of the four particle-nucleotide conjugates are labeled. In some embodiments in which two of the four particle-nucleotide conjugates are labeled, the unlabeled conjugate or the identity of the nucleotide corresponding to the conjugate is identified by the absence of a signal or by monitoring the presence of the unlabeled complex, such as by sequencing. This can be established by the identification of "dark" spots or unlabeled regions in the reaction. In some embodiments in which two of the four particle-nucleotide conjugates are labeled, the four particle-nucleotide conjugates may be exposed or contacted with the target nucleic acid sequentially, individually, or in groups of two or three. In some embodiments, two of the four particle-nucleotide conjugates may share a common label, and the four particle-nucleotide conjugates are exposed to the target nucleic acid sequentially, individually, or in groups of two or three, or contacted, and each such contacting step represents a distinction between two or more different bases such that the identity of all unknown bases after two, three, four or more such contacting steps is determined.

1E depicts images captured on a surface after a binding or incorporation complex is formed between a polymerase, a target nucleic acid, and a particle-nucleotide conjugate having a nucleotide complementary to the next base of the primed target nucleic acid. The captured image comprises four binding or incorporation complexes (107a), (107b), (107c), and (107d) formed on the surface, each binding or incorporation complex comprising a label (e.g., For example, they have different nucleotides that can be distinguished according to the color of their fluorescence emission. The use of particle-nucleotide conjugates greatly enhances the sequencing signal because binding or incorporation signals from a given sequence occur within cluster regions containing multiple copies of the target sequence. Although FIG. 1E includes four particle-nucleotide conjugates each having a different type of nucleotide, some methods may use 1, 2, or 3 particle-nucleotide conjugates, each having a different type of nucleotide and label. In some embodiments, each different type of particle-nucleotide conjugate may be labeled with the same label, or each with a label corresponding to the identity of the conjugated nucleotide. In some embodiments, three of the four types of nucleotide conjugates may be labeled and the fourth type is conjugated to or to an unlabeled or undetectable label. In some embodiments, 1, 2, 3, or 4 particle-nucleotide conjugates may be labeled with separate labels, each of which 3, 2, 1 conjugate, or any particle-nucleotide conjugate is labeled. undetectable or conjugated to an undetectable label. In some embodiments, the detecting step may include simultaneous and/or serial excitation of up to four different excitation wavelengths, e.g., fluorescence imaging of each of the possible base pairs (A, G, C, or T). This is done by detecting single and/or multiple fluorescence emission bands that uniquely classify. In some embodiments, four different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analog, can be used to determine the identity of a terminal nucleotide, wherein the four or three different nucleic acid binding or incorporation compositions one is labeled with a first fluorophore, one is labeled with a second fluorophore, one is labeled with both the first and second fluorophores, and one is unlabeled or absent, wherein the detecting step comprises a first Simultaneous excitation at an excitation wavelength and a second excitation wavelength and images are acquired at a first fluorescence emission wavelength and a second fluorescence emission wavelength.

When the multivalent binding or incorporation composition is used for the replacement of a single unconjugated or untethered nucleotide to form a binding or incorporation complex with a polymerase and a primed target nucleic acid, the local concentration of the nucleotide is multiplied, As a result, the signal strength is improved. The binding or incorporation complexes formed also have a longer duration, which helps to shorten the imaging step as a result. The high signal intensity results from the high binding or incorporation avidity of the polymer nucleotide conjugates (which may also include multiple fluorophores or other labels), thus forming complexes that remain stable during the entire binding or incorporation and imaging steps. do. Strong binding or incorporation between the polymerase, the primed target strand, and the polymer-nucleotide or nucleotide analog conjugate will also ensure that the thus formed multivalent binding or incorporation complex will remain stable during the washing step, and other reaction mixture components and correspondence This means that the signal intensity remains high when the nucleotide analogues that do not are washed out. After the imaging step, the binding or incorporation complex can be destabilized (eg, by changing the buffer composition) and the primed target nucleic acid can then be extended for one base.

The sequencing method may further comprise incorporating N+1 or terminal nucleotides into the primed strand as shown in FIG . 1F . In FIG. 1F , the primer strand of the primed target nucleic acid 108 can be extended by one base to form an extended nucleic acid 109 . The extension step may occur after or concurrently with the destabilization of the multivalent binding or incorporation complex. The primed target nucleic acid 108 uses complementary nucleotides attached to the particle of a particle-nucleotide conjugate, or uses unconjugated or untethered free nucleotides that are provided after the multivalent binding or incorporation composition has been removed. can be extended by

After the extension step, the contact step can be performed again to form binding or incorporation complexes and mimic the next sequencing cycle, as shown in Figure 1G. The contacting, detecting, and extending steps may be repeated for one or more cycles to determine the sequence of the target nucleic acid molecule. For example, FIG. 1H depicts surface images obtained after performing multiple sequencing cycles, which images can be processed to determine the sequence of the target nucleic acid molecule.

Extension of the primed target nucleic acid can be prevented or inhibited due to the use of blocked nucleotides or catalytically inactive polymerases on the strand. If the nucleotide of the polymer-nucleotide conjugate has a blocking group that prevents elongation of the nucleic acid, incorporation of the nucleotide can be accomplished by removing the blocking group from the nucleotide (e.g., by separation of the nucleotide from a polymer, branched polymer, dendrimer, particle, etc.) ) can be achieved. When extension of the primed target nucleic acid is inhibited due to the use of a catalytically inactive polymerase, incorporation of nucleotides can be achieved by provision of an activator or cofactor such as a metal ion.

Also disclosed herein is a system configured to perform any nucleic acid sequencing or nucleic acid analysis method disclosed herein. The system comprises a fluid flow controller and/or fluid distribution system configured to sequentially and repeatedly contact a primed target nucleic acid molecule attached to a solid support with a disclosed polymerase and a multivalent binding or incorporation composition and/or reagent. may include. The contacting step may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of a system. In some cases, the flow cell may be a removable and/or disposable component of the system.

A sequencing system can include an imaging module, i.e., one or more light sources, one or more optics for imaging and detection of binding or incorporation of the disclosed nucleic acid binding or incorporation composition to a target nucleic acid molecule tethered inside a solid support or flow cell. components, and one or more image sensors. The disclosed compositions, reagents, and methods can be used in any of a variety of nucleic acid sequencing and analytical applications. Examples include, but are not limited to, DNA sequencing, RNA sequencing, whole genome sequencing, targeted sequencing, exome sequencing, genotyping, and the like.

The sequencing system may also include a computer control system programmed to implement the method of the present invention. The computer system is programmed or configured to implement the methods of the present invention and includes methods for sequencing nucleic acids, interpretation of nucleic acid sequencing data and analysis of cellular nucleic acid RNA (eg, mRNA), and characterization of cells from the sequencing data. The computer system may be a user's electronic device or a computer system remotely located with respect to the electronic device. The electronic device may be a mobile electronic device.

2 is a flowchart schematically illustrating the steps of sequencing a target nucleic acid. (201) describes the step of attaching a target library sequence to a solid support surface by hybridizing the target nucleic acid molecule to a complementary adapter on the substrate surface. The target nucleic acid molecule may be single-stranded or partially double-stranded. (201) Previously, nucleic acid molecules of a target library may have been prepared to contain fragments complementary to adapter sequences through ligation or other methods. (202) describes a clonal amplification step to generate clusters of target nucleic acid molecules on the surface. (203) describes the step of hybridizing a sequencing primer in a target nucleic acid to a complementary primer binding or incorporation sequence to form a primed target nucleic acid. 204 describes combining a polymerase, a composition comprising a labeled (eg, fluorescently-labeled) particle-nucleotide conjugate as a multivalent binding or incorporation composition, and a primed target nucleic acid. 204 may also include washing or removing unbound reagents, including polymerase and particle-nucleotide conjugates.

Referring again to FIG. 2 , when the nucleotide of the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (205), the particle-nucleotide conjugate, the polymerase, and the primed target nucleic acid are ternary binding ) or incorporation complexes, which can be detected by detection methods compatible with labeling of particle-nucleotide conjugates (eg, fluorescence imaging). (205) may also include determining the duration of the ternary bond or incorporation complex. At (206), the binding or incorporation complex is destabilized to eliminate binding or incorporation of the particle-nucleotide conjugate and the polymerase. Dissociation can be achieved by placing the binding or incorporation complex into conditions that alter the conformation of the polymerase and destabilize the binding or incorporation (eg, addition of strontium ions). 206 may also include washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. (207) describes extending the primed strand of the primed target nucleic acid by a single base addition reaction. After single base extension, steps 204, 205, 206, and 207 may be repeated multiple cycles to determine the sequence of the target nucleic acid.

3 is another flow chart schematically outlining the steps of sequencing a target nucleic acid, comprising cleaving nucleotides from a particle-nucleotide conjugate and incorporating the cleaved nucleotides. (301) describes the step of attaching the target library sequence to the solid support surface by hybridizing the target nucleic acid molecule to a complementary adapter on the substrate surface. The target nucleic acid molecule may be single-stranded or partially double-stranded. (301) Previously, nucleic acid molecules of the target library may have been prepared to contain fragments complementary to adapter sequences through ligation or other methods. 302 describes a clonal amplification step to generate clusters of target nucleic acid molecules on a surface. (303) describes the step of hybridizing a sequencing primer in a target nucleic acid to a complementary primer binding or incorporation sequence to form a primed target nucleic acid. 304 describes combining a polymerase, a composition comprising a labeled (eg, fluorescently-labeled) particle-nucleotide conjugate as a multivalent binding or incorporation composition, and a primed target nucleic acid. In particle-nucleotide conjugates, the nucleotides are attached to the particle through chemical bonds or interactions that can later be cleaved. 404 may also include washing or removing unbound reagents, including polymerase and particle-nucleotide conjugates.

Referring back to FIG. 3 , when the nucleotide of the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (305), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid are ternary bond or incorporation complexes , which can be detected by a detection method (eg, fluorescence imaging) compatible with labeling of particle-nucleotide conjugates. (305) can also include determining the duration of the ternary bond or incorporation complex. At (306), the polymerase is subjected to conditions that are catalytically activated to incorporate the nucleotide. Such conditions may include exposing the polymerase to Mg or Mn ions in the reaction solution. The nucleotides bound to the polymerase and the primed target nucleic acid are then cleaved from the particle and incorporated into the primed strand of the primed target nucleic acid. The binding or incorporation complex is destabilized. 306 may also include washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. After extension, steps 304, 305, 306, and 307 may be repeated multiple cycles to determine the sequence of the target nucleic acid.

B. Detection of Target Nucleic Acid Molecules

4A-4B illustrate exemplary methods in which multivalent binding or incorporation compositions are used to detect target nucleic acids. As shown in FIG. 4A , a polymer-nucleotide conjugate 401 is placed in contact with a polymerase 406 , a first nucleic acid molecule 404 and a second nucleic acid molecule 405 . Polymer-nucleotide conjugate (401) has multiple polymeric branches radiating from the core, some branches are attached to nucleotides or oligonucleotides (402), and some branches are attached to label (403). When the nucleotides or oligonucleotides 402 of the polymer-nucleotide conjugate 401 are complementary to at least a fraction of the first nucleic acid 404, a multivalent binding or incorporation complex is formed, as shown in FIG . 4B, with strong binding or The incorporation signal may assist in detecting a target nucleic acid having a complementary or partially complementary sequence to a nucleotide or oligonucleotide of the polymer-nucleotide conjugate. In some cases, at least one of a polymerase, a nucleic acid molecule, and a polymer-nucleotide conjugate is attached to a solid support.

The multivalent binding or incorporation compositions described herein can be used in methods of detecting a target nucleic acid in a sample. Also disclosed herein is a system configured to perform any of the disclosed nucleic acid analysis methods. The system may include a fluid flow controller and/or fluid distribution system configured to sequentially and repeatedly contact the nucleic acid molecule with the disclosed polymerase and the multivalent binding or incorporation composition and/or reagent. The contacting step may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of a system. In some cases, the flow cell may be a removable and/or disposable component of the system. The system may also include a cartridge comprising a sample collection unit and an assay assembly, wherein the sample collection unit is configured to collect a sample, wherein the assay assembly comprises a multivalent binding or incorporation composition configured to interact with the analyte. A predetermined sample portion comprising at least one reaction site reacts with an assay reagent included within the assay assembly to produce a signal indicative of the presence of the analyte in the sample, and detect a signal generated from the analyte.

III. Multivalent Bonding or Incorporating Compositions

The present invention relates to multivalent binding or incorporation compositions having a plurality of nucleotides conjugated to a particle (eg, a polymer, branched polymer, dendrimer, or equivalent structure). Contacting the multivalent binding or incorporation composition with the polymerase and multiple copies of the primed target nucleic acid can result in the formation of a detectable ternary complex, resulting in more accurate base determination of the target nucleic acid.

When the multivalent binding or incorporation composition is used for the replacement of unconjugated or untethered single nucleotides to form a complex with the polymerase and one or more copies of the target nucleic acid, the local concentration of nucleotides, as well as the binding avidity of the complex. (when a complex comprising two or more target nucleic acid molecules is formed) is multiplied, resulting in improved signal strength, specifically accurate signal-to-discordance. The multivalent binding or incorporation compositions described herein may comprise at least one particle-nucleotide conjugate, each particle-nucleotide conjugate comprising multiple copies of a single nucleotide moiety, to interact with a target nucleic acid. The multivalent composition may also include 2, 3, or 4 different particle-nucleotide conjugates, each having a different nucleotide conjugated to the particle.

The multivalent binding or incorporation composition may comprise 1, 2, 3, 4 or more types of particle-nucleotide conjugates, each particle-nucleotide conjugate comprising a different type of nucleotide. A first type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. A second type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. A fourth type of particle-nucleotide conjugate may comprise a nucleotide selected from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, each particle-nucleotide conjugate is each ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP , CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and a single type of nucleotide corresponding to one or more nucleotides selected from the group consisting of dGMP. Each multivalent binding or incorporation composition may further comprise one or more labels, each corresponding to a particular nucleotide conjugated to an individual conjugate. Examples of such labels include fluorescent labels, colorimetric labels, electrochemical labels (eg, glucose or other reducing sugars, or thiols or other reducing active moieties), luminescent labels, chemiluminescent labels, spin labels, radioactive labels, stereoscopic labels. structural labels, affinity tags, and the like.

A. Particle-Nucleotide Conjugates

In particle-nucleotide conjugates, multiple copies of the same nucleotide may be covalently or non-covalently bound to the particle. Examples of particles include branched polymers; dendrimers; crosslinked polymer particles such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles, or any other particles known in the art (eg, nanoparticles, microparticles, etc.). In a preferred embodiment, the particles are branched polymers.

In some cases, a particle-nucleotide conjugate (eg, a polymer-nucleotide conjugate) has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 tethered to the particle. nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues of

The nucleotide may be linked to the particle via a linker, and the nucleotide may be attached to one end or position of the polymer. The nucleotide may be conjugated to the particle via the 5' end of the nucleotide. In some particle-nucleotide conjugates, one nucleotide is attached to one end or position of the polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to one end or position of the polymer. Conjugated nucleotides may have conformational access to one or more proteins, one or more enzymes, and nucleotide binding or incorporation moieties. In some embodiments, the nucleotides may be provided separately from a nucleotide binding or incorporation moiety such as a polymerase. In some embodiments, the linker does not comprise a light emitting or light absorbing group.

Particles may also have binding or incorporation moieties. In some embodiments, particles are capable of self-associating without the use of separate interacting moieties. In some embodiments, the particle is, for example, of calcium mediated interactions of hydroxyapatite particles, lipid or polymer mediated interactions of micelles or liposomes, or salt mediated aggregation of metal (such as iron or gold) nanoparticles. Self-association may occur due to buffer conditions or salt conditions as in the case.

The particle-nucleotide conjugate may have one or more labels. Examples of labels include fluorophores, spin labels, metal or metal ions, colorimetric labels, nanoparticles, PET labels, radiolabels, or macromolecules or molecules that would make the composition detectable by methods known in the art. including, but not limited to, other indicia that may be The label may be on the nucleotide (e.g., attached to the 5' phosphate moiety of the nucleotide), on the particle itself (e.g., on the PEG subunit), at the end of the polymer, on a central moiety, or known in the art or It can be attached to any other location within the polymer-nucleotide conjugate recognized by one of ordinary skill in the art to be sufficient to provide the composition detectable by the methods described elsewhere herein, such as to the particle. In some embodiments, one or more labels are provided to correspond to or differentiate a particular particle-nucleotide conjugate.

In some embodiments, the label is a fluorophore. Non-limiting examples of fluorescent moieties include, but are not limited to: fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachloro Fluorescein (hexachlorofluorescein), carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein Rescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine ( rhodamine) and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR- (lissamine rhodamine B) sulfonyl chloride), lissamine rhodamine B sulfonyl hydrazine, Texas red sulfonyl chloride, Texas red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo- NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY 530/550 C3 hydrazide, BODIPY 493/ 503 C3 HYDRAZIDE, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine Blue cadaverine), Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as lucifer yellow iodoacetamide, lucifer yellow CH, cyanine and derivatives For example, indolium-based cyanine dye, benzo-indolium-based cyanine dye, pyridium-based cyanine dye, thiozolium-based cyanine dye, quinolinium ( quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelate, Terbium chelate, Alexa Fluor dye , DyLight dye, Atto dye, LightCycler Red dye, CAL Flour dye, JOE and derivatives thereof, Oregon Green dye, WellRED dye, IRD dye, phycoerythrin and phycobilin dye, Malachite green , stilbene, DEG dye, NR dye, near infrared dye and Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition], or derivatives thereof, or any combination thereof. Cyanine dyes exist in sulfonated or unsulfonated forms, and are two indolenines, benzo-indolium, pyridium, thiozolium, and/or quinolinium separated by a polymethine bridge between two nitrogen atoms. may be composed of groups. Commercially available cyanine fluorophores include, for example, Cy3, (which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3) -{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-yl den}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxo Hexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3 -dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 ( It is 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6 -((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-diene -1-yl)-3,3-dimethyl-3H-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)- 2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-di methyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-ium-5-sulfonate present), and Cy7 (which is 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-yl) den)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1- ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate may), where "Cy" stands for 'cyanine', and the first number indicates the number of carbon atoms between two indolenine groups. Exceptions to this rule are the non-indolenine oxazole derivatives Cy2, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5.

In some embodiments, the detection label may be a FRET pair, allowing multiple classifications under a single excitation and imaging unit. As used herein, FRET may include excitation exchange (Forster) transport, or electron-exchange (Dexter) transport.

B. Polymer-Nucleotide Conjugates

One example of a particle-nucleotide conjugate is a polymer-nucleotide conjugate. Some non-limiting examples of polymer-nucleotide conjugates are shown in Figures 5A-5C . For example, FIG. 5A depicts polymer-nucleotide conjugates having various configurations, e.g., a “starburst” configuration with a fluorescently-labeled streptavidin core and molecular weights ranging from 1 K Daltons to 10 K 4 nucleotides linked to the core via a biotinylated, linear PEG linker in the dalton range; 5B shows, for example, a polymer-nucleotide conjugate having a dendrimer-type core having 12, 24, 48, or 96 arms and a linear PEG linker with a molecular weight ranging from 1 K Daltons to 10 K Daltons radiating from the center. shows; 5C depicts an example of a polymer-nucleotide conjugate comprising a network of, for example, a streptavidin core linked together with a branched PEG linker comprising a binding or incorporation moiety such as biotin.

Examples of suitable linear or branched polymers include linear or branched polyethylene glycol (PEG), linear or branched polypropylene glycol, linear or branched polyvinyl alcohol, linear or branched polylactic acid, linear or branched polyglycolic acid, linear or branched polyglycine, linear or branched polyvinyl acetate, dextran, or other polymers, or copolymers, incorporating any two or more of the foregoing polymers or incorporating other polymers known in the art do. In one embodiment, the polymer is PEG. In another embodiment, the polymer may have PEG branches.

Suitable polymers may be characterized by repeating units incorporating functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. A polymer may also have one or more pre-derivatized substituents such that one or more specific subunits incorporate a derivatization site or branching site, regardless of whether other subunits incorporate the same site, substituent, or moiety. Prederivatized substituents include, for example, nucleotides, nucleosides, nucleotide analogs, labels such as fluorescent labels, radioactive labels, or spin labels, interacting moieties, additional polymeric moieties, etc., or any of the foregoing. It may include a combination, or may further include.

In polymer-nucleotide conjugates, the polymer may have a plurality of branches. Branched polymers can have a variety of configurations, including, but not limited to, stellate (“starburst”) shapes, aggregated star (“helter skelter”) shapes, bottle brush shapes, or dendrimer shapes. Branched polymers may radiate from a central point of attachment or central moiety, or may incorporate multiple branching points, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more branching points. can In some embodiments, each subunit of the polymer may optionally constitute a separate branch point.

The length and size of the branches can vary depending on the type of polymer of the type. In some branched polymers, the branches have a length of 1-1,000 nm, 1-100 nm, 1-200 nm, 1-300 nm, 1-400 nm, 1-500 nm, 1-600 nm, 1-700 nm, 1 to 800 nm, or 1 to 900 nm, or more, or a length within or between any value disclosed herein.

In some polymer-nucleotide conjugates, the polymer core is 1 K Da, 2 K Da, 3 K Da, 4 K Da, 5 K Da, 10 K Da, 15 K Da, 20 K Da, 30 K Da, 50 K Da , 80 K Da, 100 K Da, or any value within the range defined by any two values described above. The apparent molecular weight of a polymer can be calculated from a representative number of subunits known molecular weights, as determined by size exclusion chromatography, by mass spectrometry, or by any other method known in the art.

In some branched polymers, branching is 1 K Da, 2 K Da, 3 K Da, 4 K Da, 5 K Da, 10 K Da, 15 K Da, 20 K Da, 30 K Da, 50 K Da, 80 K Da, 100 K Da, or any value within the range defined by any two values described above. The apparent molecular weight of a polymer can be calculated from a representative number of subunits known molecular weights, as determined by size exclusion chromatography, by mass spectrometry, or by any other method known in the art. The polymer may have multiple branches. The number of branches in the polymer can be at least 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128, or a number falling within the range defined by any two of the above values. .

For example, in the case of a polymer-nucleotide conjugate comprising a branched polymer of a branched PEG comprising 4, 8, 16, 32, or 64 branches, the polymer nucleotide conjugate comprises a nucleotide attached to the end of the PEG branch. and, accordingly, 0, 1, 2, 3, 4, 5, 6 or more nucleotides may be attached to each end. In one non-limiting example, a branched PEG polymer of 3 to 128 PEG arms may have one or more nucleotides attached to the ends of the polymer branches, thus 0, 1, 2, 3, 4, 5 at each terminus. , 6 or more nucleotides or nucleotide analogues may be attached. In some embodiments, the branched polymer or dendrimer has an even number of arms. In some embodiments, the branched polymer or dendrimer has an odd number of arms.

In some cases, the length of the linker (eg, PEG linker) can range from about 1 nm to about 1,000 nm. In some cases, the length of the linker is at least 1 nm, at least 10 nm, at least 25 nm, at least 50 nm, at least 75 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, or at least 1,000 nm. In some cases, the length of the linker may range between any two of the values in this paragraph. For example, in some cases, the length of the linker may range from about 75 nm to about 400 nm. In some cases, one of ordinary skill in the art will recognize that the length of the linker can be any value within the range of values in this paragraph, eg, 834 nm.

In some cases, the length of the linker may vary in length of different nucleotides (including deoxyribonucleotides and ribonucleotides), nucleotide analogues (including deoxyribonucleotide analogues and ribonucleotide analogues), nucleosides (including deoxyribonucleosides or ribonucleosides), or nucleosides. It is different depending on the cleoside analogue (including deoxyribonucleoside analogue or ribonucleoside analogue). In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyadenosine, and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyguanosine, and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, thymidine, and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxyuridine and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, deoxycytidine and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, adenosine, and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, guanosine, and the linker is between 1 and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, 5-methyl-uridine, and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, uridine and the linker is between 1 nm and 1,000 nm in length. In some cases, one of the nucleotides, nucleotide analogues, nucleosides, or nucleoside analogues comprises, for example, cytidine and the linker is between 1 nm and 1,000 nm in length.

In polymer-nucleotide conjugates, a moiety comprising a nucleotide (eg, an adenine, thymine, uracil, cytosine, or guanine residue or derivative or mimic thereof) is attached to each branch or subset of branches of the polymer. and such moieties may be bound or incorporated into a polymerase, reverse transcriptase, or other nucleotide binding or incorporation domain. Optionally, a moiety can be incorporated into the extending nucleic acid chain during the polymerase reaction. In some cases, the moiety may be blocked from incorporation into the extending nucleic acid chain during the polymerase reaction. In some other cases, the moiety can be reversibly blocked until the blocking is removed such that it cannot be incorporated into the extending nucleic acid chain during the polymerase reaction, after which the moiety is not incorporated into the extending nucleic acid chain during the polymerase reaction. can be incorporated into

The nucleotide may be conjugated to the polymer branch via the 5' end of the nucleotide. In some cases, nucleotides may be modified to inhibit or prevent incorporation of nucleotides into the extending nucleic acid chain during the polymerase reaction. As an example, the nucleotide may be 3' deoxyribonucleotide, 3' azidonucleotide, 3'-methyl azido nucleotide, or known per se or in the art such that it cannot be incorporated into the extending nucleic acid chain during the polymerase reaction. It may contain other nucleotides. In some embodiments, the nucleotide is a 3′-O-azido group, 3′-O-azidomethyl group, 3′-phosphorothioate group, 3′-O-malonyl group, 3′-O- an alkyl hydroxylamino group, or a 3′-O-benzyl group. In some embodiments, the nucleotide does not have a 3' hydroxyl group.

The polymer may additionally have a binding or incorporation moiety in a branch or subset of branches, respectively. Some examples of binding or incorporation moieties include biotin, avidin, streptavidin, etc., polyhistidine domains, complementary paired nucleic acid domains, G-quadruplex forming nucleic acid domains, calmodulin, maltose-binding proteins, cellulases , maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine and its derivatives, antibodies, epitopes, protein A, protein G including, but not limited to. A binding or incorporation moiety is any interaction known in the art to bind, or facilitate an interaction, between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interaction domains or moieties. It may be a molecule or a fragment thereof.

In some embodiments, a composition provided herein may include one or more elements of a complementary interacting moiety. Non-limiting examples of complementary interacting moieties include, for example, biotin and avidin; SNAP-benzylguanosine; antibodies or FABs and epitopes; IgG FC and Protein A, Protein G, Protein A/G, or Protein L; maltose binding protein and maltose; lecithin and homopolysaccharides; ion chelating moieties, complementary nucleic acids, nucleic acids capable of forming triple or triple helix interactions; nucleic acids capable of forming a G-quartet, and the like. Those skilled in the art will readily recognize that many pairs of moieties exist and are commonly used for properties that interact strongly and specifically with each other; Accordingly, any such complementary pair or set is considered suitable for this purpose in envisioning or implementing the compositions of the present invention. In some embodiments, the compositions disclosed herein have one element of the complementary interacting moiety attached to one molecule or multivalent ligand and the other element of the complementary interacting moiety attached to a separate molecule or multivalent ligand. composition may be included. In some embodiments, a composition disclosed herein may include a composition in which both or all elements of a complementary interacting moiety are attached to a single molecule or multivalent ligand. In some embodiments, a composition disclosed herein may comprise a composition in which both or all elements of a complementary interacting moiety are attached to separate arms, or positions, of a single molecule or multivalent ligand. In some embodiments, a composition disclosed herein may comprise a composition in which both or all elements of a complementary interacting moiety are attached to the same arm, or position, of a single molecule or multivalent ligand. In some embodiments, a composition comprising one element of a complementary interacting moiety and a composition comprising another element of a complementary interacting moiety may be mixed simultaneously or sequentially. In some embodiments, interactions between molecules or particles disclosed herein cause multiple molecules or particles to associate or aggregate, eg, an increase in detectable signal. In some embodiments, the fluorescence, colorimetric, or radioactive signal is enhanced. In other embodiments, other interacting moieties disclosed herein or known in the art are contemplated. In some embodiments, a composition provided herein comprises one or more molecules comprising a first interacting moiety, e.g., one or more imidazole or pyridine moieties, and a second interacting moiety, e.g., histidine One or more additional molecules comprising moieties may be provided to be mixed simultaneously or sequentially. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6 or more imidazole or pyridine moieties. In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6 or more histidine residues. In such embodiments, interactions between a given molecule or particle may be facilitated by the presence of a divalent cation such as nickel, manganese, magnesium, calcium, strontium, and the like. In some embodiments, for example, a (His) ₃ group can interact with _{a (His) 3} group of another molecule or particle through coordination of a nickel or manganese ion.

The multivalent binding or incorporation composition may include one or more buffers, salts, ions, or additives. In some embodiments, representative additives may include, but are not limited to: betaine, spermidine, detergent such as Triton X-100, Tween 20, SDS, or NP-40 , ethylene glycol, polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol, methylcellulose, heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, DMSO, N,N,N-trimethylglycine , ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymers such as pluronic (r) series polymers, arginine, histidine, imidazole, or any combination thereof, or a DNA "relaxer in the art" Any substance known as "altering the persistence length of DNA, altering the number of junctions or crossovers within a polymer, or altering the conformational dynamics of a DNA molecule so that the accessibility of an intrastrand site to a DNA binding or incorporation moiety is compounds having an increasing effect).

The multivalent bonding or incorporation composition may include a zwitterionic compound as an additive. Additional representative additives are described in Lorenz, T.C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which relates to the disclosure of additives for the promotion of nucleic acid binding or kinetics, or for the facilitation of processes involving the manipulation, use or storage of nucleic acids. and is incorporated herein by reference. In some embodiments, representative cations may include, but are not limited to: sodium, magnesium, strontium, potassium, manganese, calcium, lithium, nickel, cobalt, or self-associated, secondary or tertiary structure. Other cations known in the art to promote nucleic acid interactions such as formation, base pairing, surface association, peptide association, protein binding, and the like.

IV. Binding between a target nucleic acid and a multivalent binding or incorporation composition

When the multivalent binding or incorporation composition is used for the replacement of unconjugated or untethered single nucleotides to form a complex with the polymerase and one or more copies of the target nucleic acid, the local concentration of nucleotides, as well as the binding avidity of the complex. (when a complex comprising two or more target nucleic acid molecules is formed) is multiplied, resulting in improved signal strength, specifically accurate signal-to-discordance. The present invention contemplates contacting a multivalent binding or incorporation composition with a polymerase and a primed target nucleic acid to determine the formation of a ternary binding or incorporation complex.

6 illustrates the use of the disclosed polymer-nucleotide conjugates to achieve increased signal intensity during binding, persistence, and wash/remove steps. Due to the increased local concentration of nucleotides in the polymer-nucleotide conjugate and/or the formation of non-covalent bonds with two or more primed target nucleic acid molecules, there is a difference between the polymerase, the primed target strand, and the polymer-conjugated nucleotide. Binding is more favorable when the nucleotide is complementary to the next base of the target nucleic acid. The binding complex formed has a longer duration and helps to increase its signal and signal and shorten the imaging step. The high signal intensities due to the use of the disclosed polymer nucleotide conjugates remain stable throughout the binding and imaging steps. The strong binding between the polymerase, the primed target strand, and the polymer-conjugated nucleotide or nucleotide analog also ensures that the binding complex thus formed will remain stable during the wash step in which other reaction mixture components and mismatched nucleotide analogs are washed out. it means that After the imaging step, the binding complex can be destabilized (eg, by changing the buffer composition) and the primed target nucleic acid can then be extended for one base. After extension, the binding and imaging steps can be repeated using the disclosed polymer nucleotide conjugates to determine the identity of the next base.

As an example, a graphical depiction of the increase in signal intensity during binding, persistence, and washing/removal of the multivalent substrates described herein is provided in FIG. 6 , indicating experimentally observed changes in signal intensity. Therefore, the compositions and methods of the present invention provide a powerful and controllable means for establishing and maintaining ternary enzyme complexes, as well as greatly improved means by which the presence of such complexes can be identified and/or measured, and the persistence of the complexes controlled. provide the means to become This provides the most important solution to problems such as determining the identity of N+1 bases in nucleic acid sequencing applications.

It has been observed that the multivalent binding composition disclosed herein associates with the polymerase nucleotide complex at a time dependent rate to form a ternary bond complex, but is substantially slower than the rate of association known to be obtainable with nucleotides in free solution, although any It is not intended to be limited to a particular theory of Accordingly, the rate of association (on-rate, K _on ) is substantially and surprisingly slower than the rate of association for a single nucleotide or nucleotide not attached to the multivalent ligand complex. However, it is important to note that the dissociation rate (off rate, K _off ) of the multivalent ligand complex is significantly slower than that observed for nucleotides in free solution. ) provides a surprising and beneficial improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes, for example, to significantly improve the imaging quality for nucleic acid sequencing applications compared to currently available methods and reagents. Importantly, this property of the multivalent binding composition disclosed herein allows for controllable formation of visible ternary complexes such that subsequent visualization, transformation, or processing steps can occur essentially independent of dissociation of the complex, i.e., the complex. can be formed, imaged, modified, or used as needed and remain stable until subjected to an aggressive dissociation step such as exposing the complex to a dissociation buffer.

In some cases, the duration of the multivalent binding complex formed using the disclosed particle-nucleotide or polymer-nucleotide conjugates can range from about 0.1 seconds to about 600 seconds under non-labile conditions. In some cases, the duration is at least 0.1 seconds, at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds , at least 20 sec, at least 30 sec, at least 40 sec, at least 50 sec, at least 60 sec, at least 120 sec, at least 180 sec, at least 240 sec, at least 300 sec, at least 360 sec, at least 420 sec, at least 480 sec, at least 540 seconds, or at least 600 seconds. In some cases, the duration may range between any two of the values in this paragraph. For example, in some cases, the duration may range from about 10 seconds to about 360 seconds. Those skilled in the art will recognize that in some cases the duration may be any value within the range of values specified in this paragraph, for example, 78 seconds.

In various embodiments, polymerases suitable for binding or incorporation interactions described herein include any polymerase that may be known per se or in the art. For example, all organisms are known to encode one or more DNA polymerases within their genome. Examples of suitable polymerases include, but are not limited to: Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase ; human alpha, delta and epsilon DNA polymerases; bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNA polymerase, Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase III alpha and epsilon; 9 degree N polymerase, reverse transcriptase such as HIV type M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, or tello meraze. In addition to the non-limiting examples of DNA polymerase various archaea in (Archaea genera), for example aeropyrum (Aeropyrum), archaeoglobus (Archaeoglobus), desulfurococcus (Desulfurococcus), pyrobaculum (Pyrobaculum ), Pyrococcus , Pyrolobus , Pyrodictium , Staphylothermus , Stetteria , Sulfolobus, Thermo Rhodococcus genus (Thermococcus), and fire Carney theta in (Vulcanisaeta) and the like, or including a polymerase derived from variants thereof, the art in a known polymerase example Vent ™, Deep Vent ™, Pfu, KOD, Pfx, Therminator ™ and Tgo polymerase. In some embodiments, the polymerase is klenow polymerase.

The ternary complex has a longer duration when the nucleotides of the polymer-nucleotide conjugate are complementary to the target nucleic acid than when they are non-complementary nucleotides. Ternary complexes also have a longer duration than conjugated or untethered complementary nucleotides when the nucleotides of the polymer-nucleotide conjugate are complementary to the target nucleic acid. For example, in some embodiments, the ternary complex has a duration of less than 1s, greater than 1s, greater than 2s, greater than 3s, greater than 5s, greater than 10s, greater than 15s, greater than 20s, greater than 30s, greater than 60s, greater than 120s, 360s greater than, greater than 3600 s, or greater, or a time within the range defined by any two of the above values.

Duration can be measured, for example, by observing the onset and/or duration of the binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in the binding complex, so that a signal from the label can be detected for the duration of the binding complex.

It has been observed that different ranges of duration can be achieved with different salts or ions, ^{indicating that, for example, complexes formed in the presence of magnesium ions (Mg 2+} ) form faster than complexes formed with other ions. , for example, ^{complexes formed in the presence of strontium ions (Sr 2+} ) are readily formed and upon recovery of ions, or one or more components of the composition, such as polymers and/or one or more nucleotides, and/or upon washing with a buffer that does not have one or more interacting moieties, or a buffer comprising, for example, a chelating agent capable of causing or accelerating the removal of a divalent cation from a multivalent reagent comprising the complex. Complete or substantially complete dissociation was observed. Accordingly, in some embodiments, the compositions of the present invention comprise Mg ²⁺ . In some embodiments, a composition of the present invention comprises Ca ²⁺ . In some embodiments, a composition of the present invention comprises Sr ²⁺ . In some embodiments, a composition of the present invention comprises cobalt ions (Co ²⁺ ). In some embodiments, a composition of the present invention comprises MgCl ₂ . In some embodiments, a composition of the present invention comprises CaCl ₂ . In some embodiments, a composition of the present invention comprises SrCl ₂ . In some embodiments, a composition of the present invention comprises CoCl ₂ . In some embodiments, the composition is free or substantially free of magnesium. In some embodiments, the composition is free or substantially free of calcium. In some embodiments, the methods of the present invention provide for contacting one or more nucleic acids with one or more compositions disclosed herein, wherein the compositions do not have either calcium or magnesium, or both calcium or magnesium. does not

The dissociation of the ternary complex can be controlled by changing the buffer conditions. After the imaging step, the signal may be attenuated or terminated, such as in transitions between one sequencing cycle and the next, using a buffer with increased salt content so that the labeled polymer-nucleotide conjugate can be washed away by causing dissociation of the ternary complex. provide the means to In some embodiments, such dissociation can be effected by washing the complex with a buffer that does not have the required metals or cofactors. In some embodiments, the wash buffer may include one or more compositions for the purpose of maintaining pH control. In some embodiments, the wash buffer may comprise one or more monovalent cations, such as sodium. In some embodiments, the wash buffer is free or substantially free of divalent cations, e.g., free or substantially free of strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further comprises a chelating agent, for example, EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, and the like. In some embodiments, the wash buffer is capable of maintaining the pH of the environment at the same level as the bound complex. In some embodiments, the wash buffer can raise or lower the pH of the environment relative to the level found in the bound complex. In some embodiments, the pH is 2-4, 2-7, 5-8, 7-9, 7-10, or a range less than 2, or greater than 10, or by any two of the values provided herein. It can be within a defined range.

Addition of certain ions may affect binding of the polymerase to the primed target nucleic acid, formation of a ternary complex, dissociation of the ternary complex, or incorporation of one or more nucleotides into the extending nucleic acid during the polymerase reaction. In some embodiments, relevant anions may include chloride, acetate, gluconate, sulfate, phosphate, and the like. In some embodiments, an ion is formed by the addition of one or more acids, bases, or salts, such as NiCl ₂ , CoCl ₂ , MgCl ₂ , MnCl ₂ , SrCl ₂ , CaCl ₂ , CaSO ₄ , SrCO ₃ , BaCl ₂ , and the like. can be incorporated into the composition. Representative salts, ions, solutions and conditions are described in Remington: The Science and Practice of Pharmacy, 20th. Edition, Gennaro, AR, Ed. (2000)] (which is incorporated herein by reference in its entirety), and in particular in Chapter 17 and the related disclosure of salts, ions, salt solutions, and ionic solutions.

The present invention contemplates contacting a multivalent binding or incorporation composition comprising at least one particle-nucleotide conjugate with one or more polymerases. Contacting may optionally occur in the presence of one or more target nucleic acids. In some embodiments, the target nucleic acid is a single stranded nucleic acid. In some embodiments, the target nucleic acid is a primed single stranded nucleic acid. In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some embodiments, said contacting comprises contacting the multivalent binding or incorporation composition with one polymerase. In some embodiments, said contacting comprises contacting said composition comprising one or more nucleotides with multiple polymerases. A polymerase may be bound to a single nucleic acid molecule.

Binding between the target nucleic acid and the multivalent binding composition can be provided in the presence of a catalytically inactive polymerase. In one embodiment, the polymerase may have been made catalytically inactive by a mutation. In one embodiment, the polymerase may have been made catalytically inactive by chemical modification. In some embodiments, the polymerase molecule may have been catalytically inactivated by the absence of an essential substrate, ion, or cofactor. In some embodiments, the polymerase enzyme may have been catalytically inactivated by the absence of magnesium ions.

Binding between the target nucleic acid and the multivalent binding composition occurs in the presence of a polymerase wherein the binding solution, reaction solution, or buffer is free of magnesium or manganese. Alternatively, binding between the target nucleic acid and the multivalent binding composition occurs in the presence of a polymerase wherein the binding solution, reaction solution, or buffer comprises calcium or strontium.

When a catalytically inactive polymerase is used to assist the nucleic acid to interact with the multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex so that the complex is fluoresced or disclosed herein. or detectable by other methods known in the art. Unbound polymer-nucleotide conjugates can optionally be washed out prior to detection of the ternary binding complex.

contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein in a solution comprising either calcium or magnesium or both calcium and magnesium. Alternatively, contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein in a solution that does not have either calcium or magnesium, or neither calcium nor magnesium, and the steps, regardless of the order of the steps, are separate steps. with either calcium or magnesium, or both calcium and magnesium, to the solution. In some embodiments, contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein in a solution free of strontium comprises adding strontium to the solution in a separate step, regardless of the order of the steps. do.

V. Use of multivalent binding or incorporation compositions in combination with low non-specific binding surfaces

Disclosed herein is a solid support comprising a low non-specific binding surface composition capable of improving nucleic acid hybridization and amplification performance. In general, the disclosed supports include a substrate (or support structure), one or more covalently or non-covalently attached low-binding layers, a chemically modified layer such as a silane layer, a polymer film, and a single-stranded target nucleic acid ( ) to the surface of the support, one or more covalently or non-covalently attached primer sequences. In some cases, the formulation of the surface, e.g., the chemical composition of one or more layers, is such that non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the support surface is minimized or reduced compared to a similar monolayer The coupling chemistry used to crosslink the above layers to the surface of the support and/or to each other, and the total number of layers may vary. Often, the formulation of the surface can be varied such that non-specific hybridization at the surface of the support is minimized or reduced relative to a similar monolayer. The formulation of the surface can be varied so that non-specific amplification at the surface of the support is minimized or reduced relative to a similar monolayer. The formulation of the surface can be varied to maximize the specific amplification rate and/or yield at the surface of the support. Amplification levels suitable for detection are achieved with no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 amplification cycles or, in some cases disclosed herein, 30 or more amplification cycles.

Examples of materials from which the substrate or support structure may be made include, but are not limited to: glass, bonded-silica, silicon, polymers (eg, polystyrene (PS), macroporous polystyrene (MPPS) ), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC) , polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

The substrate or support structure may be of any of a variety of geometries and sizes known to those skilled in the art, and may comprise any of a variety of materials known to those skilled in the art. For example, in some cases the substrate or support structure may be topically planar (eg, including a microscope slide or the surface of a microscope slide). Overall, the matrix or support structure may be cylindrical (e.g., comprising a capillary or the inner surface of a capillary), spherical (e.g., comprising the outer surface of a non-porous bead), or irregular (e.g., irregularly shaped, (including the outer surface of non-porous beads or particles). In some cases, the surface of a substrate or support structure used for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some cases, the surface of a substrate or support structure used for nucleic acid hybridization and amplification may be porous, whereby the coating described herein penetrates the porous surface, and nucleic acid hybridization and amplification reactions performed thereon occur within the pores. can happen

A substrate or support structure comprising one or more chemically modified layers, eg, a layer of a low non-specific binding polymer, may be independent or integrated into another structure or assembly. For example, in some cases, a substrate or support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may include one or more surfaces within a microplate format, for example, the bottom surface of a well of a microplate. As noted above, in some preferred embodiments the substrate or support structure comprises the inner surface of the capillary (eg, the lumen surface). In an alternative preferred embodiment, the substrate or support structure comprises an inner surface (eg, a lumen surface) of a capillary etched with a planar chip.

As mentioned, the low non-specific binding scaffolds of the present invention exhibit reduced non-specific binding of proteins, nucleic acids, and other components of hybridization and/or amplification formulations used for solid phase nucleic acid amplification. The degree of non-specific binding exhibited by a given support surface can be assessed qualitatively or quantitatively. For example, in some cases, a fluorescent dye (eg, cyanine such as Cy3, or Cy5, etc., fluorescein, coumarin, rhodamine, etc. or other dyes disclosed herein), fluorescence in a standardized set of conditions -expose the surface to labeled nucleotides, fluorescently-labeled oligonucleotides, and/or fluorescently-labeled proteins (e.g., polymerases), followed by a designated cleaning protocol and fluorescence imaging to include different surface formulations It can be used as a qualitative tool to compare non-specific binding on supports. In some cases, after exposing the surface to a fluorescent dye, fluorescently-labeled nucleotide, fluorescently-labeled oligonucleotide, and/or fluorescently-labeled protein (eg, polymerase) under a standardized set of conditions, followed by a designated wash The protocol and fluorescence imaging can be performed and used as a qualitative tool to compare non-specific binding on supports containing different surface formulations, provided that the fluorescence signal is proportional to the number of fluorophores on the surface of the support (e.g., the number of fluorophores Care was taken to ensure that fluorescence imaging was performed under conditions in which signal saturation and/or self-quenching were linearly related (or related in a predictable manner) under non-problematic conditions, and suitable calibration standards were used. In some cases, other techniques known to those of skill in the art, such as radioisotope labeling and counting methods, can be used to quantitatively assess the extent to which non-specific binding is exhibited by different support surface formulations of the present invention.

Some surfaces disclosed herein have a specific to non-specific binding ratio 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 encompassed by the scope of this specification. Some surfaces disclosed herein have a specific to non-specific fluorescence ratio 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 encompassed by the scope of this specification.

As mentioned, in some cases, the extent of non-specific binding exhibited by the disclosed low-binding scaffolds can be determined using standardized protocols, using a standardized protocol to coat 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 standardized set of incubation and washing conditions; The amount of label remaining on the surface can be detected and the resulting signal can be evaluated against an appropriate calibration standard. In some cases, the label may include a fluorescent label. In some cases, the label may include a radioactive isotope. In some cases, the label may include any other detectable label known to those of skill in the art. In some cases, the degree of non-specific binding exhibited by a given support surface formulation can thus be assessed in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some cases, the lower of the invention-binding support μm ² per less than 0.001 molecule, μm ² is less than 0.01 of sugar molecules, μm ² is less than 0.1 molecules per, μm ² is less than 0.25 of sugar molecules, μm ² is less than 0.5 molecules per, μm ² per one molecule is less than, μm less than 10 molecules per ^second, μm non-specific protein binding of less than 100 molecules, or μm ² per 1000 molecules per ^second (or other specified molecule, (e.g., Cy3, or Cy5, rather than between, such as dye, fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein)). One of ordinary skill in the art will recognize that a given support surface of the present invention may exhibit nonspecific binding, eg, less than 86 molecules per ^{μm 2 , falling anywhere within this range.} For example, some modified surfaces disclosed herein were contacted with a 1 μM solution of Cy3-labeled streptavidin (GE Amersham) for 15 min in phosphate buffered saline (PBS) buffer, followed by washing with deionized water three times. , indicates non-specific protein binding of less than 0.5 molecules/μm ^{2 .} Some modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 molecules of Cy3 dye molecules per ^{μm 2 .} In an independent non-specific binding assay, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO- Rho11 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM 7-propargylamine-7-diaza-dGTP-Cy5 (Jena Biosciences, and 10 μM 7-propargylamine- 7-diaza-dGTP-Cy3 (Jena Biosciences) was incubated in a low binding substrate for 15 minutes in a 384 well plate format at 37° C. Each well was washed 2-3 times with 50 ul deionized RNase/DNase free water and 25 Wash 2-3 times with mM ACES buffer (pH 7.4).The 384-well plate was placed on a GE Typhoon instrument with a PMT gain of 800 (depending on the dye test performed) using the manufacturer-specified Cy3, AF555, or Cy5 filter set. gain) setting and a resolution of 50-100 μm For high-resolution imaging, on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA), a total internal fluorescence fluorescence (TIRF) objective (100X, 1.5 NA, Olympus) ), CCD camera (eg, Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or Olympus DP80 color and monochrome camera), light source (eg, Olympus 100W Hg lamp, Olympus 75W Xe lamp, or Olympus U-HGLGPS fluorescence light source), and an excitation wavelength of 532 nm or 635 nm A dichroic mirror was obtained from Semrock (IDEX Health & Science, LLC, Rochester, New York), for example, , 405, 488, 532, or 633 nm dichroic reflector/beamsplitters were purchased, and the bandpass filter was selected with 532 LP or 645 LP matching the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 molecules of dye molecules per ^{μm 2 .}

In some cases, some surfaces disclosed herein have a specific to nonspecific binding ratio 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 encompassed by the scope of this specification. In some cases, some surfaces disclosed herein have a ratio of specific 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 greater than 100, or any intermediate value encompassed by the scope of the present specification.

Low-background surfaces consistent with the disclosure herein have at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 attached specific dye molecules per non-specifically adsorbed molecule. , 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50, specific dye attachment (eg, Cy3 attachment) versus non-specific dye adsorption (eg, , Cy3 dye adsorption) ratio. Similarly, when excitation energy is applied, a low-background surface to which a fluorophore, e.g., Cy3, is attached, consistent with the disclosure herein, produces a specific fluorescent signal (e.g., Cy3 attached to the surface). -occurring from labeled oligonucleotide) to non-specific absorbed dye fluorescence signal ratio 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.

In some cases, the disclosed degree of support surface hydrophilicity (or "wettability" to aqueous solutions) can be assessed, for example, by measuring the water contact angle, which places a droplet on the surface, for example, optical tension. Measure the contact angle with the surface using a meter. In some cases, a static contact angle may be determined. In some cases, the advancing or receding contact angle may be measured. In some cases, the water contact angle for the hydrophilic, low-bonding support surface disclosed herein can range from about 0 degrees to about 30 degrees. In some cases, water contact angles for hydrophilic, low-bonding support surfaces disclosed herein are 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 or less. In many cases the contact angle is less than 40 degrees. One of ordinary skill in the art will recognize that a given hydrophilic, low-binding support surface herein can exhibit a water contact angle having any value within the above range.

In some cases, the hydrophilic surfaces disclosed herein facilitate reduced wash times for bioassays, often due to reduced non-specific binding of biomolecules to low-binding surfaces. In some cases, suitable washing steps may be performed in less than 60, 50, 40, 30, 20, 15, 10 seconds, or less than 10 seconds. For example, in some cases a suitable washing step may be performed in less than 30 seconds.

Some low-bonding surfaces herein exhibit significant improvements in stability or durability over prolonged exposure to solvents and high temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of a disclosed surface is determined by fluorescently labeling functional groups on the surface, or biomolecules tethered to the surface (eg, oligonucleotide primers), prolonged exposure to solvents and high temperatures, or solvent exposure. or by monitoring the fluorescence signal before, during, and after repeated cycles of temperature change. 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 , 1%, 2%, 3%, 4% over a period of 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours , less than 5%, 10%, 15%, 20%, or 25% (or any combination of the above percentages measured over the period). In some cases, the degree of change in fluorescence used to evaluate the quality of the surface is 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles of repeated exposure to solvent changes and/or temperature changes. 1%, 2%, 3%, over a cycle, 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; less than 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of the above percentages measured over the above cycle range).

In some cases, the surfaces disclosed herein may exhibit a high ratio of specific to non-specific signals or other backgrounds. For example, when used for nucleic acid amplification, some surfaces have at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100-fold, or 100-fold greater than the amplification signal. Similarly, some surfaces may at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times, or 100 times the signal of an adjacent amplified nucleic acid dense region of the surface. - Represents an amplified signal that is over-fold.

In some cases, the disclosed fluorescence images of low background surfaces are used in nucleic acid hybridization or amplification applications to generate clusters of hybridized or clonally amplified nucleic acid molecules (eg, directly or indirectly labeled with a fluorophore). When, 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 a contrast-to-noise ratio (CNR) greater than 250.

One or more types of primers may be attached or tethered to the surface of the support. In some cases, one or more types of adapters or primers include a spacer sequence, an adapter sequence for hybridizing to an adapter-ligation target library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or a molecular barcoding sequence, or a sequence thereof. It may include any combination. In some cases, one primer or adapter sequence may 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 primer or adapter sequences may be tethered to at least one layer of the surface.

In some cases, the tethered adapter and/or primer sequences may range from about 10 nucleotides to about 100 nucleotides in length. In some cases, the tethered adapter and/or primer sequence may 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 length. have. In some cases, the tethered adapter and/or primer sequences 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 in length. have. Any lower and lower limits set forth in this paragraph may be combined to form ranges encompassed within this specification, for example, in some cases the length of the tethered adapter and/or primer sequence ranges from about 20 nucleotides to about 80 nucleotides. It can be in the range of nucleotides. One of ordinary skill in the art will recognize that the length of the tethered adapter and/or primer sequence can be any value within this range, for example, about 24 nucleotides.

In some cases, the resulting surface density of the primer on the low binding surface of the support of the present invention may be about 100 μm ² to primer molecules in the range of about 100,000 molecules per primer per μm ^2. In some cases, the resulting surface density of the primer on the low binding surface of the support of the present invention may be from about 1.000 μm to about 1,000,000 primer molecules range of primer molecules per ^second per μm ^2. In some cases, the surface density of the primer may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules per ^{μm 2 .} In some cases, the surface density of the primer may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules per ^{μm 2 .} Any lower limit value and the lower limit value described in this paragraph may be combined to form a range included in the present specification, for example, the surface density of the primer in some cases from about 10,000 per μm ² of about 100,000 molecules to μm ² per It can be a range of molecules. One of ordinary skill in the art will recognize that the surface density of primer molecules can have any value within this range, for example, about 455,000 molecules per ^{μm 2 .} In some cases, the surface density of the target library nucleic acid sequence initially hybridized to the adapter or primer sequence on the surface of the support may be less than or equal to that indicated for the surface density of the tethered primer. In some cases, the surface density of the cloned amplified target library nucleic acid sequence hybridized to the adapter or primer sequence on the surface of the support may span the same range as indicated for the surface density of the tethered primer.

Since the local densities listed above do not exclude density variations across the surface, the surface comprises, for example, a ^{region having an oligo density of 500,000/μm 2} , while also comprising at least a second region having a substantially different local density. can do.

VI. Exemplary Alternative Embodiments

The disclosed method for determining the sequence of a target nucleic acid comprises the steps of: a) a double-stranded or partially double-stranded target nucleic acid molecule comprising a template strand to be sequenced and a primer strand to be extended into one or more disclosed nucleic acid binding compositions contacting with; and b) detecting binding of the nucleic acid binding composition to the nucleic acid molecule, such that the presence of one of said one or more nucleic acid binding compositions on said nucleic acid molecule and the next nucleotide (i.e., N+1 or terminal nucleotide) to be incorporated into the complementary strand determining the substance.

The sequencing method further comprises incorporating N+1 or terminal nucleotides into the primer strand and then repeating the contacting, detecting, and incorporation steps for one or more additional repeats to determine the sequence of the template strand of the nucleic acid molecule. can do. After detecting the ternary binding complex, the primed strand of the primed target nucleic acid is extended for one base before another round of analysis is performed. The primed target nucleic acid can be extended using conjugated nucleotides attached to a polymer in the multivalent binding composition or using unconjugated or untethered free nucleotides that are provided after the multivalent binding composition is removed.

Extension of the primed target nucleic acid can be prevented or inhibited due to the use of blocked nucleotides or catalytically inactive polymerases on the strand. If the nucleotide of the polymer-nucleotide conjugate has a blocking group that prevents elongation of the nucleic acid, incorporation of the nucleotide can be accomplished by removing the blocking group from the nucleotide (e.g., by separation of the nucleotide from a polymer, branched polymer, dendrimer, particle, etc.) ) can be achieved. When extension of the primed target nucleic acid is inhibited due to the use of a catalytically inactive polymerase, incorporation of nucleotides can be achieved by provision of an activator or cofactor such as a metal ion.

Detection of the ternary complex is accomplished prior to, concurrently with, or after incorporation of the nucleotide residues. In some embodiments, a primed target nucleic acid may comprise a target nucleic acid having multiple primed positions for attachment of a polymerase and/or nucleic acid binding moiety. In some embodiments, multiple polymerases may be attached to a single target nucleic acid molecule, such as at multiple sites within the target nucleic acid molecule. In some embodiments, multiple polymerases may be bound to a multivalent binding composition disclosed herein comprising multiple nucleotides. In some embodiments, the target nucleic acid molecule is used for strand displacement synthesis, rolling circle amplification, concatenation or fusion of multiple sequences of a query sequence, or for generating a nucleic acid molecule comprising multiple copies of the same sequence known in the art. or the product of other methods disclosed elsewhere herein. Thus, in some embodiments, multiple polymerases may be attached at multiple identical or substantially identical positions within a target nucleic acid comprising multiple identical or substantially identical copies of a query sequence. In some embodiments, the multiple polymerases may be involved in the interaction with more than one multivalent binding complex; However, in a preferred embodiment, the number of binding sites in the target nucleic acid is at least two and the number of nucleotides or substrate moieties present in the particle-nucleotide conjugates such as polymer-nucleotide conjugates is also two or more.

It may be advantageous to provide a multivalent binding composition in combination with other elements, such as to provide an optimized signal, for example, to provide for identification of a nucleotide at a particular position in a nucleic acid sequence. In some embodiments, the compositions disclosed herein are provided in combination with a surface that provides low background binding or low levels of protein binding, particularly a hydrophilic or polymer coated surface. Representative surfaces can be found, for example, in US Patent Application Serial No. 16/363,842, the content of which is incorporated herein by reference in its entirety.

In some cases, the nucleic acid molecule is tethered to the surface of the solid support, for example, via hybridization of a template strand to an adapter nucleic acid sequence or a primer nucleic acid sequence tethered to the solid support. In some cases, the solid support comprises a glass, bonded-silica, silicon, or polymer matrix. In some cases, the solid support comprises a low non-specific binding coating comprising at least one hydrophilic polymer layer (e.g., a PEG layer), wherein at least one of the hydrophilic polymer layers comprises a branched polymer molecule (e.g., 4, branched PEG molecules comprising 8, 16, or 32 branches).

The solid support is an oligonucleotide tethered to a surface density of about 1,000,000 primer per molecule ranges from about 1,000 to primer molecules per μm ² μm ^2, at least one hydrophilic polymer layer comprises a nucleotide primer or adapter. In some cases, the surface density of the oligonucleotide primer may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules per ^{μm 2 .} In some cases, the surface density of the oligonucleotide primers may be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules per ^{μm 2 .} Any lower limit value and the lower limit value described in this paragraph may be combined to form a range included in the present specification, for example, in some cases the surface density is about 100,000 per approximately 10,000 molecules to μm ² per μm ² of the primers It can be a range of molecules. One of ordinary skill in the art will recognize that the surface density of primer molecules can have any value within this range, for example, about 455,000 molecules per ^{μm 2 .}

One skilled in the art will recognize that in a series of iterative sequencing reactions, sometimes one or more sites fail to incorporate nucleotides during a given cycle, resulting in one or more sites being unsynchronized with the bulk of the extending nucleic acid chain. In conditions where the sequencing signal is derived from a reaction that occurs in a single copy of the target nucleic acid, such incorporation failures yield individual errors in the output sequence. The purpose of this specification is to describe a method for reducing this type of error in a sequencing reaction. For example, the use of multivalent substrates capable of incorporation into the extending strand by providing increased likelihood of recombination upon premature dissociation of the ternary polymerase complex may reduce the frequency of "skipped" cycles in which no base is incorporated. can Accordingly, in some embodiments, the present invention is encompassed within nucleotides having a 5' phosphate, diphosphate, or triphosphate moiety, wherein the nucleoside moiety is free or reversibly modified, wherein such nucleotides are unstable or Or contemplate the use of a polyvalent substrate disclosed herein that is linked to a particle or polymer disclosed herein via a cleavable linkage. In some embodiments, the present specification contemplates a reduction in intrinsic error rates due to skipped incorporation as a result of using the multivalent substrates disclosed herein.

The present invention also contemplates sequencing reactions in which a sequencing signal from or related to a given sequence originates from or within a definable region comprising multiple copies of the target sequence. Sequencing methods that incorporate multiple copies of a target sequence offer the advantage that the signal is amplified due to the presence of multiple simultaneous sequencing reactions within a defined region, each providing a unique signal. The presence of multiple signals within the defined region also reduces the effect of any single skipped cycle due to the fact that the signal of a large number of correct base confirmations can overwhelm the signal of a small number of skips or false confirmations. The present invention further contemplates the inclusion of free, unlabeled nucleotides during the extension reaction, or during a separate portion of the extension cycle, to provide incorporation at sites that may have been skipped in previous cycles. For example, during or after the incorporation cycle, unlabeled blocked nucleotides can be added to allow incorporation at the skipped site. Unlabeled blocked nucleotides may be of the same type or types as the multivalent binding substrate or nucleotides attached to the substrates that were or were present during a particular cycle, or of 1, 2, 3, 4 or more types of unlabeled blocked nucleotides. Mixtures may be included.

If each sequencing cycle goes through perfectly, each reaction within the defined region will give the same signal. However, as noted elsewhere herein, in a series of iterative sequencing reactions, sometimes one or more sites fail to incorporate nucleotides during a given cycle, causing one or more sites to become out of synchronization with the bulk of the extending nucleic acid chain. will recognize that it is not This problem, also referred to as “phasing,” leads to degradation of the sequencing signal as the signal is contaminated with spurious signals from sites having one or more cycles skipped. This in turn creates a possibility for errors in base identification. The gradual accumulation of skipped cycles through multiple cycles also reduces the effective read length as the sequencing signal gradually degrades with each cycle. It is additionally an object of the present invention to provide a method for reducing paging error and/or improving read length in a sequencing reaction.

A sequencing method may comprise contacting a target nucleic acid or multiple target nucleic acids, comprising multiple linked or unlinked copies of a target sequence, with a multivalent binding composition described herein. Contacting the target nucleic acid comprising multiple linked or unlinked copies of the target sequence, or multiple target nucleic acids with one or more particle-nucleotide conjugates can substantially increase the local concentration of the correct nucleotides investigated in a given sequencing cycle, and , thus inhibiting signals from inappropriately incorporated or paged nucleic acid chains (ie, extending nucleic acid chains with one or more skipped cycles).

A method of obtaining nucleic acid sequence information may comprise contacting a target nucleic acid, or multiple target nucleic acids, with one or more particle-nucleotide conjugates, wherein the target nucleic acid or multiple target nucleic acids are multiple linked or unlinked of the target sequence. Includes unpublished copies. This method reduces the error rate of sequencing as indicated by a reduction in base misidentification, non-existent base reporting, or failure to report correct bases. In some embodiments, the reduction in the error rate of said sequencing is compared to an error rate observed using a monovalent ligand, including free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides. 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more.

A method of obtaining nucleic acid sequence information may comprise contacting a target nucleic acid, or multiple target nucleic acids, with one or more particle-nucleotide conjugates, wherein the template nucleic acid or multiple target nucleic acids are multiple linked or unlinked of the target sequence. Includes unpublished copies. This method compares the average read length to 5 observed using a monovalent ligand, including free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides. %, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more.

Disclosed herein is a method of obtaining nucleic acid sequence information, the method comprising contacting a target nucleic acid, or multiple target nucleic acids, with one or more particle-nucleotide conjugates, wherein the target nucleic acid or multiple target nucleic acids are It includes multiple linked or unlinked copies of a sequence. This method compares the average read length with the average read length observed using monovalent ligands, including free nucleotides, labeled free nucleotides, protein or peptide bound nucleotides, or labeled protein or peptide bound nucleotides, to 10 nucleotides (NT), 20 NT, 25 NT, 30 NT, 50 NT, 75 NT, 100 NT, 125 NT, 150 NT, 200 NT, 250 NT, 300 NT, 350 NT, 400 NT, 500 NT.

In some cases, the disclosed compositions and methods can result in average read lengths ranging from 100 nucleotides to 1,000 nucleotides for sequencing applications. In some cases, the average read length is at least 100 nucleotides, at least 200 nucleotides, at least 225 nucleotides, at least 250 nucleotides, at least 275 nucleotides, at least 300 nucleotides, at least 325 nucleotides, at least 350 nucleotides, at least 375 nucleotides. nucleotides, at least 400 nucleotides, at least 425 nucleotides, at least 450 nucleotides, at least 475 nucleotides, at least 500 nucleotides, at least 525 nucleotides, at least 550 nucleotides, at least 575 nucleotides, at least 600 nucleotides, at least 625 nucleotides nucleotides, at least 650 nucleotides, at least 675 nucleotides, at least 700 nucleotides, at least 725 nucleotides, at least 750 nucleotides, at least 775 nucleotides, at least 800 nucleotides, at least 825 nucleotides, at least 850 nucleotides, at least 875 nucleotides nucleotides, at least 900 nucleotides, at least 925 nucleotides, at least 950 nucleotides, at least 975 nucleotides, or at least 1,000 nucleotides. In some cases, the average read length is a range bounded by any two values within the range, For example, it can be an average read length of 375 nucleotides to 825 nucleotides. One of ordinary skill in the art will recognize that, in some cases, the average read length may have any value within the range specified in this paragraph, eg, 523 nucleotides.

The use of a multivalent binding composition for sequencing effectively shortens the sequencing time. The sequencing reaction cycle, including the contacting, detecting, and incorporation steps, is performed for a total time ranging from about 5 minutes to about 60 minutes. In some cases, the sequencing reaction cycle is performed within at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some cases, the sequencing reaction cycle is performed within at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any lower and lower limits set forth in this paragraph may be combined to form ranges encompassed within this specification, for example, in some cases the sequencing reaction cycle may be performed for a total time ranging from about 10 minutes to about 30 minutes. have. One of ordinary skill in the art will recognize that the sequencing cycle time can be any value within the above range, for example, about 16 minutes.

In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, over the course of a sequencing run, will provide an average base determination accuracy that is at least 99.5%, at least 99.8%, or at least 99.9% accurate. In some cases, at least 80%, at least 85%, at least 90%, at least every 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases called, disclosed compositions and methods for sequencing nucleic acids 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% accurate mean base determination accuracy.

The use of multivalent binding compositions for sequencing provides more accurate base reads. The disclosed compositions and methods for sequencing nucleic acids will provide an average Q-score ranging from about 20 to about 50 for base determination accuracy over a sequencing run. In some cases, the average Q-score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. One of ordinary skill in the art will recognize that the average Q-score can be any value within this range, for example, about 32.

In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 of the identified terminal (or N+1) nucleotides. %, at least 98%, or at least 99% will provide a Q-score greater than 30. In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 of the identified terminal (or N+1) nucleotides. %, at least 98%, or at least 99% will provide a Q-score greater than 35. In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 of the identified terminal (or N+1) nucleotides. %, at least 98%, or at least 99% will provide a Q-score greater than 40. In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 of the identified terminal (or N+1) nucleotides. %, at least 98%, or at least 99% will provide a Q-score greater than 45. In some cases, the disclosed compositions and methods for sequencing nucleic acids comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95 of the identified terminal (or N+1) nucleotides. %, at least 98%, or at least 99% will provide a Q-score greater than 50.

The disclosed low non-specific binding scaffolds and related nucleic acid hybridization and amplification methods can be used for the analysis of nucleic acid molecules derived from any of a variety of different cells, tissues, or sample types known to those of skill in the art. For example, nucleic acids can be extracted from a cell, or tissue sample, including one or more types of cells derived from eukaryotes (eg, animals, plants, fungi, protists), archaea, or eubacteria. In some cases, nucleic acids may be extracted from prokaryotic or eukaryotic cells, adherent or non-adherent eukaryotic cells. Nucleic acids are variously extracted from, for example, primary or immortalized rodent, pig, cat, dog, bovine, horse, primate or human cell lines. Nucleic acids can be used in any of a variety of different cell, organ, or tissue types (eg, leukocyte cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, germ cells). or cells from the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon or small intestine). Nucleic acids can be extracted from normal or healthy cells. Alternatively or in combination, the nucleic acid is extracted from a diseased cell, such as a cancer cell, or from a pathogenic cell that infects a host. A distinct subset of some nucleic acid cell types, e.g., immune cells (such as T cells, cytotoxic (killer) T cells, helper T cells, alpha beta T cells, gamma delta T cells, T cell progenitors, B cells) , B-cell progenitor cells, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and/or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells induced to differentiate, rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts). Nucleic acids may further include nucleic acids derived from viral samples and subviral pathogens such as viroids and infectious RNA. Nucleic acids can be derived from clinical or other samples such as sputum, saliva, ocular fluid, synovial fluid, blood, feces, urine, tissue exudates, sweat, pus, drainage, and the like. The nucleic acid may further be derived from a plant or fungal sample, such as from a leaf, cambium, root, meristem, pollen, egg, seed, spore, inflorescence, mycelium, and the like. Nucleic acids can also be derived from environmental or industrial samples, such as water, air, dust, food, and the like. Other cells, tissues, and samples are contemplated and consistent with the present disclosure.

Extraction of nucleic acids from cells or other biological samples can be performed using any of a variety of techniques known to those of skill in the art. For example, a DNA extraction procedure may include the following steps: (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane to release DNA and other cytoplasmic components (i.e., cell lysis) , (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, centrifuging to isolate the precipitated proteins, lipids, and RNA, and (iv) purifying DNA from the supernatant; Removal of detergents, proteins, salts, or other reagents used in the cell membrane lysis step.

A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the present disclosure. Examples include QIAamp kit (isolation of genomic DNA from human samples) and DNAeasy kit (isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or Maxwell® and ReliaPrep™ series kits from Promega (Madison, WI) However, it is not limited thereto.

VII. system

System Module: As noted above, also disclosed herein is a system configured to perform any nucleic acid sequencing or nucleic acid detection and analysis method disclosed herein. In some cases, the disclosed systems may include one or more multivalent binding compositions described herein, one or more buffers, and/or one or more nucleic acid molecules tethered to a solid support.

In some cases, the system is configured to sequentially and repeatedly contact a tethered template nucleic acid molecule hybridized to a nucleic acid molecule (eg, adapter or primer) tethered to a solid support with a disclosed multivalent binding composition and/or reagent. It may further include a configured fluid flow controller and/or fluid distribution system. In some cases, the contacting step may be performed in one or more flow cells. In some cases, the flow cell may be a stationary component of a system. In some cases, the flow cell may be a removable and/or disposable component of the system.

In some cases, the system may further comprise an imaging module, wherein the imaging module is configured to image and detect binding of a disclosed multivalent binding composition to a target (or template) nucleic acid molecule tethered inside a solid support or flow cell. For example, one or more light sources, one or more optical components (eg, lenses, mirrors, prisms, optical filters, color glass filters, narrowband interference filters, broadband interference filters, dichroic reflectors, diffraction gaps, apertures) , optical fibers, or optical waveguides, etc.), and one or more image sensors (eg, charge-coupled device (CCD) sensors or cameras, complementary metal-oxide-semiconductor (CMOS) ) image sensor or camera, or negative-channel metal-oxide semiconductor (NMOS) image sensor or camera).

Processors and computer systems: One or more processors may be used to implement systems for nucleic acid sequencing or other nucleic acid detection and analysis methods disclosed herein. The one or more processors may include a hardware processor, such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit, or a computing platform. The one or more processors may include any of a variety of suitable integrated circuits (eg, purpose-built integrated circuits (ASICs) specifically designed to implement deep learning network architectures), or to accelerate computation time, etc., and/or facilitate deployment. It may be composed of a field-programmable gate array (FPGA)), a microprocessor, an emerging next-generation microprocessor design (eg, a memristor-based processor), a logic device, and the like. Although this specification is described with reference to a processor, other types of integrated circuits and logic elements may also be applied. The processor may have any suitable data operation function. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. The one or more processors may be single-core or multi-core processors, or a plurality of processors configured for parallel processing.

The one or more processors or computers used to implement the disclosed methods may be part of a larger computer system and/or are operable to a computer network (“network”) with the aid of a communication interface to facilitate the transfer and sharing of data. can be connected The network may be a local area network, an intranet and/or extranet, an intranet and/or extranet that communicates with the Internet, or the Internet. The network is in some cases a telecommunications and/or data network. A network may include one or more computer servers, which in some cases enable distributed computing, such as cloud computing. The network, in some cases with the aid of a computer system, may implement a peer-to-peer network, which enables a device connected with the computer system to act as either a client or a server.

The computer system also communicates with memory or memory locations (eg, random-access memory, read-only memory, flash memory, Intel® Optane™ technology), electronic storage devices (eg, hard disk), and one or more other systems. communication interfaces (eg, network adapters), and peripherals such as caches, other memory, data storage, and/or electronic display adapters. Memory, storage, interfaces, and peripherals may communicate with one or more processors, eg, a CPU, via a communication bus, eg, as found on a motherboard. The storage device(s) may be data storage device(s) (or data storage) for storing data.

One or more processors, eg, a CPU, execute a series of machine readable instructions, which are implemented in a program (or software). Instructions are stored in memory locations. The instructions are directed to the CPU, which subsequently programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of operations performed by the CPU include fetching, decoding, executing, and rewriting. The CPU may be part of a circuit such as an integrated circuit. One or more other components of the system may be included in the circuitry. In some cases, the circuit is an application specific integrated circuit (ASIC).

The storage device stores files such as drivers, libraries and stored programs. The storage device stores user data, such as user-specified preferences and use-specified programs. The computer system may in some cases include one or more additional data storage devices located external to the computer system, such as on a remote server that communicates with the computer system via an intranet or the Internet.

Some aspects of the methods and systems provided herein may be implemented via machine (e.g., processor) executable code stored in electronic storage, e.g., memory, or electronic storage of a computer system. . The machine-executable or machine-readable code may be provided in the form of software. During use, the code is executed by one or more processors. In some cases, the code is retrieved from the storage device and stored in memory for immediate access by one or more processors. In some circumstances, electronic storage is excluded, and machine-executed instructions are stored in memory. The code may be precompiled and configured for use with a machine having one or more processors configured to execute the code, or may be compiled at runtime. The code may be provided in a programming language of choice to be run precompiled or in an as-compiled fashion.

Various aspects of the technology are often referred to as "products" or "articles of manufacture", e.g., "computer programs or software products," in the form of machine- (or processor-) executable code and/or related data stored on a type of machine-readable medium. may be considered, wherein executable code includes a plurality of instructions for controlling a computer or computer system to perform one or more of the methods disclosed herein. The machine-executable code may be stored on an optical storage device including an optically readable medium such as an optical disc, CD-ROM, DVD or Blu-ray disc. The machine-executable code may be stored in memory (eg, read-only memory, random access memory, flash memory) or an electronic storage device such as a hard disk. "Storage" tangible media includes some or all of the tangible memory of a computer, processor, etc. or tangible memory of various semiconductor memory chips, optical drives, tape drives, disk drives, etc. may provide temporary storage for software that encodes

All or part of the software code may be communicated from time to time over the Internet or various other communication networks. For example, such communication may enable loading of software from one computer or processor to another computer or processor, eg, from a management server or host computer to a computer platform of an application server. Accordingly, other types of media used to convey software-encoded instructions include optical, electrical, and electromagnetic waves such as those used over physical interfaces between local devices, wired and optical wired networks, and various atmospheric links. Physical elements that carry these waves, such as wired or wireless links, optical links, etc., are also considered media carrying software encoded instructions for performing the methods disclosed herein. As used herein, unless limited to a temporary tangible "storage" medium, terms such as computer or machine "readable medium" mean any medium that participates in providing instructions to a processor for execution.

A computer system often includes or may communicate with an electronic display, for example, to provide an image captured by a machine vision system. A display may often provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, and the like.

System Control Software: In some cases, the disclosed systems provide a user interface, as well as manual, semi-automatic, or fully automatic control of all system functions, eg, a fluid flow controller and/or a fluid distribution system (or sub-system), temperature control. a computer (or processor) and computer-readable medium comprising code for providing control of a system (or sub-system), an imaging system (or sub-system), and the like . In some cases, the system computer or processor may be an integrated component of a device system (eg, a microprocessor or motherboard embedded within the device). In some cases, the system computer or processor may be a standalone module, such as a personal computer or laptop computer. Examples of fluid flow control functions that may be provided by instrument control software include, but are not limited to, volumetric fluid flow rates, fluid flow rates, timing and duration for sample and reagent additions, cleaning steps, and the like. Temperature control functions that may be provided by the instrument control software include, but are not limited to, specifying temperature setpoint(s) and timing, duration, and ramp rate for temperature changes. Examples of imaging system control functions that may be provided by instrument control software include, but are not limited to, autofocus functions, illumination or excitation light exposure time and intensity control, image acquisition rate control, exposure time, data storage options, etc. does not

Image processing software: In some cases of the disclosed systems, the system can further include a computer readable medium comprising code for providing image processing and analysis capabilities. Image processing and analysis capabilities that may be provided by the software include manual, semi-automatic, or fully automatic image exposure adjustment (eg, white balance, contrast adjustment, signal-averaging and other noise reduction capabilities, etc.), manual, semi-automatic, or fully automatic edge detection and object identification (eg, identification of clusters of template nucleic acid molecules amplified on a substrate surface), manual, semi-automatic, or fully automatic signal intensity measurement and/or one or more detection channels (eg, one or more fluorescence emission) in the channel), manual, semi-automated, or fully automated statistical analysis (eg, comparing signal intensity with a reference value for base determination purposes).

In some cases, system software provides integrated real-time image analysis and instrument control so that sample loading, reagent addition, washing, and/or imaging/base determination steps are required, for example, until optimal base determination results are achieved. may be extended, modified, or repeated according to Any of a variety of image processing and analysis algorithms known to those of ordinary skill in the art may be used to implement real-time or post-processed image analysis capabilities. Examples of such algorithms include Canny edge detection method, Canny-Derich edge detection method, first-order gradient edge detection method (e.g., Sobel operator), second-order differential edge detection methods, phase matching (phase-consistent) edge detection methods, other image segmentation algorithms (e.g., intensity threshold setting, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., arbitrary generalized Hough transform, circular Hough transform, etc.), and mathematical analysis algorithms (eg, Fourier transform, Fast Fourier transform, wavelet analysis, autocorrelation, etc.) , or a combination thereof.

In some cases, the system control and image processing/analysis software may be written as separate software modules. In some cases, system control and image processing/analysis software may be included in an integrated software package.

VIII. Example

1. Preparation of Multivalent Binding Compositions

One type of multi-arm substrate shown in Figure 5A was prepared by reacting propargylamine dNTPs with biotin-PEG-NHS. This aqueous reaction was completed and purified; A pure biotin-PEG-dNTP species was generated. In a separate reaction, several different PEG lengths were used, with average molecular weights varying from 1 K Da to 20 K Da. Biotin-PEG-dNTP species were mixed with freshly prepared or commercially-supplied dye-labeled streptavidin (SA) in a Dye:SA ratio of 3-5:1. Mixing of biotin-PEG-dNTPs with dye-labeled streptavidin was performed in the presence of excess biotin-PEG-dNTPs to ensure saturation of the biotin binding sites of each streptavidin tetramer. The complete complex was purified from excess biotin-PEG-dNTP by size exclusion chromatography. Each nucleotide type was individually conjugated and purified and then mixed together to create a 4-base mixture for sequencing.

Another type of multi-arm substrate shown in Figure 5A was prepared by reacting multi-arm PEG NHS with excess dye-NH _{2 and propargylamine dNTPs in a single pot.} Various multi-arm PEG NHS variants were used in the 4-16 arm range and molecular weight range from 5 K Da to 40 K Da. After the reaction, excess small molecule dye and dNTPs were removed by size exclusion chromatography. Each nucleotide type was independently conjugated and purified and then mixed together to create a 4-base mixture for sequencing.

The class II substrate shown in Figure 5B was prepared using a one pot reaction to conjugate the dye and dNTPs simultaneously. Alkyne-PEG-NHS was reacted with an excess of propargylamine dNTPs. This product (alkyne-PEG-dNTP) was purified to homogeneity by chromatography. Multiple PEG lengths were used and the average molecular weight varied between 1 K Da and 20 K Da. A dendrimer core containing a variable and discrete number (12, 24, 48, 96) of azide conjugation sites was used. Conjugation of alkyne-dye and alkyne-PEG-dNTP to the dendrimeric core occurred in a one pot reaction involving excess dye and dNTP species via copper mediated click chemistry. After the reaction, excess small molecule dye and dNTPs were removed by size exclusion chromatography. Each nucleotide type was independently conjugated and purified and then mixed together to create a 4-base mixture for sequencing. This plan focuses on dextran, other polymers that can immediately replace the core of alternatives, such as proteins.

The class III polymer-nucleotide conjugates shown in Figure 5C were constructed by reacting 4- or 8-arm PEG NHS with a saturated mixture of biotin and propargylamine dNTPs. The reaction was purified by size exclusion chromatography. This reaction resulted in a multi-arm PEG with separate distributions of biotin and nucleotides. This heterogeneous population was reacted with dye-labeled streptavidin and purified by size exclusion chromatography. Each nucleotide type was independently conjugated and purified and then mixed together to create a 4-base mixture for sequencing. Note that the distribution of biotin and nucleotides is tunable by the input ratio of _{biotin-NH 2 to propargylamine dNTPs.}

2. Detection of ternary complexes

Binding reactions using multivalent binding compositions with PEG polymer-nucleotide conjugates were analyzed to detect possible formation of ternary binding complexes, fluorescence images of various stages are illustrated in Figures 7A-7J. In FIG. 7A , red and green fluorescence images show DNA rolling circle application (RCA) templates (G and A first bases) for 500 nM base labeled nucleotides (A-Cy3 and G-Cy5) with 20 nM Klenow polymerase and After exposure in exposure buffer containing 2.5 mM Sr ^{+2 .} Polyvalent PEG-substrate compositions were prepared using various ratios of 4-arm EG-amine (4ArmPEG-NH), biotin-PEG-amine (biotin-PEG-NH), and cleotide (Nuc): Sample PB1 and PB5, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.25: 1: 0.5; Sample PB2, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.125: 0.5: 0.25; Sample PB3, 4ArmPEG-NH: Biotin-PEG-NH: Nuc = 0.25: 1: 0.5. Images were collected after washing with an imaging buffer of the same composition as the exposure buffer but without nucleotides or polymerase.

Contrast was adjusted to maximize visualization of the faintest signal, but the signal did not persist after washing with imaging buffer ( Figure 7A , inset). In Figures 7B-7E , the fluorescence images show the multivalent PEG-nucleotide (base-labeled) ligand at 500 nM after mixing in exposure buffer and imaging in the same imaging buffer as above ( Figure 7B : PB1; Figure 7C : PB2; Figure 7C: PB2; 7D : PB3; Fig. 7E : PB5). Figure 7F : Fluorescence image showing the multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uM after mixing in exposure buffer and imaging in the same imaging buffer as above. In Figures 7G-7I , the fluorescence images show additional base discrimination by multivalent ligand exposure to an inactive mutant of Klenow polymerase ( Figure 7G : D882H; Figure 7H : D882E; Figure 7I : D882A, and wild-type Klenow (control)) enzymes as shown in Figure 7J ).

Multivalent ligand formulations can be used to allow for base identification by providing polymerase-ligand interactions with increased binding. Furthermore, we show that increasing the concentration of multivalent ligands can provide a higher signal, as well as various Klenow mutations that knock out catalytic enzyme activity that can be used for avidity-based sequencing.

3. Sequencing of Target Nucleic Acid Molecules Using Ternary Complexes

To demonstrate multivalent ligand reporter-based sequencing, four known templates were amplified using the RCA method in low binding substrates. Successive cycles were exposed to exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr ^{+2, washed with imaging buffer and imaged.} After imaging, the substrate was washed with wash buffer (EDTA and high salt) and blocked nucleotides were added to proceed to the next base. The cycle was repeated for 5 cycles. Spots were detected using standard imaging processing and spot detection, and sequences were called using a two-color green and red scheme (G-Cy3 and A-Cy5) to identify cycled templates. As shown in Figures 8A and 8B , multivalent ligands can provide base discrimination through all five sequencing cycles.

4. Control of Nucleotide Dissociation from Ternary Complexes

Ternary complexes were prepared and imaged as in Example 2. To demonstrate the persistence of the ternary complex, image the complex over various lengths of time (e.g., 60 s). After a period of time, the same buffer as used to form the complex, but without any divalent cations, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% Triton X100 (without SrOAc) Wash the complex with a buffer, or, alternatively, any divalent cation including a chelating agent, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, identical to the buffer used to form the complex. , 0.016% Triton X100 (without SrOAc), and 100 nm-100 mM EDTA free buffer. Fluorescence from the complex is observed over time, allowing observation and quantification of dissociation of the ternary complex. A representative time course of this dissolution is shown in FIG. 6 .

5. Extension of the target nucleic acid complementary sequence

After preparing, imaging, and dissociating the ternary complex as in Example 4, a blocking moiety such as an O-azidomethyl group, an O-alkyl hydroxylamino group, or an O- Sufficient unblocking solution to remove the amino group is introduced into the chamber containing the bound DNA molecule. Accordingly or concurrently, an extension solution is introduced into the chamber containing the bound DNA molecules. The extension solution contains a buffer, a divalent cation sufficient to aid support polymerase activity, an active polymerase, and an appropriate amount of all four nucleotides, wherein the nucleotides are added to the extending DNA strand after addition of a single nucleotide, such as 3' An —O-azidomethyl group, a 3′-O-alkyl hydroxylamino group, or a 3′-O-amino group is blocked from further extension by incorporation. The extended strand is thus extended by only one base, and binding of the catalytically inactive polymerase and the multivalent binding substrate can be used to call the next base in the cycle.

Alternatively, nucleotides attached to the multivalent substrate are attached via labile bonds and buffer into a chamber containing bound DNA molecules containing sufficient divalent cations or other cofactors to render the polymerase catalytically active. This can be imported. Before, after, or concurrently with this, conditions may be provided sufficient to cleave the base from the multivalent substrate for incorporation into the extending strand. This cleavage and incorporation results in dissociation of the label and polymer backbone of the multivalent substrate, extending the extending DNA strand by exactly one base. Washing to remove the used polymer backbone is performed, and a new polyvalent substrate is introduced into the chamber containing the bound DNA molecule, and a new base is called up as in Example 1.

6. Use of Polymer-Nucleotide Conjugates with PEG Branches of Various Lengths

Polymer-nucleotide conjugates with various PEG arm lengths described in Example 3 were subjected to a single sequencing cycle and imaged as described in Example 1. As shown in Figures 9A-9J , increasing the length of the PEG branch increased the signal up to a length corresponding to the average PEG MW of 5 K Da ( Figures 9A-9D ). Using a longer PEG arm reduced the fluorescence signal for both Cy3-A and Cy5-G ( FIGS. 9E-9G ). A quantitative measure of signal strength is graphically shown in FIG . 10 .

7. Enhancement of Multivalent Substrate Binding by Addition of Detergent

Multivalent substrates were prepared and assembled into binding complexes in the presence and absence of detergent: one set using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0% TritonX100 (Condition A), and one set The set used 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0.016% Triton X100. Figure 11 shows the normalized fluorescence from the multivalent substrate bound to the DNA cluster, the substrate complex formed in the presence of Triton-X100 (0.016%) (condition B) clearly shows enhanced fluorescence intensity.

8. Evaluation of Multivalent Substrate Binding Time Course

A multivalent substrate was prepared and assembled into a binding complex as in Example 2. Complexes were also formed under the same buffer conditions using labeled free nucleotides. The complexes were imaged over the course of 60 minutes to characterize the duration of the complexes. 12A-12B show representative results. The multivalent binding complex is stable over a >60 min time scale ( FIG. 12B ) while labeled free nucleotides dissociate in less than 1 min ( FIG. 12A ).

VIII. conclusion

The present invention provides greatly improved methods and compositions for DNA sequencing and biosensor applications. It is to be understood that the above description is for purposes of illustration and not limitation. Many embodiments will be apparent to those skilled in the art upon review of the above description. By way of example, while the concepts of the present invention have been described with respect to the use of polymer-nucleotide conjugates, it will be readily appreciated by those skilled in the art that other types of particle-nucleotide conjugates may also be used. For example, in some embodiments quantum dots; liposomes; Alternatively, it may be desirable to use a particle-nucleotide conjugate comprising emulsion particles. Alternatively, conjugation may be accomplished by non-covalent bonding, such as hydrogen bonding or other interactions. Therefore, the scope of the disclosed inventive concept should be determined without reference to the foregoing description, but instead with reference to the appended claims and the full scope of equivalents to which such claims are granted.

Claims (73)

A method for determining the identity of a nucleotide in a target nucleic acid sequence, comprising:
ai two or more copies of the target nucleic acid sequence;
ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and
iii. two or more polymerase molecules
providing a composition comprising;
b. Under conditions sufficient to form a multivalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence of the composition of (a), the composition with the polymer nucleotide conjugate, wherein the polymer-nucleotide conjugate comprises at least two copies of a nucleotide moiety and optionally at least one detectable label; and
c. detecting the multivalent binding complex to determine the identity of the nucleotide in the target nucleic acid sequence;
A decision method comprising The method of claim 1 , wherein the target nucleic acid sequence is DNA. The method according to claim 1 or 2, wherein the step of detecting the multivalent binding complex is carried out in the absence of an unbound or solution-borne polymer nucleotide conjugate. 4. The method according to any one of claims 1 to 3, wherein the target nucleic acid sequence has been replicated or amplified or produced by replication or amplification. 5. The method according to any one of claims 1 to 4, wherein the at least one detectable label is a fluorescent label. 6. The method according to any one of claims 1 to 5, wherein the step of detecting the multivalent complex comprises fluorescence measurement. 7. A method according to any one of claims 1 to 6, wherein the contacting step comprises the use of one type of polymer-nucleotide conjugate. 8. The method according to any one of claims 1 to 7, wherein the contacting step comprises the use of two or more types of polymer-nucleotide conjugates. 9. The method of claim 8, wherein each type of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. 10. The method of claim 9, wherein the contacting step comprises the use of three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. How to decide. 11. The method of any one of claims 1-10, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. The method of claim 11 , wherein the blocked nucleotides are 3′-O-azidomethyl nucleotides, 3′-O-methyl nucleotides, or 3′-O-alkyl hydroxylamine nucleotides. 13. A method according to any one of claims 1 to 12, wherein said contacting occurs in the presence of ions stabilizing said multivalent binding complex. 14. The method of any one of claims 1-13, wherein the contacting step is in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. 15. The method according to any one of claims 1 to 14, wherein the polymerase molecule is catalytically inactive. 16. The method according to any one of claims 1 to 15, wherein the polymerase molecule has been made catalytically inactive by mutation or chemical modification. 17. The method according to any one of claims 1 to 16, wherein the polymerase molecule is made catalytically inactive in the absence of the required ion or cofactor. 18. The method of any one of claims 1-17, wherein the polymerase molecule is catalytically active. 19. The method according to any one of claims 1 to 18, wherein the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. 20. The method according to any one of claims 1 to 19, wherein the multivalent binding complex has a duration of greater than 2 seconds. 21. The method according to any one of claims 1 to 20, which can be carried out at a temperature within the range from 25°C to 62°C. 22. The method of any one of claims 1-21, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and the two or more copies of the target nucleic acid sequence are deposited, attached or hybridized to the surface, wherein the surface wherein the fluorescence image of the multivalent binding complex on the phase has a contrast to noise ratio greater than 20 in the detection step. 23. The method according to any one of claims 1-22, wherein the composition of (a) is deposited on the surface using a buffer incorporating a polar aprotic solvent. 24. The method according to any one of claims 1 to 23, wherein the contacting step stabilizes the multivalent binding complex when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence, and wherein the nucleotide moiety is the target nucleic acid The method of claim 1 , wherein the method is carried out under conditions that destabilize the multivalent binding complex when not complementary to the next base of the sequence. 25. The crystal of any one of claims 1-24, wherein said polymer-nucleotide conjugate comprises a polymer having a plurality of branches, and wherein said two or more nucleotide moieties are attached to said branches. Way. 26. The method of claim 25, wherein the polymer has a star, comb, cross-linked, bottle brush, or dendrimer morphology. 27. The method of any one of claims 1-26, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. 28. The method of any one of claims 1 to 27, further comprising a dissociation step destabilizing the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, wherein the dissociation step comprises the polymer-nucleotide conjugate. A method of determining which enables removal of the nucleotide conjugate. 29. The method of claim 28, further comprising an extension step of incorporating into said two or more primer nucleic acid molecules a nucleotide complementary to the next base of the target nucleic acid sequence. 30. The method of claim 29, wherein the extending step occurs concurrently with or after the dissociating step. A method for determining the identity of a nucleotide in a target nucleic acid sequence, comprising:
ai two or more copies of the target nucleic acid sequence;
ii. two or more primer nucleic acid molecules complementary to one or more regions of the target nucleic acid sequence; and
iii. two or more polymerase molecules
providing a composition comprising;
b. contacting said composition with said polymeric nucleotide conjugate under conditions sufficient to allow a multivalent binding complex to form between said polymer-nucleotide conjugate and said two or more copies of said target nucleic acid sequence of said composition of (a); , wherein the polymer-nucleotide conjugate comprises at least two copies of a reversibly terminated nucleotide moiety and optionally at least one cleavable and detectable label; and
c. detecting the multivalent complex to determine the identity of the nucleotide in the target nucleic acid sequence;
A decision method comprising The method of claim 31 , wherein the target nucleic acid sequence is DNA. 33. The method of claim 31 or 32, wherein after detection of the multivalent binding complex, the composition of (a) is combined with a reversibly terminated nucleotide or a polymer-nucleotide conjugate comprising two or more copies of the reversibly terminated nucleotide; The method of determining further comprising the step of contacting. 34. The method according to any one of claims 31 to 33, wherein the target nucleic acid sequence has been replicated or amplified or produced by replication or amplification. 35. The method of any one of claims 31-34, wherein the at least one detectable label is a fluorescent label. 36. The method according to any one of claims 31 to 35, wherein the step of detecting the multivalent complex comprises fluorescence measurement. 37. The method according to any one of claims 31 to 36, wherein the contacting step comprises the use of one type of polymer-nucleotide conjugate. 38. The method according to any one of claims 31 to 37, wherein the contacting step comprises the use of two or more types of polymer-nucleotide conjugates. 39. The method of claim 38, wherein each type of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. 40. The method of claim 39, wherein the contacting step comprises the use of three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. How to decide. 41. The method of any one of claims 31-40, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. 42. The method of claim 41, wherein the blocked nucleotide is 3'-0-azidomethyl, 3'-0-methyl, or 3'-0-alkyl hydroxylamine. 43. The method according to any one of claims 31 to 42, wherein said contacting occurs in the presence of ions stabilizing said multivalent binding complex. 44. The method of any one of claims 31-43, wherein the polymerase molecule is catalytically inactive. 45. The method according to any one of claims 31 to 44, wherein the polymerase molecule has been made catalytically inactive by mutation or chemical modification. 46. The method of any one of claims 31-45, wherein the polymerase molecule is catalytically active. 47. The method of any one of claims 31-46, wherein the polymer-nucleotide conjugate does not comprise a blocked nucleotide moiety. 48. The method according to any one of claims 31 to 47, which can be carried out at a temperature within the range of 25°C to 80°C. 49. The method of any one of claims 31-48, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and wherein the two or more copies of the target nucleic acid sequence are deposited, attached or hybridized to the surface, wherein the surface wherein the fluorescence image of the multivalent binding complex on the phase has a contrast-to-noise ratio greater than 20 in the detection step. a) i) a substrate comprising multiple copies of a target nucleic acid sequence tethered to the surface of the substrate, a reagent comprising a polymerase and one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence; contacting to form a primed target nucleic acid sequence;
ii) contacting the substrate surface with a reagent comprising the polymer nucleotide conjugate under conditions sufficient to permit the formation of a multivalent binding complex between the polymer-nucleotide conjugate and two or more copies of the primed target nucleic acid sequence; , wherein the polymer-nucleotide conjugate comprises at least two copies of a known nucleotide moiety and a detectable label;
iii) acquiring and processing an image of the substrate surface to detect the multivalent binding complex, thereby determining the identity of the nucleotide in the target nucleic acid sequence;
one or more computer processors individually or collectively programmed to perform a method comprising
a system containing 51. The system of claim 50, further comprising a fluid module configured to deliver the series of reagents to the substrate surface in a designated order and for a designated time interval. 52. The system of claim 50 or 51, further comprising an imaging module configured to acquire an image of the substrate surface. 53. The system of any one of claims 50-52, wherein steps (ii) and (iii) are repeated two or more times to determine the identity of a series of two or more nucleotides in the target nucleic acid sequence. 54. The method of any one of claims 50-53, wherein the series of steps further comprises a dissociation step destabilizing the multivalent binding complex, wherein the dissociation step enables removal of the polymer-nucleotide conjugate. in system. 55. The system of claim 54, wherein the series of steps further comprises an extension step of incorporating into said two or more primer nucleic acid molecules a nucleotide complementary to the next base of the target nucleic acid sequence. 56. The system of claim 55, wherein the extending step occurs concurrently with or after the dissociating step. 57. The system of any one of claims 50-56, wherein the detectable label comprises a fluorophore and the image comprises a fluorescence image. 58. The method of claim 57, wherein the fluorescence image of the multivalent conjugate complex on the surface of the substrate is a dichroic mirror, wherein the fluorophore is cyanine dye 3 (Cy3), 20X objective, NA = 0.75, optimized for 532 nm light. , using an inverted fluorescence microscope equipped with a bandpass filter optimized for cyanine dye-3 emission, and a camera, images were taken under non-signal saturation conditions while the surface was immersed in 25 mM ACES, pH 7.4 buffer. If obtained, the system has a contrast-to-noise ratio greater than 20. 59. The system of any one of claims 50-58, wherein the series of steps are completed in less than 60 minutes. 60. The system of any one of claims 50-59, wherein the series of steps are completed in less than 30 minutes. 61. The system of any one of claims 50-60, wherein the series of steps are completed in less than 10 minutes. 62. The system of any one of claims 50-61, wherein the accuracy of base-calling is a Q-score greater than 25 for at least 80% of the determined nucleotide entities. 63. The system of any one of claims 50-62, wherein the accuracy of base determination is a Q-score greater than 30 for at least 80% of the determined nucleotide entities. 64. The system of any one of claims 50-63, wherein the accuracy of base determination is a Q-score greater than 40 for at least 80% of the determined nucleotide entities. a) a polymer core; and
b) two or more nucleotides, nucleotide analogues, nucleosides, or nucleoside analogue moieties attached to the polymer core
As a composition comprising a,
wherein the length of the linker is dependent on the nucleotide, nucleotide analogue, nucleoside, or nucleoside analogue moiety attached to the polymer core. a) each polymer-nucleotide conjugate is
i) a polymer core; and
ii) two or more nucleotides, nucleotide analogues, nucleosides, or nucleoside analogue moieties attached to the polymer core
A mixture of polymer-nucleotide conjugates comprising
As a composition comprising a,
the length of the linker depends on the nucleotide, nucleotide analogue, nucleoside, or nucleoside analogue moiety attached to the polymer core;
wherein the mixture comprises a polymer-nucleotide conjugate having at least two different types of attached nucleotides, nucleotide analogues, nucleosides, or nucleoside analogue moieties. 67. The method of claim 65 or 66, wherein the polymeric core comprises a polymer having a plurality of branches, wherein two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties are attached to the branches. composition. 68. The composition of claim 67, wherein the polymer has a star, comb, crosslinked, bottle brush, or dendrimer shape. 69. The composition of any one of claims 65-68, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. 70. The composition of any one of claims 65-69, wherein the polymeric core comprises branched polyethylene glycol (PEG) molecules. 71. The composition of any one of claims 65-70, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. 72. The composition of claim 71, wherein the blocked nucleotides are 3'-0-azidomethyl nucleotides, 3'-0-methyl nucleotides, or 3'-0-alkyl hydroxylamine nucleotides. 73. The composition of any one of claims 65-72, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels.

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