KR102607124B1 - Multivalent binding compositions for nucleic acid analysis - Google Patents

Abstract

A multivalent binding composition comprising a particle-nucleotide conjugate having multiple copies of a nucleotide attached to the particle is described. Multivalent binding compositions allow localization of a detectable signal to the active site of a biochemical interaction, e.g., protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzyme reaction, polymerase reaction. It can be used to identify base incorporation sites within an extending nucleic acid chain and 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 on September 23, 2019, U.S. Provisional Patent Application No. 62/897,172, filed on September 6, 2019, and U.S. Provisional Patent Application No. 1, filed on May 24, 2019. 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 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 increases the local concentration of nucleotides and enhances the binding signal. Multivalent binding compositions can be applied, for example, in sequencing and biosensor microarray fields.

Nucleic acid sequencing can be used to obtain information in a variety of biomedical contexts, including diagnosis, prognosis, 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 existing technologies.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method comprising the following steps: 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 cause the formation of a multivalent binding complex between a polymer nucleotide conjugate and the two or more copies of the target nucleic acid sequence of the composition (a), the composition contacting said 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 and determining 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 multivalent binding complexes is performed in the absence of unbounded or solution-borne polymer nucleotide conjugates. In some embodiments, the target nucleic acid sequence has been cloned or amplified, or was produced by cloning or amplification. In some embodiments, the one or more detectable labels are fluorescent labels. In some embodiments, the step of detecting the multivalent complex includes measuring fluorescence. In some embodiments, the contacting step includes the use of one type of polymer-nucleotide conjugate. In some embodiments, the contacting step involves 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 includes the use of three types of polymer-nucleotide conjugates, where 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 a 3'-O-azidomethyl nucleotide, a 3'-O-Methyl nucleotide, or a 3'-O-alkyl nucleotide. It is a 3'-O-alkyl hydroxylamine nucleotide. In some embodiments, the contacting step occurs in the presence of an ion that stabilizes the multivalent bond complex. In some embodiments, the contacting step occurs 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 rendered catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is rendered catalytically inactive by the absence of a necessary ion or cofactor. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not include a blocked nucleotide moiety. In some embodiments, the multivalent binding complex has a duration of 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, and two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to a surface, wherein a fluorescent image of the multivalent binding complex on the surface is detected. At this stage, the contrast to noise ratio is greater than 20. 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 when the nucleotide moiety is complementary to the next base of the target nucleic acid sequence and the nucleotide moiety is not complementary to the next base of the target nucleic acid sequence. If the multivalent bond is carried out under conditions that destabilize the complex. In some embodiments, the polymer-nucleotide conjugate comprises a polymer having a plurality of branches, wherein at least two nucleotide moieties are attached to the 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 to destabilize the multivalent bond complex formed between the composition of step (a) and the polymer-nucleotide conjugate, wherein the dissociation step causes removal of the polymer-nucleotide conjugate. Make it possible. In some embodiments, the method further comprises an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extension step occurs simultaneously with or after the dissociation step.

Disclosed herein is a method for determining the identity of a nucleotide in a target nucleic acid sequence, the method comprising the following steps: 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 the composition with the polymer nucleotide conjugate under conditions sufficient to cause the formation of 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), , wherein the polymer-nucleotide conjugate comprises two or more copies of a reversibly terminated nucleotide moiety and optionally one or more cleavable and detectable labels; and c. Detecting the multivalent complex and determining 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 the step of contacting the composition of step (a) after detection of the multivalent binding complex with a reversibly terminated nucleotide or a polymer-nucleotide conjugate comprising two or more copies of a reversibly terminated nucleotide. Includes additional In some embodiments, the target nucleic acid sequence has been cloned or amplified, or was produced by cloning or amplification. In some embodiments, the one or more detectable labels are fluorescent labels. In some embodiments, the step of detecting the multivalent complex includes measuring fluorescence. In some embodiments, the contacting step includes the use of one type of polymer-nucleotide conjugate. In some embodiments, the contacting step involves 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 includes the use of three types of polymer-nucleotide conjugates, where 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'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkyl hydroxylamine. In some embodiments, the contacting step occurs in the presence of an ion that stabilizes the multivalent bond complex. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule has been rendered 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 include 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, and two or more copies of the target nucleic acid sequence are deposited, attached, or hybridized to a surface, wherein a fluorescent image of the multivalent binding complex on the surface is detected. The contrast-to-noise ratio at stage 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 following steps: i) tethered to the surface of a substrate; Contacting a substrate comprising multiple copies of a target nucleic acid sequence with a polymerase and a reagent comprising 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 cause 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 images 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 fluidic module configured to deliver a series of reagents to the substrate surface in a specified order and over a specified time interval. In some embodiments, the system further includes an imaging module configured to acquire images 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 series of steps further comprises a dissociation step to destabilize the multivalent binding complex, wherein the dissociation step allows removal of the polymer-nucleotide conjugate. In some embodiments, the series of steps further comprises an extension step that incorporates a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. In some embodiments, the extension step occurs simultaneously with or after the dissociation step. In some embodiments, the detectable label comprises a fluorophore and the image comprises a fluorescence image. In some embodiments, fluorescence images of the multivalent conjugate complex on a substrate surface are obtained using a dichroic mirror where the fluorophore is cyanine dye 3 (Cy3), a 20X objective, NA = 0.75, and a dichroic mirror optimized for 532 nm light, cyanine dye- 3 Contrast-to-noise ratio when images were acquired under non-signal saturated conditions, with the surface immersed in 25 mM ACES, pH 7.4 buffer, using an inverted fluorescence microscope equipped with an emission-optimized bandpass filter and a camera. It is over 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 is completed in less than 10 minutes. In some embodiments, the accuracy of base determination is characterized by a Q-score greater than 25 for at least 80% of the nucleotide entities determined. In some embodiments, the accuracy of base determination is characterized by a Q-score greater than 30 for at least 80% of the nucleotide entities determined. In some embodiments, the accuracy of base determination is characterized by a Q-score greater than 40 for at least 80% of the nucleotide entities determined.

Disclosed herein are compositions comprising: a) a polymer core; and b) two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core; The length of the linker herein depends on the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moiety attached to the polymer core. Also disclosed herein are compositions comprising: a) a mixture of polymer-nucleotide conjugates, wherein each polymer-nucleotide conjugate comprises: i) a polymer core; and ii) a conjugate comprising two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to a polymer core, wherein the length of the linker is equal to the length of the nucleotide, nucleotide analog, nucleoside, or nucleoside analog moiety attached to the polymer core. relies on a cleoside, or nucleoside analog moiety; wherein the mixture comprises a polymer-nucleotide conjugate having at least two different types of attached nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties. In some embodiments, the polymer 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, cross-linked, bottlebrush, or dendrimeric form. 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 polymer core comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. In some embodiments, the blocked nucleotide is a 3'-O-azidomethyl nucleotide, a 3'-O-methyl nucleotide, or a 3'-O-alkyl hydroxylamine nucleotide. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels.

In some embodiments, the disclosure provides a method of determining the identity of a nucleotide in a target nucleic acid, the method comprising the following steps, without regard to any particular order of operations: 1) providing a composition comprising: Step: Target nucleic acid containing 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 the composition with a multivalent binding or incorporating composition comprising a polymer-nucleotide conjugate under conditions sufficient to cause the formation of a binding or incorporating complex between the polymer-nucleotide conjugate and the composition of step (a). wherein the polymer-nucleotide conjugate comprises two or more copies of the nucleotide and optionally one or more detectable labels; and 3) detecting the bound or incorporated complex to establish 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 the target nucleic acid is subjected to, for example, rolling circle amplification, bridge amplification, helicase dependent amplification ( Any commonly practiced DNA cloning or amplification method, such as helicase dependent amplification, isothermal bridge amplification, rolling circle multiple displacement amplification (RCA/MDA), and/or recombinase It is cloned by a recombinase-based cloning or amplification method. In some additional embodiments, the methods provided herein are wherein the detectable label is a fluorescent label and/or the step of detecting the complex comprises measuring fluorescence. In some further embodiments, the methods provided herein include wherein the multivalent linkage composition comprises one type of polymer-nucleotide conjugate, and the multivalent linkage composition comprises two or more types of polymer-nucleotide conjugate. , and/or two or more types of polymer-nucleotide conjugates, each type comprising a different type of nucleotide. In some embodiments, the methods provided herein provide that the binding complex or incorporated complex further comprises a blocked nucleotide, in particular the blocked nucleotide is 3'-O-azidomethyl nucleotide, 3'-O- It is an alkyl hydroxylamino nucleotide, or 3'-O-methyl nucleotide. In some additional embodiments, the methods provided herein are wherein the contacting step occurs in the presence of strontium ions, barium, magnesium ions, and/or calcium ions. In some embodiments, the methods provided herein are such that the polymerase molecule is catalytically inactive, such as when the polymerase molecule is catalytically inactive, such as by mutation, chemical modification, or absence of a required ion or cofactor. It has become inactive. In some embodiments, the methods also provided herein are such that the polymerase molecule is catalytically active and/or the binding complex does not include blocked nucleotides. In some embodiments, the methods provided herein comprise the binding complex having a duration of greater than 2 seconds, and/or the method comprising: , is performed or can be performed at a temperature of 42°C or higher, 55°C or higher, 60°C or higher, or 72°C or higher, or within a range defined by any of the foregoing. In some embodiments, the methods provided herein are such that the binding complex is deposited, attached, 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 such that the composition is deposited under buffered conditions incorporating a polar aprotic solvent. In some embodiments, the methods provided herein stabilize 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 methods provided herein include wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches, wherein the plurality of copies of the first nucleotide are attached to the branch, and in particular wherein the first nucleotide is attached to the branch. 1 Polymers may have star, comb, cross-linked, bottlebrush, or dendrimer-type morphologies. In some embodiments, the methods provided herein are such that the polymer-nucleotide conjugate comprises one or more linking groups selected from the group consisting of avidin, biotin, affinity tag, and combinations thereof. In some embodiments, the methods provided herein further include a dissociation step to destabilize the binding complex formed between the composition of step (a) and the polymer-nucleotide conjugate, thereby removing the polymer-nucleotide conjugate. It includes. In some embodiments, the methods provided herein further comprise an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid into the primer nucleic acid, optionally the extension step being performed concurrently with the dissociation step or It happens after that.

In some embodiments, provided herein are compositions comprising a branched polymer having two or more branches and two or more copies of a nucleotide, wherein the nucleotides are attached to a first plurality of the branches or arms, and , optionally, one or more interaction moieties are attached to said branches or arms of the second plurality. In some embodiments, the composition may further include one or more labels on the polymer. In some embodiments, the compositions provided herein are such that the nucleosides have a surface density of at least 4 nucleotides per polymer. In some embodiments, the compositions provided herein include or incorporate nucleotides or nucleotide analogs that are modified to prevent incorporation into the extending nucleic acid chain during the polymerase reaction. In some embodiments, the composition may include or incorporate a nucleotide or nucleotide analog that is reversibly modified to prevent incorporation into the nucleic acid chain that extends during the polymerase reaction. In some embodiments, the compositions provided herein are wherein one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the composition has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more branches or arms, or 2, 4, 8, It may contain 16, 32, 64 or more branches or arms. In some embodiments, branches or arms can radiate from a central moiety. In some embodiments, the composition may comprise one or more interacting moieties, wherein the interacting moiety is avidin or streptavidin; biotin moiety; affinity tag; Enzymes, antibodies, minibodies, receptors, or other proteins; Non-protein tag; It may include 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 include wherein the linker comprises PEG, wherein the PEG linker moiety has an average molecular weight of 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 a deoxyribonucleotide, ribonucleotide, deoxyribonucleoside, or ribonucleoside; and/or wherein the nucleotide or nucleotide analog is conjugated to a linker through the 5' end of the nucleotide or nucleotide analog. In some embodiments, the compositions provided herein are wherein one of the nucleotides or nucleotide analogs is deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5- includes methyl-uridine, and/or cytidine; Here, the length of the linker is 1 nm to 1,000 nm. In some embodiments, the compositions provided herein comprise at least one nucleotide or nucleotide analog modified to inhibit elongation during a polymerase reaction or sequencing reaction, wherein at least one nucleotide or nucleotide analog is a nucleotide, such as at least one nucleotide or nucleotide analog that is modified to inhibit elongation during a polymerase reaction or sequencing reaction. nucleotides that do not have hydroxyl groups; a nucleotide modified to contain 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 It is a nucleotide modified with a group, or a 3'-O-benzyl group. In some embodiments, the compositions provided herein are those in which at least one nucleotide or nucleotide analog is a nucleotide that is not modified at the 3' position.

In some embodiments, the disclosure provides a method of determining the sequence of a nucleic acid molecule, the method comprising the following steps, without regard to any particular order 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 a 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 disclosure provides a method of determining the sequence of a nucleic acid molecule, the method comprising the following steps, without regard to any particular order 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 into the nucleic acid molecule, and 4) identifying the identity of the terminal nucleotide that will be incorporated into the complementary strand of the nucleic acid molecule from partial or complete incorporation of the embodiments described herein. decision step. In some embodiments, the methods provided herein comprise the steps of incorporating the terminal nucleotide into the complementary strand, and repeating the contacting, detection, and incorporation steps for one or more additional repetitions of the nucleic acid molecule. It further includes the step of determining the sequence of the template strand. In some embodiments, the methods provided herein include wherein the nucleic acid molecule is tethered to a solid support; In particular, the solid support is one comprising a glass or polymer substrate, at least one hydrophilic polymer coating layer, and a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer. In some embodiments, the methods provided herein include wherein at least one hydrophilic polymer coating layer comprises PEG; and/or at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least eight branches. In some embodiments, the methods provided herein comprise a plurality of oligonucleotide molecules of 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 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, the methods provided herein are wherein the nucleic acid molecule is clonally amplified on a solid support. In some embodiments, the methods provided herein include clonal amplification using polymerase chain reaction (PCR), multiple displacement 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-stranded binding (SSB) ) protein-dependent amplification, or any combination thereof. In some embodiments, the methods provided herein include wherein one or more nucleic acid binding compositions are labeled with a fluorophore and the detecting step includes the use of fluorescence imaging; In particular, fluorescence imaging includes dual-wavelength excitation/four-wavelength emission fluorescence imaging. In some embodiments, the methods provided herein comprise four different nucleic acid binding compositions, each composition comprising a different nucleotide or nucleotide analog, used to determine the identity of a terminal nucleotide, the four different nucleic acid binding compositions are labeled with distinct individual fluorophores, and the detection step involves simultaneous excitation at a wavelength sufficient to excite all four fluorophores and imaging the fluorescence emission at a wavelength sufficient to detect each individual fluorophore. will be. In some embodiments, the methods provided herein comprise four different nucleic acid binding compositions, each composition comprising a different nucleotide or nucleotide analog, used to determine the identity of a terminal nucleotide, the four different nucleic acid binding compositions are labeled with cyanine dye 3 (Cy3), cyanine dye 3.5 (Cy3.5), cyanine dye 5 (Cy5), and cyanine dye 5.5 (Cy5.5), and the detection steps are 532 nm and 568 nm, respectively. Simultaneous excitation at any two wavelengths of nm and 633 nm, and imaging fluorescence emission at about 570 nm, 592 nm, 670 nm, and 702 nm; and/or fluorescence imaging includes dual wavelength excitation/dual 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, used to determine the identity of the terminal nucleotide, wherein 1, 2, 3 , or four different nucleic acid binding compositions, each labeled with a separate fluorophore or set of fluorophores, and the detection step is simultaneous at a wavelength sufficient to excite one, two, three, or four fluorophores or sets of fluorophores. This involves excitation and imaging of the fluorescence emission at a wavelength sufficient to detect each individual fluorophore. In some embodiments, the methods provided herein include three different nucleic acid binding or incorporation compositions, each comprising a different nucleotide or nucleotide analog, used to determine the identity of the terminal nucleotide, wherein 1, 2 , or three different nucleic acid binding or incorporating compositions, each labeled with a separate fluorophore or set of fluorophores, and the detection step is simultaneous at a wavelength sufficient to excite one, two, or three fluorophores or sets of fluorophores. excitation, and imaging the fluorescence emission at a wavelength sufficient to detect each individual fluorophore, wherein detection of the fourth nucleotide is determined by reference to a “dark” or unlabeled spot or target nucleotide; or You can decide. In some embodiments, the methods provided herein provide that the multivalent linkage or incorporation composition may comprise three types of polymer-nucleotide conjugates, wherein each type of the three types of polymer-nucleotide conjugates is different. It contains nucleotides of the type. In some embodiments, the methods provided herein are such that detection of binding or incorporation complexes is performed in the absence of unbound or solution-type polymer 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 composition comprising a different nucleotide or nucleotide analog, to determine the identity of a terminal nucleotide. wherein one of 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 detection step includes simultaneous excitation at a first excitation wavelength and a second excitation wavelength and images are 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 such that the detection label comprises one or more portions of a fluorescence resonance energy transfer (FRET) pair, thereby allowing multiple fractionation to be performed under a single excitation and imaging step. In some embodiments, the methods provided herein are such that the sequencing reaction cycle comprising contacting, detection, and incorporation/extension 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 confirmation accuracy across sequencing runs of at least 30, and/or at least 40. In some embodiments, the methods provided herein are such that 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 40. is to have. In some embodiments, the methods provided herein are such that at least 95% of the terminal nucleotides identified have a Q-score greater than 30.

In some embodiments, the disclosure provides reagents comprising one or more nucleic acid binding compositions disclosed herein and a buffer. For example, in some embodiments, the disclosure provides reagents, wherein the reagent comprises 1, 2, 3, 4 or more nucleic acid binding or incorporation compositions, wherein each nucleic acid binding or incorporation composition is a single type of nucleic acid binding or incorporation composition. It contains nucleotides. In some embodiments, a reagent herein comprises a composition of 1, 2, 3, 4 or more nucleic acid binding or incorporation, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog, and wherein the nucleotide or The nucleotide analogs may respectively correspond to: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), and one or more 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 one or more 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 monophosphate one or more 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. One or more 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. It includes a nucleotide or a nucleotide analog, wherein the nucleotide or nucleotide analog 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.

Disclosed herein are nucleic acid binding or incorporation compositions of any of the embodiments disclosed herein and/or reagents of any of the embodiments disclosed herein, and/or one or more buffers; A kit comprising and instructions for use thereof is disclosed.

Disclosed herein are systems for performing the methods of any of the embodiments disclosed herein, comprising nucleic acid binding or incorporation compositions of any of the embodiments disclosed herein, and/or of any of the embodiments disclosed herein. Contains reagents. In some embodiments, the system comprises sequential contact of a tethered, primed nucleic acid molecule with the nucleic acid binding or incorporation composition and/or the reagent; and repeatedly detecting 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 are compositions comprising particles (e.g., nanoparticles or polymer cores), wherein the particles comprise a plurality of enzyme or protein bound or incorporated substrates, wherein the enzyme or protein bound or An incorporation substrate binds to one or more enzymes or proteins to form one or more binding or incorporation complexes (e.g., multivalent binding or incorporation complexes), wherein said binding or incorporation refers to the location, presence, or presence of the one or more binding or incorporation complexes. Alternatively, 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, nucleoside, nucleotide analog, or nucleoside analog. In some embodiments, the enzyme or protein binding or incorporating substrate may comprise an agent capable of binding the polymerase. In some embodiments, the enzyme or protein may include a polymerase. In some embodiments, said observation of the location, presence, or persistence of one or more binding or incorporation complexes may include fluorescence detection. In some embodiments, the disclosure provides compositions comprising a plurality of separate particles as disclosed herein, wherein each particle comprises a single type of nucleoside or nucleoside analog, and each of these nucleosides The side or nucleoside analog is detectably associated with a fluorescent label of a different emission or excitation wavelength. In some embodiments, the compositions provided herein further comprise one or more labels on the particles, such as a fluorescent label. 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 particles tethered to the particles. It includes nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs. In some embodiments, the compositions provided herein contain nucleosides or nucleoside analogs at a concentration of 0.001 to 1,000,000 per μm ² , 0.01 to 1,000,000 per μm ² , 0.1 to 1,000,000 per μm ² , ¹ to 1,000,000 per μm 2 , 10 to 1,000,000 per μm ² , 100 to 1,000,000 per μm ² , 1,000 to 1,000,000 per μm ² , 1,000 to 100,000 per μm ^{2 ,} 10,000 to 100,000 per μm ² , or 50,000 per μm ² to 100,000, or any of the preceding values. It exists at a surface density within the range defined by the two values of . In some embodiments, the compositions provided herein are those in which the nucleoside or nucleoside analog is present in a nucleotide or nucleotide analog. In some embodiments, the compositions provided herein include or incorporate nucleotides or nucleotide analogs that are modified to prevent incorporation into the nucleic acid chain that extends during the polymerase reaction. In some embodiments, the compositions provided herein include or incorporate a nucleotide or nucleotide analog that has been reversibly modified to prevent incorporation into the nucleic acid chain that extends during the polymerase reaction. In some embodiments, the compositions provided herein are wherein one or more labels comprise a fluorescent label, a FRET donor, and/or a FRET acceptor. In some embodiments, the compositions provided herein are those in which a substrate (e.g., a nucleotide, nucleotide analog, nucleoside, or nucleoside analog) is attached to the particle via a linker. In some embodiments, the compositions provided herein include at least one nucleotide or nucleotide analog that is modified to inhibit elongation during a polymerase reaction or sequencing reaction, such as, for example, a nucleotide that does not have a 3' hydroxyl group. nucleotide; a nucleotide modified to contain 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'-O-benzyl group; and/or is a nucleotide that is not modified at the 3' position.

In some embodiments, the disclosure provides a method of determining the sequence of a nucleic acid molecule, the method comprising the following steps in any order: 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 a nucleic acid binding or incorporation composition to a nucleic acid molecule, and 4) determining the identity of a terminal nucleotide to be incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the method comprises incorporating the terminal nucleotide into the complementary strand, and repeating the contacting, detection, and incorporation steps for one or more additional repetitions to determine the sequence of the template strand of the nucleic acid molecule. An additional decision step may be included. In some embodiments, the methods provided herein are wherein the nucleic acid molecule is clonally amplified on a solid support. In some embodiments, the methods provided herein include clonal amplification using polymerase chain reaction (PCR), multiple displacement 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 This includes the use of amplification, or any combination thereof. In some embodiments, the methods provided herein are such that the sequencing reaction cycle comprising contacting, detection, and incorporation 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 confirmation accuracy across sequencing runs 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 has a Q-score greater than 40. In some embodiments, the methods provided herein are such that at least 95% of the terminal nucleotides identified have a Q-score greater than 30.

In some embodiments, the disclosure provides reagents comprising one or more nucleic acid binding or incorporation compositions disclosed herein, and a buffering agent. In some embodiments, the reagent provided herein comprises a nucleic acid binding or incorporation composition of 1, 2, 3, 4, or more, wherein each nucleic acid binding or incorporation composition is a single type of nucleotide or nucleotide analog. Including, wherein the nucleotide or nucleotide analog includes a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog. In some embodiments, the methods provided herein include the reagent comprising a nucleic acid binding or incorporation composition of 1, 2, 3, 4 or more nucleic acids, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog. and wherein the nucleotide or nucleotide analog is one or more 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 the reagent comprising a nucleic acid binding or incorporation composition of 1, 2, 3, 4 or more nucleic acids, wherein each nucleic acid binding or incorporation composition comprises a single type of nucleotide or nucleotide analog. Including, 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, this disclosure provides for any composition disclosed herein; and/or any reagent disclosed herein; one or more buffering agents; and instructions for use thereof.

In some embodiments, the disclosure provides a system for performing any of the methods disclosed herein; wherein the method comprises any composition disclosed herein; and/or any reagent disclosed herein; It may comprise the use of one or more buffering agents, and one or more nucleic acid molecules optionally tethered or attached to a solid support, wherein the system comprises sequential contact of the nucleic acid molecules with the composition and/or the reagents; and configured to repeatedly detect binding or incorporation of the nucleic acid binding or incorporation composition to one or more nucleic acid molecules.

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

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

In some embodiments, the disclosure 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 to a surface. Provided are compositions disclosed herein for:

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

In some embodiments, the disclosure provides compositions disclosed herein for use in increasing specificity, accuracy, or read length in sequencing by binding or incorporation, sequencing by synthesis, single molecule sequencing, or ensemble sequencing methods. .

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

In some embodiments, the disclosure 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 to a surface. Provided are reagents disclosed herein for:

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

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

Inclusion by reference

All publications and patent applications mentioned herein 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 case of conflict between terms in this specification and terms in incorporated references, the terms in this specification control.

The patent or application file contains at least one drawing drawn in color. Copies of this published patent or patent application with color drawing(s) will be available from the Patent and Trademark Office upon request and payment of the necessary fee.
The novel features of the inventive concept disclosed herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed configurations, methods and systems will be obtained by reference to the following detailed description and accompanying drawings, which illustrate illustrative embodiments in which the principles of the inventive concepts are utilized.
Figures 1A-1H depict steps for utilizing a non-limiting example of a multivalent binding composition for sequencing a target nucleic acid: Figure 1A shows a non-limiting example 4 of attaching a target nucleic acid to a surface; Figure 1B depicts target nucleic acids clonally to form clusters of amplified target nucleic acid molecules; Figure 1C shows a non-limiting example of priming a target nucleic acid to generate a primed target nucleic acid; Figure 1D shows a non-limiting example of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; Figure 1E shows a non-limiting example of an image of a binding complex captured on a surface; Figure 1F shows a non-limiting example of extending a primer strand by one nucleotide; Figure 1G shows a non-limiting example of another cycle of contacting a primed target nucleic acid with a multivalent binding composition and a polymerase to form a binding complex; Figure 1H shows a non-limiting example of an image of a binding complex captured on a surface in a subsequent sequencing cycle.
Figure 2 depicts a flow chart outlining the steps of sequencing a target nucleic acid and extending primer strands through single base addition.
Figure 3 shows a flow chart outlining the steps for sequencing a target nucleic acid and extending the primer strand by incorporating nucleotides into a particle-nucleotide conjugate.
Figures 4A-4B illustrate non-limiting examples of target nucleic acid detection using polymer-nucleotide conjugates. Figure 4A depicts the steps in which a polymerase and a polymer-nucleotide conjugate contact some nucleic acid molecules; Figure 4B depicts the binding complex formed between polymerase, polymer-nucleotide conjugate, and target nucleic acid molecule.
Figures 5A-5C schematically depict non-limiting examples of various configurations of polymer-nucleotide conjugates: Figure 5A shows polymer-nucleotide conjugates having various multi-arm structures; Figure 5B shows a polymer-nucleotide conjugate with polymer branches radiating from the center; Figure 5C depicts a polymer-nucleotide conjugate with the binding moiety biotin.
Figure 6 shows a generalized graphical depiction of the observed increase in signal intensity during binding, persistence, and washing and removal of the multivalent substrate.
Figures 7A-7J show fluorescence images of steps in a sequencing reaction using a multivalent PEG-based composition. Figure 7A : DNA RCA template (G and A first bases) was exposed to 500 nM base labeled nucleotides (A-Cy3 and G-Cy5) in exposure buffer containing -20 nM Klenow polymerase and 2.5 mM Sr ⁺² . Red and green fluorescence images after exposure. Images were collected after washing with imaging buffer, which had the same composition as the exposure buffer but did not contain 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). Figures 7B-7E : Multivalent PEG-nucleotide (base-labeled) ligands PB1 ( Figure 7B ), PB2 ( Figure 7C ), PB3 ( Figure 7D ) at 500 nM after mixing in exposure buffer and imaging in 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 imaging buffer as above. Figures 7G-7I : Inactive mutants of Klenow polymerase ( Figure 7G :D882H; Figure 7H :D882E; Figure 7I :D882A), versus wild-type Klenow (control) enzyme ( Figure 7J ) showing additional base discrimination by multivalent ligand exposure. Fluorescence image shown.
Figures 8A-8B depict the efficacy of a multivalent reporter composition in determining the base sequence of a DNA sequence over five sequencing cycles: Figure 8A shows images and expected sequences for templates taken after each sequencing cycle; Figure 8B shows aligned sequencing results using the image captured in Figure 8A .
Figures 9A-9J show polyvalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates with an effective nucleotide concentration of 500 nM and various PEG branch lengths, containing 20 nM Klenow polymerase and 2.5 mM Sr ⁺² . Shown is a fluorescence image after contacting a support surface containing a DNA template (with G or A as the first base; prepared using rolling circle amplification (RCA)) in exposure buffer. Images were acquired after washing with imaging buffer, which had the same composition as the exposure buffer but did not contain nucleotides and polymerase. The panels show images obtained using multivalent PEG-nucleotide ligands with 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. Figure 9G shows images obtained using inactive klenow polymerase containing 10 K PEG and mutant D882H. Figure 9H shows images obtained using inactive klenow polymerase containing 10 K PEG and mutant D882E. Figure 9I shows images obtained using inactive klenow polymerase containing 10 K PEG and mutant D882A. Figure 9J shows 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 traces corresponding to the red label (Cy3 label; A base) and green traces corresponding to the green label (Cy5 label; G base). Blue traces, identified by color value.
Figure 11 shows normalized fluorescence from multivalent substrates bound to DNA clusters as described in Figures 7A-7J , with substrate complexes formed in the presence (condition B) and absence (condition A) of Triton-X100 (0.016%). do.
Figures 12A-12B show plots of normalized fluorescence intensities measured for multivalent polymer-nucleotide conjugates and free nucleotides. Figure 12A : Two replicates of the multivalent polymer-nucleotide conjugate bound to a DNA cluster (conditions A and B) versus the binding complex using labeled free nucleotides (condition C) after 1 minute; Figure 12B : Time course of fluorescence from multivalent substrate complex over 60 minutes.

details

I. Definition

As used herein, a “nucleic acid” (also referred to as a “polynucleotide”, “oligonucleotide”, ribonucleic acid (RNA), or deoxyribonucleic acid (DNA)) is a nucleic acid consisting of two or more nucleic acids linked by covalent internucleoside linkages. It is a linear polymer of nucleotides, or a variant or functional fragment thereof. In naturally occurring examples of nucleic acids, the internucleoside linkage is a phosphodiester bond. However, other examples may optionally include other internucleoside linkages, such as phosphorothiolate linkages, or may not include phosphate groups. Nucleic acids include double- and single-stranded DNA, as well as double- and single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids (PNAs), 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, nucleoside, or 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; deoxyadenin, 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 containing or ribonucleosides containing D-ribose).

As used herein, “complementary” 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 contact surface properties are complementary to each other.

As used herein, “branched polymer” refers to a polymer that has multiple functional groups that help conjugate biologically active molecules, such as nucleotides, where these functional groups are located in the side chains of the polymer or in 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 emanating from the backbone for conjugation. Branched polymers can also be polymers with one or more side chains, which have sites suitable for conjugation. Examples of functional groups include hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfone, hydrazide, thiol, alkanoic acid, acid halide, Isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate. Including, but not limited to.

As used herein, “polymerase” refers to an enzyme that contains a nucleotide binding moiety and assists 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. No. Polymerases may include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain nucleotide binding or incorporation moieties.

As used herein, “duration time” refers to the time during which the binding complex formed between the target nucleic acid, polymerase, and conjugated or unconjugated nucleotides remains stable without any binding components dissociating from the binding complex. refers to the length of The 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 signals from labeled components of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in a binding complex, such that a signal from the label can be detected for the duration of the binding complex. A non-limiting example of one of the labels is a fluorescent label.

II. Methods for analyzing target nucleic acids

Disclosed herein are multivalent linkage or incorporation compositions and their use in the analysis of nucleic acid molecules, including sequencing or other bioassay applications. Increased binding or incorporation of a nucleotide into an enzyme (e.g., polymerase) or enzyme complex can be affected by increasing the effective concentration of the nucleotide. This increase can be achieved by increasing the concentration of the nucleotide in free solution, or by increasing the amount of nucleotide 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, thereby making these structures unconjugated, untethered, or otherwise It can bind or incorporate into a binding or incorporation site with an apparent avidity higher than that observed for unrestricted individual nucleotides. One non-limiting means of achieving this limitation is to provide multivalent binding or incorporating compositions in which multiple nucleotides are bound to particles, including polymers, branched polymers, dendrimers, micelles, liposomes, microparticles, nanoparticles. , quantum dots, or other suitable particles known in the art.

The multivalent linkage or incorporation compositions disclosed herein may include at least one particle-nucleotide conjugate, which 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 is a bond formed using a single unconjugated or untethered nucleotide. or exhibit increased stability and longer duration than incorporated complexes. Each nucleotide moiety of the multivalent linkage composition binds to the complementary N+1 nucleotide of the primed target nucleic acid molecule, thereby forming two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and the multivalent linkage composition (e.g. For example, a polymer-nucleotide conjugate) may be formed. Each nucleotide moiety of the multivalent linkage composition binds to the complementary N nucleotide of the primed target nucleic acid molecule, thereby forming two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and the multivalent linkage composition (e.g. , polymer-nucleotide conjugate) can form a multivalent binding complex. Nucleotides from the bound complex can be probed for complementary bases before 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 base before and after deprotection. In this way, the overall accuracy of base determination can be improved by performing error checking and reading the probe twice. An important distinguishing factor from traditional methods is that binding is used to search for matching bases, whereas staging or incorporation steps are used only 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 interaction, such as a protein-nucleic acid interaction, nucleic acid hybridization reaction, or site of an enzymatic reaction such as 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 polymerase reactions 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 improves the signal, greatly improving base determination accuracy and shortening imaging time.

Additionally, the use of multivalent binding compositions allows sequencing signals from a given sequence to occur within a cluster region containing 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 a defined region also reduces the impact of any single skipped cycle, due to the fact that signals from a large number of correct base confirmations can overwhelm signals from a small number of skipped or incorrect base confirmations, resulting in phasing errors ( A method for reducing phasing error and/or improving read length in a sequencing reaction is provided.

The multivalent binding compositions disclosed herein and their uses result in one or more of the following: (i) stronger signals for better base confirmation accuracy compared to conventional nucleic acid amplification and sequencing methodologies; ii) allows greater discrimination 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) reducing paging errors, and (vi) improving 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 can comprise two or more nucleic acid molecules. In some embodiments, a target nucleic acid may comprise two or more nucleic acid molecules with the same sequence.

A. Targeted nucleic acid sequencing

Figures 1A-1H illustrate exemplary methods in which multivalent binding compositions are used to sequence target nucleic acids. As shown in Figure 1A , target nucleic acid 102 can be tethered to a solid support surface 101. Target nucleic acids can be attached directly or indirectly to the surface. Although not shown in Figure 1A , target nucleic acid 102 can hybridize to an adapter that is attached to the surface through covalent or non-covalent bonds. When one or more adapters are used to attach a target nucleic acid to a surface, the target surface may include a fragment that is complementary to the adapter and therefore hybridizes to the adapter. In some cases, one adapter sequence may be tethered to the surface. In some cases, multiple adapter sequences may be tethered to the surface. In some cases, the target nucleic acid 102 can also be attached directly to the solid-support surface without using adapters. The solid support may have a low non-specific binding surface.

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

Figure 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 Figure 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. 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, multiple different primer sequences may be used in the hybridization step.

As shown in Figure 1D , primed target nucleic acid 104 is combined with a multivalent binding or incorporation composition and polymerase 106 to form a binding or incorporation complex. Non-limiting examples of multivalent linkage or incorporation compositions of Figure 1D include four particle-nucleotide conjugates (105a), (105b), (105c), and (105d). Each particle-nucleotide conjugate has multiple copies of a nucleotide attached to the particle, with four particle-nucleotide conjugates covering 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 linkage or incorporation composition may include 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 can be labeled and the fourth type is unlabeled or conjugated to a 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 identity of its conjugated nucleotide, 3, 2, 1, respectively. The particle-nucleotide conjugate, or any conjugate, may be unlabeled, or may remain conjugated to or with an undetectable label. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using four-color detection, such that conjugates corresponding to all four nucleotides are present in the sample, and each conjugate is It has a separate label corresponding to the nucleotide to which it is conjugated. In some embodiments, four particle-nucleotide conjugates can be simultaneously exposed or contacted to a target nucleic acid; In some other embodiments, the four particle-nucleotide conjugates may be exposed or contacted sequentially, individually, or in groups of two or three, to the target nucleic acid. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using three-color detection, such that conjugates corresponding to three of the four nucleotides are present in the sample, and three The conjugates have separate labels corresponding to the nucleotides to which they are conjugated 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 to which they are conjugated. With , one conjugate is absent. In some embodiments, the identity of the nucleotide corresponding to the unlabeled or nucleotide-free conjugate can be determined in conjunction with the identification of a known target nucleic acid position and/or a “dark” spot or position that does not exhibit a fluorescent signal. In some embodiments, the methods provided herein are such that detection of binding or incorporation complexes is performed in the absence of unbound or solution-type polymer nucleotide conjugates.

In some embodiments where 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 indicated in the absence of a signal. This can be established by 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, such that conjugates corresponding to two of the four nucleotides are present in the sample, and two The conjugate has a separate label corresponding to the nucleotide to which it is conjugated and the two conjugates are conjugated to an unlabeled or undetectable label. 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 identity of the unlabeled conjugate or the nucleotide corresponding to the conjugate can be determined by the absence of a signal or by monitoring the presence of the unlabeled complex, such as by sequencing. This can be established by identification of “dark” spots or unlabeled areas in the reaction. In some embodiments where two of the four particle-nucleotide conjugates are labeled, 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, 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 Contact is made, and each such contact step represents a distinction between two or more different bases such that after two, three, four or more such contact steps the identity of all unknown bases is determined.

Figure 1E shows an image captured on a surface after a binding or incorporation complex is formed between the polymerase, a target nucleic acid, and a particle-nucleotide conjugate with a nucleotide complementary to the next base of the primed target nucleic acid. The captured image includes four binding or incorporation complexes (107a), (107b), (107c), and (107d) formed on the surface, each binding or incorporation complex containing a label (e.g. For example, the fluorescence has different nucleotides that can be distinguished based on their emission color). The use of particle-nucleotide conjugates greatly enhances the sequencing signal because the binding or incorporation signal from a given sequence occurs within a cluster region containing multiple copies of the target sequence. Although Figure 1E includes four particle-nucleotide conjugates, each with a different type of nucleotide, some methods may use one, two, or three particle-nucleotide conjugates, each with 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 with a label that corresponds to the identity of each conjugated nucleotide. In some embodiments, three of the four types of nucleotide conjugates can be labeled and the fourth type is unlabeled or conjugated to a undetectable label. In some embodiments, 1, 2, 3, or 4 particle-nucleotide conjugates can be labeled with a separate label, and each of 3, 2, 1 conjugates, or any particle-nucleotide conjugate can be labeled with a separate label. It is not activated or is conjugated to an undetectable label. In some embodiments, the detection step may include simultaneous and/or serial excitation of up to four different excitation wavelengths, such as fluorescence imaging, for each possible base pair (A, G, C, or T). This is accomplished by detecting single and/or multiple fluorescent emission bands that uniquely classify them. 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 four or three different nucleic acid binding or incorporation compositions one is labeled with the first fluorophore, one is labeled with the second fluorophore, one is labeled with both the first and second fluorophores, and one is unlabeled or absent, wherein the detection step is It includes 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 a multivalent binding or incorporation composition is used for 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 increased several fold; As a result, signal strength is improved. The binding or incorporation complexes formed also have a longer duration, thereby helping to shorten the imaging steps. The high signal intensity results from the high binding or incorporation avidity of the polymer nucleotide conjugate (which may also contain multiple fluorophores or other labels), thus forming a complex that remains stable throughout the entire binding or incorporation and imaging steps. do. The strong binding or incorporation between the polymerase, the primed target strand, and the polymer-nucleotide or nucleotide analog conjugate also ensures that the multivalent binding or incorporation complex thus formed will remain stable during the washing steps and will be consistent with other reaction mixture components. This means that the signal intensity remains high when unwanted nucleotide analogs are washed away. 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 one base.

The sequencing method may further include the step of incorporating N+1 or terminal nucleotides into the primed strand, as shown in Figure 1F . In Figure 1F , the primer strand of the primed target nucleic acid 108 can be extended by one base to form the extended nucleic acid 109. The extension step may occur simultaneously with or subsequent to destabilization of the multivalent bond or incorporation complex. Primed target nucleic acid 108 may be formed using complementary nucleotides attached to the particle of a particle-nucleotide conjugate, or using unconjugated or untethered free nucleotides provided after the multivalent binding or incorporating composition has been removed. It can be extended.

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

Extension of the primed target nucleic acid can be prevented or inhibited by blocked nucleotides on the strand or by the use of a catalytically inactive polymerase. When a nucleotide in a 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 the polymer, branched polymer, dendrimer, particle, etc. ) can be achieved. If elongation 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 are systems configured to perform any of the nucleic acid sequencing or nucleic acid analysis methods disclosed. The system includes 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 incorporating 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 the system. In some cases, the flow cell may be a removable and/or disposable component of the system.

The sequencing system may include an imaging module, i.e., one or more light sources, one or more optics for imaging and detecting binding or incorporation of the disclosed nucleic acid binding or incorporation composition to a target nucleic acid molecule tethered within a solid support or flow cell. It may include 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 analysis applications. Examples include, but are not limited to, DNA sequencing, RNA sequencing, whole genome sequencing, targeted sequencing, exome sequencing, genotyping, etc.

The sequencing system may also include a computer control system programmed to implement the methods of the invention. A computer system is programmed or configured to implement the methods of the invention, including nucleic acid sequencing methods, interpretation of nucleic acid sequencing data and analysis of cellular nucleic acid RNA (e.g., mRNA), and characterization of cells from sequencing data. The computer system may be a user's electronic device or a computer system remotely located in relation to the electronic device. The electronic device may be a mobile electronic device.

Figure 2 is a flow chart schematically explaining the steps for 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, the nucleic acid molecules of the target library may have been prepared to contain fragments complementary to the adapter sequence through ligation or other methods. (202) describes a clonal amplification step to generate clusters of target nucleic acid molecules on a surface. (203) describes the steps of hybridizing a sequencing primer to a complementary primer binding or incorporating sequence in a target nucleic acid to form a primed target nucleic acid. (204) describes the step of combining a polymerase, a composition comprising a labeled (e.g., fluorescently-labeled) particle-nucleotide conjugate as a multivalent linkage 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 back to Figure 2 , if the nucleotide of the particle-nucleotide conjugate is complementary to the next base of the primed target nucleic acid (205), the particle-nucleotide conjugate, polymerase, and primed target nucleic acid form a ternary binding. ) or form an incorporation complex, which can be detected by a detection method (e.g., fluorescence imaging) compatible with labeling of the particle-nucleotide conjugate. (205) may also include measuring the duration of the ternary bond or incorporation complex. (206), the binding or incorporation complex is destabilized, eliminating binding or incorporation of the particle-nucleotide conjugate and polymerase. Dissociation can be achieved by subjecting the binding or incorporation complex to conditions that alter the conformation of the polymerase and destabilize the binding or incorporation (e.g., addition of strontium ions). (206) may also include washing or removing the dissociated particle-nucleotide conjugate and/or polymerase. (207) describes the step of extending the primed strand of a primed target nucleic acid by a single base addition reaction. After single base extension, steps (204), (205), (206), and (207) can be repeated in multiple cycles to determine the sequence of the target nucleic acid.

Figure 3 is another flow diagram schematically illustrating the steps for sequencing a target nucleic acid, including cleaving nucleotides from a particle-nucleotide conjugate and incorporating the cleaved nucleotides. (301) describes 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. (301) Previously, the nucleic acid molecules of the target library may have been prepared to contain fragments complementary to the adapter sequence 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 to a complementary primer binding or incorporating sequence in a target nucleic acid to form a primed target nucleic acid. 304 describes the step of combining a polymerase, a composition comprising a labeled (e.g., fluorescently-labeled) particle-nucleotide conjugate as a multivalent binding or incorporation composition, and a primed target nucleic acid. In particle-nucleotide conjugates, 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 Figure 3 , when a 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 form a three-way association or integration complex. , which can be detected by labeling of the particle-nucleotide conjugate and a detection method compatible with it (e.g., fluorescence imaging). 305 may also include measuring the duration of the ternary bond or incorporation complex. At (306), the polymerase is placed in conditions where it is catalytically active to incorporate nucleotides. These 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 can be repeated in multiple cycles to determine the sequence of the target nucleic acid.

B. Detection of target nucleic acid molecules

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

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

III. Multivalent Linkage or Incorporation Composition

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

When a multivalent binding or incorporation composition is used for replacement of a single unconjugated or untethered nucleotide to form a complex with a polymerase and one or more copies of a target nucleic acid, the local concentration of the nucleotide, as well as the binding avidity of the complex. (when a complex containing two or more target nucleic acid molecules is formed) increases several fold, resulting in improved signal intensity, specifically accurate signal to mismatch. The multivalent binding or incorporation compositions described herein may include at least one particle-nucleotide conjugate (each particle-nucleotide conjugate comprising multiple copies of a single nucleotide moiety) for interacting 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 linkage or incorporation composition may include one, two, three, four or more types of particle-nucleotide conjugates, with each particle-nucleotide conjugate comprising a different type of nucleotide. The first type of particle-nucleotide conjugate may include nucleotides selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. The second type of particle-nucleotide conjugate may comprise nucleotides 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 nucleotides selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. A fourth type of particle-nucleotide conjugate may include nucleotides selected from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, each particle-nucleotide conjugate is ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, respectively. , CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP. Each multivalent linkage or incorporation composition may further include one or more labels corresponding to the specific nucleotides each conjugated to the respective conjugate. Examples of such labels include fluorescent labels, colorimetric labels, electrochemical labels (e.g., glucose or other reducing sugars, or thiols or other reductive moieties), luminescent labels, chemiluminescent labels, spin labels, radioactive labels, and stereogenic labels. Including, but not limited to, structural labels, affinity tags, etc.

A. Particle-Nucleotide Conjugate

In a particle-nucleotide conjugate, multiple copies of the same nucleotide may be covalently or non-covalently linked to the particle. Examples of particles include branched polymers; dendrimer; crosslinked polymer particles such as agarose, polyacrylamide, acrylates, methacrylates, cyanoacrylates, methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dot; liposome; Emulsion particles, or any other particles known in the art (e.g., nanoparticles, microparticles, etc.). In a preferred embodiment, the particles are branched polymers.

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

The nucleotides may be linked to the particle through a linker, and the nucleotides may be attached to one end or location of the polymer. Nucleotides can be conjugated to the particle through the 5' end of the nucleotide. In some particle-nucleotide conjugates, one nucleotide is attached to one end or location of the polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to one end or location of the polymer. The conjugated nucleotide may be conformationally accessible to one or more proteins, one or more enzymes, and a nucleotide binding or incorporation moiety. In some embodiments, the nucleotide may be provided separately from a nucleotide binding or incorporation moiety such as a polymerase. In some embodiments, the linker does not include a light-emitting or light-absorbing group.

Particles may also have bonding or incorporating moieties. In some embodiments, particles can self-associate without using separate interaction moieties. In some embodiments, the particles are subject to, for example, 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 buffering or salt conditions, such as in the case of

The particle-nucleotide conjugate may have one or more labels. Examples of labels include fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or labels that make the composition detectable by methods known in the art for detection of macromolecules or molecular interactions. Including, but not limited to, other signs that may be used. The label may be attached to the nucleotide (e.g., attached to the 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to the PEG subunit), to the end of the polymer, to the central moiety, or known in the art. It may be attached to a particle, such as at any other location within the polymer-nucleotide conjugate recognized by those skilled in the art as sufficient to provide the composition detectable by the methods described elsewhere herein. In some embodiments, one or more labels are provided to correspond or differentiate to 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, hexachlorofluorescein, Hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein fluorescein 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, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow Iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes dyes, quinolinium cyanine dyes, imidazolium cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates, terbium ( Terbium) chelate, Alexa Fluor dye, DyLight dye, Atto dye, LightCycler Red dye, CAL Flour dye, JOE and its derivatives, 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 nonsulfonated forms, consisting of two indolenines, benzo-indolium, pyridium, thiozolium, and/or quinolinium separated by a polymethine bridge between the two nitrogen atoms. It can 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 ( This 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 May include methyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-ium-5-sulfonate. ), 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 can), where "Cy" represents 'cyanine', and the first number represents the number of carbon atoms between two indolenine groups. Cy2, a non-indolenine oxazole derivative, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the detection label can be a FRET pair, which allows multiple classifications to be performed under a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer, or electron-exchange (Dexter) transfer.

B. Polymer-Nucleotide Conjugate

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, Figure 5A depicts polymer-nucleotide conjugates with various configurations, such as a “starburst” configuration with a fluorescently-labeled streptavidin core and a molecular weight ranging from 1 K daltons to 10 K daltons. It contains four nucleotides linked to the core via a biotinylated, linear PEG linker in the dalton range; Figure 5B shows, for example, a polymer-nucleotide conjugate having a dendrimeric core with 12, 24, 48, or 96 arms and a linear PEG linker with a molecular weight ranging from 1 K Dalton to 10 K Dalton radiating from the center. shows; Figure 5C shows an example of a polymer-nucleotide conjugate comprising a network of, e.g., streptavidin cores, linked together with branched PEG linkers containing binding or incorporation moieties 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, Including 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 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 feature repeating units incorporating functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl, or allyl groups. The polymer may also have one or more pre-derivatized substituents such that one or more particular 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, interaction moieties, additional polymeric moieties, etc., or any of the foregoing. It may include a combination, or may include additionally.

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

The length and size of the branches may vary depending on the type of polymer. In some branched polymers, the branches have lengths of 1 to 1,000 nm, 1 to 100 nm, 1 to 200 nm, 1 to 300 nm, 1 to 400 nm, 1 to 500 nm, 1 to 600 nm, 1 to 700 nm, It may be a length between 1 and 800 nm, or between 1 and 900 nm, or greater, or within or between any of the values 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 of the preceding values. The apparent molecular weight of a polymer can be calculated from a representative number of subunits of known molecular weight, as determined by size exclusion chromatography, by mass spectrometry, or by any other method known in the art.

In some branched polymers, the branches are 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 It may have a size corresponding to an apparent molecular weight of Da, 100 K Da, or any value within the range defined by any two of the preceding values. The apparent molecular weight of a polymer can be calculated from a representative number of subunits of known molecular weight, 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 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number that falls within a range defined by any two of the above values. .

For example, for polymer-nucleotide conjugates comprising branched polymers of branched PEG containing 4, 8, 16, 32, or 64 branches, the nucleotides attached to the ends of the polymer nucleotide conjugate PEG branches are It can have 0, 1, 2, 3, 4, 5, 6 or more nucleotides 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 end. , six or more nucleotides or nucleotide analogs can 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 (e.g., PEG linker) may 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. 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, those skilled in the art will recognize that the length of the linker may be any value within the range of values in this paragraph, for example, 834 nm.

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

In a polymer-nucleotide conjugate, a moiety comprising a nucleotide (e.g., an adenine, thymine, uracil, cytosine, or guanine residue or a derivative or mimetic thereof) is attached to each branch or subset of branches of the polymer. This moiety may be bound to or incorporated into a polymerase, reverse transcriptase, or other nucleotide binding or incorporation domain. Optionally, the moiety can be incorporated into the extending nucleic acid chain during the polymerase reaction. In some cases, the moiety may be blocked so that it cannot be incorporated into the extending nucleic acid chain during the polymerase reaction. In some other cases, the moiety may 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 may be incorporated into the extending nucleic acid chain during the polymerase reaction. may be mixed in.

Nucleotides can be conjugated to polymer branches through the 5' end of the nucleotide. In some cases, nucleotides may be modified to inhibit or prevent incorporation of the nucleotide into the extending nucleic acid chain during the polymerase reaction. By way of example, the nucleotide may be a 3' deoxyribonucleotide, a 3' azidonucleotide, a 3'-methyl azidonucleotide, or as such, or as may be known in the art, so that they cannot be incorporated into the extending nucleic acid chain during the polymerase reaction. It may contain another nucleotide. In some embodiments, the nucleotide is a 3'-O-azido group, a 3'-O-azidomethyl group, a 3'-phosphorothioate group, a 3'-O-malonyl group, a 3'-O- It may contain 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 bonding or incorporating moieties on each branch or subset of branches. Some examples of binding or incorporating moieties include biotin, avidin, streptavidin, etc., polyhistidine domains, complementary pairing nucleic acid domains, G-quadruplex forming nucleic acid domains, calmodulin, maltose-binding protein, cellulase. , 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 to or promote 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, compositions provided herein may include one or more elements of a complementary interaction moiety. Non-limiting examples of complementary interaction 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 cognate polysaccharides; Ionic chelating moieties, complementary nucleic acids, nucleic acids capable of forming triple or triple helix interactions; Includes nucleic acids capable of forming a G-quartet, etc. Those skilled in the art will readily recognize that many pairs of moieties exist and are commonly used for their properties that interact strongly and specifically with each other; Accordingly, any such complementary pair or set is considered suitable for this purpose in conceiving or implementing the compositions of the present invention. In some embodiments, the compositions disclosed herein have one element of the complementary interaction moiety attached to one molecule or multivalent ligand and the other element of the complementary interaction moiety attached to a separate molecule or multivalent ligand. may include a composition. In some embodiments, compositions disclosed herein may include compositions in which both or all elements of the complementary interaction moiety are attached to a single molecule or multivalent ligand. In some embodiments, compositions disclosed herein may include compositions in which both or all elements of complementary interaction moieties are attached to separate arms, or positions, of a single molecule or multivalent ligand. In some embodiments, compositions disclosed herein may include compositions in which both or all elements of complementary interaction moieties 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 interaction moiety and a composition comprising another element of a complementary interaction 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, for example, resulting in increased detectable signal. In some embodiments, fluorescent, colorimetric, or radioactive signals are enhanced. In other embodiments, other interaction moieties disclosed herein or known in the art are contemplated. In some embodiments, the compositions provided herein comprise 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 the residues may be provided for simultaneous or sequential mixing. 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 provided molecules or particles may be promoted by the presence of divalent cations such as nickel, manganese, magnesium, calcium, strontium, etc. In some embodiments, for example, a (His) ₃ group may interact with a (His) ₃ group of another molecule or particle through coordination of a nickel or manganese ion.

The multivalent linkage 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, detergents 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 DNA "relaxers" within the art. "Any substance known as "changes the persistence length of DNA, alters the number of junctions or crossovers in the polymer, or alters the conformational dynamics of the DNA molecule, thereby increasing the accessibility of a site within the strand to a DNA binding or incorporation moiety. compounds that have an increasing effect).

The multivalent bond or incorporation composition may include a zwitterionic compound as an additive. Additionally representative additives are described in Lorenz, T.C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which discloses additives for promoting nucleic acid binding or dynamics, or for facilitating processes involving the manipulation, use or storage of nucleic acids. It is therefore 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-associating, secondary or tertiary structures. Other cations known in the art to promote nucleic acid interactions such as formation, base pairing, surface association, peptide association, protein binding, etc.

IV. Binding between target nucleic acid and multivalent or incorporated composition

When a multivalent binding or incorporation composition is used for replacement of a single unconjugated or untethered nucleotide to form a complex with a polymerase and one or more copies of a target nucleic acid, the local concentration of the nucleotide, as well as the binding avidity of the complex (when a complex containing two or more target nucleic acid molecules is formed) is increased several fold, resulting in improved signal intensity, specifically the correct signal to mismatch. 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.

Figure 6 illustrates the use of the disclosed polymer-nucleotide conjugates to achieve increased signal intensity during binding, persistence, and washing/removal steps. Due to the increased local concentration of nucleotides in the polymer-nucleotide conjugate and/or forming non-covalent bonds with two or more primed target nucleic acid molecules, the interaction between the polymerase, the primed target strand, and the polymer-conjugated nucleotides occurs. Binding becomes more favorable when the nucleotide is complementary to the next base of the target nucleic acid. The formed binding complex has a longer duration, which helps to increase the signal and shorten the imaging step. The high signal intensity resulting from the use of the disclosed polymer nucleotide conjugates remains stable throughout the binding and imaging steps. The strong bond 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 away. It means that it is. 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 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.

By way of 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 Figure 6 , which represents the change in signal intensity observed experimentally. Therefore, the compositions and methods of the present invention provide a powerful and controllable means of establishing and maintaining a three-way enzyme complex, as well as a greatly improved means by which the presence of the complex can be identified and/or measured, and the persistence of the complex can be controlled. Provides the means to do so. 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 linkage compositions disclosed herein associate with the polymerase nucleotide complex at a time-dependent rate to form a ternary linkage complex, but substantially slower than the rate of association known to be obtainable with nucleotides in free solution. It is not intended to be limited to a specific theory. Accordingly, the association rate (K _on ) is substantially and surprisingly slower than the association rate for a single nucleotide or a nucleotide not attached to a multivalent ligand complex. However, importantly, the dissociation rate (K _off ) of the multivalent ligand complex is significantly slower than the rate observed for nucleotides in free solution. Therefore, the multivalent ligand complexes herein (in particular those formed with free nucleotides) provides a surprising and beneficial improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes and, for example, allows significantly improved imaging quality for nucleic acid sequencing applications compared to currently available methods and reagents. Importantly, this property of the multivalent binding compositions disclosed herein allows the formation of visible ternary complexes to be controllable so that subsequent visualization, modification, or processing steps can occur essentially independent of dissociation of the complex, i.e., complex can be formed, imaged, modified, or used as needed, and remains stable until an active dissociation step is performed, such as exposing the complex to a dissociation buffer.

In some cases, the duration of multivalent binding complexes formed using the disclosed particle-nucleotide or polymer-nucleotide conjugates can range from about 0.1 seconds to about 600 seconds under non-destabilizing conditions. In some cases, the duration is at least 0.1 second, 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, or at least 10 seconds. , at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, at least 120 seconds, at least 180 seconds, at least 240 seconds, at least 300 seconds, at least 360 seconds, at least 420 seconds, at least 480 seconds, at least It could be 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 per se or any polymerase that may be known 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 polymerase; 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 telomerase. Meraje. Additional non-limiting examples of DNA polymerases include those from various Archaea genera, such as Aeropyrum , Archaeoglobus , Desulfurococcus , and Pyrobaculum . ), Pyrococcus , Pyrolobus , Pyrodictium , Staphylothermus , Stetteria , Sulfolobus , Thermo Polymerases known in the art, including polymerases derived from Thermococcus , Vulcanisaeta , etc., or variants thereof, such as Vent™, Deep Vent™, Pfu, KOD, Pfx, Therminator™, and May include Tgo polymerase. In some embodiments, the polymerase is klenow polymerase.

The ternary complex lasts longer when the nucleotides of the polymer-nucleotide conjugate are complementary to the target nucleic acid than when the nucleotides are non-complementary. Ternary complexes also have a longer duration when the nucleotides of the polymer-nucleotide conjugate are complementary to the target nucleic acid than when the complementary nucleotides are not conjugated or tethered. For example, in some embodiments, the ternary complex has a duration of less than 1 s, greater than 1 s, greater than 2 s, greater than 3 s, greater than 5 s, greater than 10 s, greater than 15 s, greater than 20 s, greater than 30 s, greater than 60 s, greater than 120 s, 360 s. It can be a time greater than, greater than or equal to 3600 s, or longer, or within a 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 the 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 a binding complex, such that a signal from the label can be detected for the duration of the binding complex.

It has been observed that different ranges of durations can be achieved with different salts or ions, showing, for example, that complexes formed in the presence of magnesium ions (Mg ²⁺ ) form more quickly than complexes formed with other ions. , for example, complexes formed in the presence of strontium ions (Sr ²⁺ ) are readily formed, upon recovery of the ions, or with 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 interaction moieties, or a buffer that contains, for example, a chelating agent capable of causing or accelerating the removal of divalent cations from the multivalent reagent comprising the complex. Complete or substantially complete dissociation has been observed. Accordingly, in some embodiments, the compositions of the present invention include Mg ²⁺ . In some embodiments, the compositions of the invention include Ca ²⁺ . In some embodiments, the compositions of the present invention include Sr ²⁺ . In some embodiments, the compositions of the present invention include cobalt ions (Co ²⁺ ). In some embodiments, the compositions of the present invention include MgCl ₂ . In some embodiments, the compositions of the present invention include CaCl ₂ . In some embodiments, the compositions of the present invention include SrCl ₂ . In some embodiments, the compositions of the present invention include CoCl ₂ . In some embodiments, the composition contains no, or substantially no, magnesium. In some embodiments, the composition contains no, or substantially no, calcium. In some embodiments, the methods of the invention provide the step of contacting one or more nucleic acids with one or more compositions disclosed herein, wherein the composition has neither calcium nor magnesium, or neither calcium nor magnesium. No.

Dissociation of the ternary complex can be controlled by varying the buffer conditions. After the imaging step, buffers with increased salt content are used to cause dissociation of the ternary complex so that the labeled polymer-nucleotide conjugate can be washed away, causing the signal to attenuate or terminate, as in the transition between one sequencing cycle and the next. Provides the means to do so. In some embodiments, this dissociation can be effected by washing the complex with a buffer that does not have the required metal or cofactor. 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 include one or more monovalent cations, such as sodium. In some embodiments, the wash buffer is free or substantially free of divalent cations, for example, free or substantially free of strontium, calcium, magnesium, or manganese. In some embodiments, the wash buffer further comprises a chelating agent, such as EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, etc. In some embodiments, the wash buffer can maintain the pH of the environment at the same level as the bound complex. In some embodiments, the wash buffer can further raise or lower the pH of the environment relative to the level found in the bound complex. In some embodiments, the pH is in the range of 2-4, 2-7, 5-8, 7-9, 7-10, or less than 2, or greater than 10, or by any two of the values provided herein. It can be within a defined range.

The addition of specific ions can 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, etc. In some embodiments, the ion is added to 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 ,} etc., as described herein. may 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 especially Chapter 17 and the related disclosure of salts, ions, salt solutions, and ionic solutions.

The present invention contemplates contacting a multivalent binding or incorporating 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, the contacting includes contacting the multivalent bond or incorporating composition with a polymerase. In some embodiments, the contacting includes contacting the composition comprising one or more nucleotides with a multiple polymerase. Polymerase can bind 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 rendered catalytically inactive by mutation. In one embodiment, the polymerase may be rendered catalytically inactive by chemical modification. In some embodiments, a polymerase molecule may be catalytically inactivated by the absence of an essential substrate, ion, or cofactor. In some embodiments, the polymerase enzyme may be 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 contains calcium or strontium.

When a catalytically inactive polymerase is used to help the nucleic acid interact with the multivalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex, causing the complex to fluorescently or as disclosed herein. or become detectable by other methods known in the art. Unbound polymer-nucleotide conjugates can optionally be washed away prior to detection of the ternary 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, the steps of contacting one or more nucleic acids with a polymer-nucleotide conjugate disclosed herein in a solution having either calcium or magnesium, or neither calcium nor magnesium, and, regardless of the order of the steps, separate steps. Adding either calcium or magnesium, or both calcium and magnesium, to the solution. In some embodiments, contacting one or more nucleic acids in a solution without strontium with a polymer-nucleotide conjugate disclosed herein includes adding strontium to the solution in a separate step, regardless of the order of the steps. do.

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

Disclosed herein are solid supports comprising low non-specific binding surface compositions that can improve nucleic acid hybridization and amplification performance. Generally, the disclosed support includes 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 ( s) may include one or more covalently or non-covalently attached primer sequences that can be used to link to the support surface. In some cases, the formulation of the surface, e.g., the chemical composition of one or more layers, 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 similar monolayers. The coupling chemistry used to crosslink one or more layers to the support surface and/or to each other, and the total number of layers can vary. Often, the formulation of the surface can be varied so that non-specific hybridization at the support surface is minimized or reduced compared to a similar monolayer. The formulation of the surface can be varied so that non-specific amplification on the support surface is minimized or reduced compared to a similar monolayer. The formulation of the surface can be varied to maximize the specific amplification rate and/or yield on the support surface. Suitable amplification levels for detection are achieved with no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or, in some cases disclosed herein, at least 30 amplification cycles.

Examples of materials from which the substrate or support structure may be made may include, but are not limited to: glass, fused-silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS) ), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin 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 can be of any of a variety of geometries and sizes known to those skilled in the art, and may include 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 locally planar (e.g., comprising a microscope slide or the surface of a microscope slide). Overall, the substrate or support structure may be cylindrical (e.g., including a capillary or the inner surface of a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregularly shaped (e.g., irregularly shaped, including the outer surface of a non-porous bead or particle). In some cases, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some cases, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous, such that the coatings described herein penetrate the porous surface and the nucleic acid hybridization and amplification reactions conducted thereon occur within the pores. It can happen.

A substrate or support structure comprising one or more chemically modified layers, for example a layer of a low non-specific binding polymer, can 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 comprise one or more surfaces within the microplate format, such as the bottom surfaces of the wells of the microplate. As mentioned above, in some preferred embodiments the substrate or support structure comprises the interior surface (e.g., lumen surface) of the capillary. In an alternative preferred embodiment, the substrate or support structure comprises the inner surface (eg, lumen surface) of the capillary etched into a planar chip.

As mentioned, the low nonspecific binding supports of the present invention exhibit reduced nonspecific 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 (e.g., cyanine such as Cy3, or Cy5, etc., fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), fluoresces under a standardized set of conditions. -Expose the surface to labeled nucleotides, fluorescently-labeled oligonucleotides, and/or fluorescently-labeled proteins (e.g., polymerase), followed by specified cleaning protocols and fluorescence imaging to cover different surface formulations. It can be used as a qualitative tool to compare non-specific binding in supporting supports. In some cases, exposure of the surface to fluorescent dyes, fluorescently-labeled nucleotides, fluorescently-labeled oligonucleotides, and/or fluorescently-labeled proteins (e.g., polymerases) under a standardized set of conditions followed by designated washing. 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 varies with the number of fluorophores on the support surface (e.g. Care was taken to ensure that fluorescence imaging was performed under conditions that were linearly related (or related in a predictable manner) (under conditions where signal saturation and/or self-quenching were not a problem) and appropriate calibration standards were used. In some cases, other techniques known to those skilled 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 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 ranges herein. 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 ranges herein.

As noted, in some cases, the degree of non-specific binding exhibited by the disclosed low-binding supports can be reduced by using standardized protocols to surface-label the 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 evaluated by comparing it to 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 skilled in the art. In some cases, the degree of non-specific binding exhibited by a given support surface formulation can therefore be assessed in terms of the number of protein molecules (or other molecules) non-specifically bound per unit area. In some cases, the low-binding supports of the invention have less than 0.001 molecules per μm ² , less than 0.01 molecules per μm ² , less than 0.1 molecules per μm ² , less than 0.25 molecules per μm ² , less than 0.5 molecules per μm ² , less than 0.5 molecules per μm ² Non-specific protein binding of less than 1 molecule, less than 10 molecules per μm ² , less than 100 molecules per μm ² , or less than 1,000 molecules per μm ² (or other specified molecules, (e.g., cyanine such as Cy3, or Cy5, etc.) dye, fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein). Those skilled in the art will recognize that a given support surface of the invention may exhibit non-specific binding anywhere within the above range, for example, less than 86 molecules per μm ² . For example, some of the modified surfaces disclosed herein are contacted with a 1 μM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes, followed by three washes with deionized water. , indicating nonspecific 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-647N (Jena Biosciences) 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-Diaz-dGTP-Cy3 (Jena Biosciences) was incubated in low binding substrate in 384 well plate format for 15 min at 37 °C. Each well was washed 2-3 times with 50 ul deionized RNase/DNase free water and incubated for 25 minutes. Washes were performed 2-3 times with mM ACES buffer (pH 7.4).384 well plates were filtered on a GE Typhoon instrument using a manufacturer-specified Cy3, AF555, or Cy5 filter set (depending on the dye test being performed) at a PMT gain of 800. gain setting and imaging at a resolution of 50-100 μm. For high-resolution imaging, an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) was used with a total internal fluorescence (TIRF) objective (100X, 1.5 NA, Olympus). ), CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus Images were collected with an Olympus U-HGLGPS fluorescence source) and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors were obtained from Semrock (IDEX Health & Science, LLC, Rochester, New York), e.g., 405, 488. , 532, or 633 nm dichroic reflectors/beamsplitters were purchased, and the bandpass filter was selected as 532 LP or 645 LP, matching the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 dye molecules per μm ² .

In some cases, 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 ranges herein. 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 ranges herein.

Low-background surfaces, consistent with the disclosure herein, have at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 specific dye molecules attached per molecule non-specifically adsorbed. , 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50, specific dye attachment (e.g., Cy3 attachment) versus non-specific dye adsorption (e.g. , Cy3 dye adsorption) ratio can be expressed. Similarly, when excitation energy is applied, consistent with the disclosure herein, a low-background surface to which a fluorophore, e.g., Cy3, is attached produces a specific fluorescence signal (e.g., Cy3 attached to the surface). -a ratio of non-specifically absorbed dye fluorescence signal (resulting from labeled oligonucleotides) to 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 degree of hydrophilicity (or “wetability” for aqueous solutions) of the disclosed support surface can be assessed, for example, by measuring the water contact angle, which places a droplet on the surface and, for example, optical tension. Measure the contact angle with the surface using a meter. In some cases, a static contact angle can be determined. In some cases, advancing or receding contact angles may be measured. In some cases, the water contact angle for the hydrophilic, low-binding support surface disclosed herein can range from about 0 degrees to about 30 degrees. In some cases, the water contact angle for the hydrophilic, low-binding support surfaces disclosed herein is 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. Those skilled in the art will recognize that a given hydrophilic, low-binding support surface herein may 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, a suitable cleaning step may be performed in less than 60, 50, 40, 30, 20, 15, 10 seconds, or in less than 10 seconds. For example, in some cases a suitable cleaning step can be performed in less than 30 seconds.

Some low-bond surfaces herein exhibit significant improvements in stability or durability against long-term exposure to solvents and high temperatures, or repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be determined by fluorescently labeling functional groups on the surface, or biomolecules tethered to the surface (e.g., oligonucleotide primers), prolonged exposure to solvents and high temperatures, or exposure to solvents. Alternatively, it can be tested by monitoring the fluorescence signal before, during, and after repeated cycles of temperature changes. In some cases, the degree of change in fluorescence used to assess the quality of a surface can be measured after 1 minute of exposure to solvent and/or high temperature. 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. , 5%, 10%, 15%, 20%, or 25% (or any combination of the above percentages measured over the above period of time). In some cases, the degree of change in fluorescence used to assess the quality of a surface is 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, or 70 cycles of repeated exposure to solvent changes and/or temperature changes. 1%, 2%, 3%, over 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, It may be 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 signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces have signals at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, greater than those of adjacent non-dense regions of the surface. It may indicate an amplified signal of 100 times or more than 100 times. Similarly, some surfaces enhance the signal of adjacent amplified nucleic acid-dense regions of the surface by at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 100 times the signal of adjacent amplified nucleic acid-dense regions of the surface. -Indicates an amplified signal exceeding twofold.

In some cases, the fluorescent images of the disclosed low background surfaces may be used in nucleic acid hybridization or amplification applications to generate clusters of hybridized or clonally amplified nucleic acid molecules (e.g., labeled directly or indirectly 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 support surface. In some cases, one or more types of adapters or primers include a spacer sequence, an adapter sequence for hybridizing to an adapter-ligated target library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or a molecular barcoding sequence, or the like. Any combination may be included. 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 can be tethered to at least one layer of the surface.

In some cases, the tethered adapter and/or primer sequence 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. there is. In some cases, the tethered adapter and/or primer sequence may be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides in length. there is. Any of the lower limits and lower limits set forth in this paragraph may be combined to form ranges encompassed within the specification, for example, in some cases the length of the tethered adapter and/or primer sequence may range from about 20 nucleotides to about 80 nucleotides. It can be a range of nucleotides. Those skilled in the art will recognize that the length of the tethered adapter and/or primer sequence may be any value within the above range, for example, about 24 nucleotides.

In some cases, the resulting surface density of primers on the low binding support surfaces of the invention may range from about 100 primer molecules per μm ² to about 100,000 primer molecules per μm ² . In some cases, the resulting surface density of primers on the low binding support surfaces of the invention may range from about 1.000 primer molecules per μm ² to about 1,000,000 primer molecules per μm ² . In some cases, the surface density of the primers 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 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 of the lower limits and lower limits set forth in this paragraph may be combined to form ranges encompassed within the specification, for example, in some cases the surface density of the primer may range from about 10,000 molecules per μm ² to about 100,000 molecules per μm ² It may be a range of molecules. Those skilled in the art will recognize that the surface density of primer molecules can have any value within the above range, for example, about 455,000 molecules per μm ² . In some cases, the surface density of target library nucleic acid sequences initially hybridized to adapter or primer sequences on the support surface may be less than or equal to that indicated for the surface density of the tethered primers. In some cases, the surface density of clonally amplified target library nucleic acid sequences hybridized to adapter or primer sequences on the support surface may span the same range as indicated for the surface density of the tethered primers.

The local densities listed above do not exclude density variations across the surface, so that the surface includes, for example, a region with an oligodensity of 500,000/μm ² while also comprising at least a second region with a substantially different local density. can do.

VI. Exemplary Alternative Embodiments

The disclosed method of determining the sequence of a target nucleic acid includes the following steps: a) binding one or more disclosed nucleic acid binding compositions to 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; contacting with; and b) detecting binding of the nucleic acid binding composition to a nucleic acid molecule, determining the presence of one of the one or more nucleic acid binding compositions on the nucleic acid molecule and the presence of the next nucleotide (i.e., N+1 or terminal nucleotide) to be incorporated into the complementary strand. The step of determining substance.

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

Extension of the primed target nucleic acid can be prevented or inhibited by blocked nucleotides on the strand or by the use of a catalytically inactive polymerase. When a nucleotide in a 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 the polymer, branched polymer, dendrimer, particle, etc. ) can be achieved. If elongation 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 before, simultaneously with, or after incorporation of the nucleotide residues. In some embodiments, a primed target nucleic acid may comprise a target nucleic acid that has multiple primed sites for attachment of polymerase and/or nucleic acid binding moieties. In some embodiments, multiple polymerases can 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 can be linked to a multivalent linkage composition disclosed herein comprising multiple nucleotides. In some embodiments, the target nucleic acid molecule can be prepared by strand displacement synthesis, rolling circle amplification, concatenation or fusion of multiple sequences of a query sequence, or any method known in the art to generate a nucleic acid molecule comprising multiple copies of the same sequence. or it may be the product of another method disclosed elsewhere herein. Therefore, in some embodiments, multiple polymerases may be attached to multiple identical or substantially identical positions within a target nucleic acid comprising multiple identical or substantially identical copies of the query sequence. In some embodiments, the multiple polymerases may be involved in interaction with one or more multivalent binding complexes; However, in a preferred embodiment, the number of binding sites in the target nucleic acid is at least 2, and the number of nucleotides or substrate moieties present in the particle-nucleotide conjugate, such as a polymer-nucleotide conjugate, is also at least 2.

It may be advantageous to provide multivalent binding compositions in combination with other elements, such as to provide optimized signals, for example to provide identification of nucleotides at specific positions in a nucleic acid sequence. In some embodiments, the compositions disclosed herein are provided in combination with surfaces that provide low background binding or low levels of protein binding, particularly hydrophilic or polymer coated surfaces. Representative surfaces can be found, for example, in US patent application Ser. No. 16/363,842, the contents of which are incorporated herein by reference in their entirety.

In some cases, the nucleic acid molecule is tethered to the surface of a solid support, for example, through 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 includes glass, fused-silica, silicon, or polymeric matrices. In some cases, the solid support comprises a low non-specific binding coating comprising one or more hydrophilic polymer layers (e.g., a PEG layer), where at least one of the hydrophilic polymer layers has branched polymer molecules (e.g., 4, branched PEG molecules containing 8, 16, or 32 branches).

The solid support includes oligonucleotide adapters or primers tethered to at least one hydrophilic polymer layer, with a surface density ranging from about 1,000 primer molecules per μm ² to about 1,000,000 primer molecules per μm ² . In some cases, the surface density of oligonucleotide primers 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 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 of the lower limits and lower limits set forth in this paragraph may be combined to form ranges encompassed within the specification, for example, in some cases the surface density of the primer may range from about 10,000 molecules per μm ² to about 100,000 molecules per μm ² It may be a range of molecules. Those skilled in the art will recognize that the surface density of primer molecules can have any value within the above range, for example, about 455,000 molecules per μm ² .

Those skilled in the art will recognize that, in a series of iterative sequencing reactions, occasionally one or more sites fail to incorporate nucleotides during a given cycle, causing one or more sites to become unsynchronized with the bulk of the extending nucleic acid chain. In conditions where the sequencing signal is derived from reactions arising from 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 methods to reduce these types of errors in sequencing reactions. For example, by providing an increased possibility of recombination upon premature dissociation of the ternary polymerase complex, the use of multivalent substrates that can incorporate into the extending strand will reduce the frequency of “skipped” cycles in which no base is incorporated. You can. Accordingly, in some embodiments, the invention encompasses a nucleoside moiety within a nucleotide having a free, or reversibly modified, 5' phosphate, diphosphate, or triphosphate moiety, wherein such nucleotide is unstable or or linked to a particle or polymer disclosed herein via a cleavable linkage. In some embodiments, the present disclosure contemplates a reduction in intrinsic error rate due to skipped incorporation as a result of using the multivalent substrates disclosed herein.

The invention also contemplates sequencing reactions in which the sequencing signal from or associated with a given sequence is derived from or within a definable region containing 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 a defined region also reduces the impact of any single cycle being skipped, due to the fact that signals from a large number of correct base confirmations can overwhelm signals from a small number of skipped or incorrect base confirmations. The present invention additionally contemplates the inclusion of free, unlabeled nucleotides, either during the elongation reaction, or during a separate portion of the elongation cycle, to provide for incorporation into 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 for incorporation into skipped sites. Unlabeled blocked nucleotides may be of the same type or types as the nucleotides attached to the multivalent binding substrate or substrate that are or were present during a particular cycle, or may be one, two, three, four or more types of unlabeled blocked nucleotides. Mixtures may be included.

If each sequencing cycle proceeds perfectly, each reaction within the defined region will give the same signal. However, as noted elsewhere herein, in a series of iterative sequencing reactions, occasionally one or more sites fail to incorporate nucleotides during a given cycle, causing one or more sites to become out of sync with the bulk of the extending nucleic acid chain. You will recognize that it is not. This problem, also called “phasing,” leads to degradation of the sequencing signal because the signal is contaminated with spurious signals from regions that have one or more skipped cycles. This, in turn, creates the potential for errors in base identification. As skipped cycles gradually accumulate over multiple cycles, the effective read length also decreases because the sequencing signal gradually degrades with each cycle. Additionally, it is an object of the present invention to provide a method for reducing paging errors and/or improving read length in sequencing reactions.

Sequencing methods may include 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 one or more particle-nucleotide conjugates with said target nucleic acid, or multiple target nucleic acids, containing multiple linked or unlinked copies of a target sequence can substantially increase the local concentration of the correct nucleotide being probed in a given sequencing cycle; , thus suppressing signals from inappropriately incorporated or phased nucleic acid chains (i.e., extending nucleic acid chains with one or more skipped cycles).

A method of obtaining nucleic acid sequence information may include 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 copies of the target sequence. Contains unused copies. These methods reduce the error rate of sequencing, as indicated by a decrease in misidentification of bases, reporting of non-existent bases, or failure to report correct bases. In some embodiments, the reduction in error rate of the sequencing is compared to the error rate observed using monovalent ligands, including free nucleotides, labeled free nucleotides, protein or peptide linked nucleotides, or labeled protein or peptide linked nucleotides. This may include reductions of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more.

A method of obtaining nucleic acid sequence information may include 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 copies of the target sequence. Contains unused copies. This method compares 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 an average read length of 5 Increase by %, 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 target nucleic acids. Contains multiple linked or unlinked copies of a sequence. This method compares 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 an average read length of 10 Increases nucleotides (NT) by 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, 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, 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 may be an average read length of 375 nucleotides to 825 nucleotides. Those skilled in the art will recognize that in some cases the average read length may have any value within the range specified in this paragraph, for example, 523 nucleotides.

The use of multivalent binding compositions for sequencing effectively shortens sequencing time. The sequencing reaction cycle, including contacting, detection, and incorporation steps, is performed with an overall 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, a sequencing reaction cycle is performed in up to 60 minutes, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 20 minutes, up to 10 minutes, or up to 5 minutes. Any of the lower limits and lower limits described in this paragraph may be combined to form ranges encompassed within the specification, for example, in some cases a sequencing reaction cycle may be performed with an overall time ranging from about 10 minutes to about 30 minutes. there is. Those skilled 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 nucleic acid sequencing provide at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, It will provide an average base determination accuracy of at least 99.5%, at least 99.8%, or at least 99.9% accurate. In some cases, the disclosed compositions and methods for nucleic acid sequencing are at least 80%, at least 85%, at least 90%, or at least every 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases. It will provide an average base determination accuracy of 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.

The use of multivalent binding compositions for sequencing provides more accurate base reads. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q-score ranging from about 20 to about 50 for base confirmation accuracy across sequencing runs. 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. Those skilled in the art will recognize that the average Q-score may be any value within the above range, for example about 32.

In some cases, the disclosed compositions and methods for nucleic acid sequencing can be used to sequence 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 nucleic acid sequencing can be used to sequence 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 nucleic acid sequencing can be used to sequence 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 nucleic acid sequencing can be used to sequence 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 nucleic acid sequencing can be used to sequence 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 supports and associated 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 skilled in the art. For example, nucleic acids can be extracted from cells, or tissue samples, including one or more types of cells derived from eukaryotes (e.g., 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, for example, from primary or immortalized rodent, porcine, cat, dog, bovine, equine, primate or human cell lines. Nucleic acids can be used in any of a variety of different cell, organ, or tissue types (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, reproductive 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 diseased cells, such as cancer cells, or from pathogenic cells that infect the host. Distinct subsets of some nucleic acid cell types, e.g., immune cells (e.g., 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 may be derived from clinical or other samples, such as sputum, saliva, ocular fluid, synovial fluid, blood, feces, urine, tissue exudates, sweat, pus, drainage fluid, etc. Nucleic acids may additionally be derived from plant or fungal samples, such as leaves, cambium, roots, meristems, pollen, ova, seeds, spores, inflorescences, mycelium, etc. Nucleic acids can also be derived from environmental or industrial samples such as water, air, dust, food, etc. Other cells, tissues, and samples are contemplated and are consistent with the present disclosure.

Nucleic acid extraction from cells or other biological samples can be performed using any of a variety of techniques known to those skilled 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 will be extracted, (ii) disrupting cell membranes (i.e., cell lysis) to release DNA and other cytosolic components. , (iii) treating the dissolved 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 the 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 the Maxwell® and ReliaPrep™ series kits from Promega (Madison, WI). However, it is not limited to this.

VII. system

System Modules: As noted above, also disclosed herein are systems configured to perform any of the nucleic acid sequencing or nucleic acid detection and analysis methods disclosed. In some cases, the disclosed systems may include one or more multivalent binding compositions described herein, one or more buffering agents, and/or one or more nucleic acid molecules tethered to a solid support.

In some cases, the system is adapted to sequentially and repeatedly contact a tethered template nucleic acid molecule hybridized to a nucleic acid molecule (e.g., an adapter or primer) tethered to a solid support with the 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 within one or more flow cells. In some cases, the flow cell may be a stationary component of the 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 include an imaging module, which may be used to image and detect binding of the disclosed multivalent binding composition to a target (or template) nucleic acid molecule tethered within a solid support or flow cell. For example, one or more light sources, one or more optical components (e.g., lenses, mirrors, prisms, optical filters, colored glass filters, narrow-band interference filters, broadband interference filters, dichroic reflectors, diffraction gaps, apertures) , optical fiber, or optical waveguide, etc.), and one or more image sensors (e.g., 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 a system 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), graphics processing unit (GPU), general purpose processing unit, or computing platform. The one or more processors may be any of a variety of suitable integrated circuits (e.g., special purpose integrated circuits (ASICs) specifically designed to implement deep learning network architectures, or to accelerate computation time, etc., and/or to facilitate deployment. It may be composed of a field-programmable gate array (FPGA), a microprocessor, an emerging next-generation microprocessor design (e.g., a memristor-based processor), a logic device, etc. Although this specification is described with reference to processors, other types of integrated circuits and logic devices may also be applied. The processor may have any suitable data computing functionality. For example, a processor can perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations. The one or more processors may be a single core or multi-core processor, or a plurality of processors configured for parallel processing.

One or more processors or computers used to implement the disclosed methods may be part of a larger computer system and/or be operable on a computer network (“network”) with the aid of communication interfaces 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 communicating 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 enables distributed computing, such as cloud computing. A network may, in some cases with the help of a computer system, implement a peer-to-peer network, allowing devices connected to the computer system to act as clients or servers.

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

One or more processors, such as a CPU, execute a series of machine-readable instructions, which are implemented in a program (or software). Instructions are stored in memory locations. Instructions are directed to the CPU, which subsequently programs or otherwise configures the CPU to implement the methods of the present disclosure. Examples of tasks performed by the CPU include fetch, decode, execute, and write back. A 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 circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

Storage devices store files such as drivers, libraries, and stored programs. The storage device stores user data, such as user-specified preferences and usage-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 in communication 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. . Machine-executable or machine-readable code may be provided in the form of software. During use, code is executed by one or more processors. In some cases, the code is retrieved from storage and stored in memory for immediate access by one or more processors. In some situations, electronic storage is excluded and machine-executed instructions are stored in memory. The code may be precompiled and configured for use with a machine that has one or more processors configured to execute the code, or it may be compiled at runtime. The code may be precompiled or provided in the programming language of your choice so that it can be executed in an as-compiled fashion.

Various aspects of the technology are often referred to as "products" or "articles of manufacture" in the form of machine- (or processor-) executable code and/or associated data stored on some type of machine-readable medium, such as a "computer program or software product". The executable code may be considered to include 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 disk, CD-ROM, DVD, or Blu-ray disk. Machine-executable code may be stored in memory (e.g., read-only memory, random access memory, flash memory) or in an electronic storage device, such as a hard disk. “Storage” tangible media includes any or all of the tangible memory of a computer, processor, etc., or related modules, such as various semiconductor memory chips, optical drives, tape drives, disk drives, etc., including the methods and algorithms disclosed herein. May provide temporary storage for software that encodes .

All or portions of the Software Code may from time to time be communicated via the Internet or various other communications networks. For example, such communication enables loading of software from one computer or processor to another computer or processor, for example 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 standby links. The physical element carrying these waves, such as a wired or wireless link, optical link, etc., is also considered a medium carrying software encoded instructions for performing the methods disclosed herein. As used herein, and unless limited to a temporary, tangible “storage” medium, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

Computer systems often include or can communicate with an electronic display to present images captured, for example, by a machine vision system. A display may often provide a user interface (UI). Examples of UI include, but are not limited to, graphical user interfaces (GUIs), web-based user interfaces, etc.

System control software: In some cases, the disclosed system includes a user interface, as well as manual, semi-automatic, or fully automatic control of all system functions, such as fluid flow controller and/or fluid distribution system (or sub-system), temperature control. may include a computer (or processor) containing code to provide control of a system (or sub-system), an imaging system (or sub-system), etc., and a computer-readable medium . In some cases, the system computer or processor may be an integrated component of the device system (e.g., a microprocessor embedded within the device or the motherboard). 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 rate, fluid flow rate, timing and duration for sample and reagent additions, cleaning steps, etc. Temperature control functions that may be provided by device 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 times, data storage options, etc. No.

Image Processing Software: In some cases of the disclosed systems, the system may further include a computer-readable medium containing code to provide image processing and analysis capabilities. Image processing and analysis capabilities that may be provided by the Software include, but are not limited to, manual, semi-automatic, or fully automatic image exposure adjustments (e.g., white balance, contrast adjustments, signal-averaging and other noise reduction capabilities, etc.); or fully automated edge detection and object identification (e.g., identification of clusters of amplified template nucleic acid molecules on a substrate surface), manual, semi-automatic, or fully automatic signal intensity measurement and/or one or more detection channels (e.g., one or more fluorescence emissions) Channel) threshold setting, manual, semi-automated, or fully automated statistical analysis (e.g., comparing signal intensity to 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, cleaning, and/or imaging/base confirmation steps are required until optimal base confirmation results are achieved, for example. It may be extended, modified, or repeated depending on. Any of a variety of image processing and analysis algorithms known to those skilled in the art may be used to implement real-time or post-processed image analysis capabilities. Examples of these algorithms include the Canny edge detection method, Canny-Deriche edge detection method, first-order gradient edge detection method (e.g., Sobel operator), and second-order differential edge detection. methods, phase-consistent (phase-consistent) edge detection methods, different image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., random generalized Hough transform, circular Hough transform, etc.), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation, etc.) to detect the shape of , or a combination thereof, but is not limited thereto.

In some cases, 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 bond composition

One type of multi-arm substrate shown in Figure 5A was prepared by reacting propargylamine dNTP with biotin-PEG-NHS. By completing and purifying this aqueous reaction; Pure biotin-PEG-dNTP species was generated. In separate reactions, 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-sourced dye-labeled streptavidin (SA) at a Dye:SA ratio of 3-5:1. Mixing of biotin-PEG-dNTPs with dye-labeled streptavidin was performed in the presence of an excess of 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 conjugated and purified individually and then mixed together to create a four-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 dNTP 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 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 four-base mixture for sequencing.

The class II substrate shown in Figure 5B was prepared using a one-pot reaction to simultaneously conjugate the dye and dNTP. Alkyne-PEG-NHS was reacted with an excess of propargylamine dNTP. 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 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 four-base mixture for sequencing. It is noted that this scheme allows the immediate replacement of alternative cores such as dextrans, other polymers , proteins, etc.

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. The reaction resulted in a multi-arm PEG containing separate distributions of biotin and nucleotides. The 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 four-base mixture for sequencing. Note that the distribution of biotin and nucleotides is tunable by the input ratio of biotin-NH ₂ to propargylamine dNTP.

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, and fluorescence images at various stages are illustrated in Figures 7A-7J . In Figure 7A , red and green fluorescence images show DNA rolling circle application (RCA) template (G and A first bases) with 20 nM Klenow polymerase and 500 nM base labeled nucleotides (A-Cy3 and G-Cy5). This is after exposure in exposure buffer containing 2.5 mM Sr ⁺² . Multivalent PEG-based compositions were prepared using the following various ratios of 4-arm EG-amine (4ArmPEG-NH), biotin-PEG-amine (Biotin-PEG-NH), and nucleotide (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 imaging buffer, which had the same composition as the exposure buffer but did not contain 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 , fluorescence images show the multivalent PEG-nucleotide (base-labeled) ligand at 500 nM after mixing in exposure buffer and imaging in imaging buffer as above ( Figure 7B : PB1; Figure 7C : PB2; Figure 7 Figure 7D : PB3; Figure 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 imaging buffer as above. In Figures 7G-7I , fluorescence images show additional base discrimination by multivalent ligand exposure for inactive mutants of Klenow polymerase ( Figure 7G : D882H; Figure 7H : D882E; Figure 7I : D882A, and wild-type Klenow (control) Enzymes are as shown in Figure 7J ).

Multivalent ligand formulations can be used to provide polymerase-ligand interactions with increased avidity, thereby enabling base identification. Additionally, we show that increasing the concentration of multivalent ligand can provide higher signal, as well as a variety of Klenow mutants that knock out catalytic enzyme activity, which 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. Subsequent 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 concentration) 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 cycling templates. As shown in Figures 8A and 8B , multivalent ligands can provide base discrimination through all five sequencing cycles.

4. Controlling nucleotide dissociation from ternary complexes

The ternary complex was 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 some time, the same buffer used for the formation of the complex but without any divalent cations, e.g. 10mM Tris pH 8.0, 0.5mM EDTA, 50mM NaCl, 0.016% Triton Wash the complex with a buffer, or alternatively, the same buffer used for complex formation, but containing a chelating agent, but containing optional divalent cations, e.g., 10mM Tris pH 8.0, 0.5mM EDTA, 50mM NaCl. Wash the complex with buffer containing no , 0.016% Triton X100 (without SrOAc), and 100nm-100mM EDTA. Fluorescence from the complex is observed over time, allowing the dissociation of the ternary complex to be observed and quantified. A representative time course of this dissolution is shown in Figure 6 .

5. Extension of 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-, is added from the 3' end of the extending DNA strand. An unblocking solution sufficient to remove amino groups is introduced into the chamber containing the bound DNA molecules. Accordingly or simultaneously, 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 the addition of a single nucleotide, e.g. It is blocked from further extension by incorporation of an -O-azidomethyl group, 3'-O-alkyl hydroxylamino group, or 3'-O-amino group. The extended strand is thus extended by just one base, and the combination of a catalytically inactive polymerase and a multivalent substrate can be used to call the next base in the cycle.

Alternatively, the nucleotides attached to the multivalent substrate are attached via labile bonds, allowing the polymerase to flow into a buffer containing the bound DNA molecules containing sufficient divalent cations or other cofactors to render them catalytically active. This may come in. Before, after or simultaneously with this, conditions may be provided sufficient to cleave the base from the multivalent substrate to allow for incorporation into the extended strand. This cleavage and incorporation causes the label and polymer backbone of the multivalent substrate to dissociate, extending the extending DNA strand by exactly one base. Washing is performed to remove the used polymer backbone, and a new multivalent substrate is introduced into the chamber containing the bound DNA molecules to call a new base 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 to a length corresponding to an average PEG MW of 5 K Da ( Figures 9A-9D ). Using longer PEG arms reduced the fluorescence signal for both Cy3-A and Cy5-G ( Figures 9E-9G ). Quantitative measurements of signal strength are shown graphically in Figure 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 10mM Tris pH 8.0, 0.5mM EDTA, 50mM NaCl, 5mM SroAc, 0% TritonX100 (condition A), and one set using The set used 10mM Tris pH 8.0, 0.5mM EDTA, 50mM NaCl, 5mM SroAc, 0.016% Triton X100. Figure 11 shows normalized fluorescence from multivalent substrates bound to DNA clusters, with substrate complexes formed in the presence of Triton-X100 (0.016%) (condition B) showing a clearly enhanced fluorescence intensity.

8. Evaluation of the time course of multivalent substrate binding

Multivalent substrates were prepared and assembled into binding complexes as in Example 2. Complexes were also formed under the same buffer conditions using labeled free nucleotides. The complex was imaged over a 60 minute period to characterize its duration. Figures 12A-12B show representative results. The multivalent complex is stable over time scales >60 minutes ( Figure 12B ), whereas the labeled free nucleotide dissociates in less than 1 minute ( Figure 12A ).

VIII. conclusion

The present invention provides greatly improved methods and compositions for DNA sequencing and biosensor applications. It should be understood that the above description is illustrative and not restrictive. Many embodiments will be apparent to those skilled in the art upon reviewing the above description. By way of example, although the inventive concept has 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; liposome; Alternatively, it may be desirable to use particle-nucleotide conjugates comprising emulsion particles. Alternatively, conjugation may be accomplished by non-covalent bonds such as hydrogen bonds or other interactions. Therefore, the scope of the disclosed inventive concept should be determined without reference to the above description, but instead with reference to the appended claims and the full scope of equivalents to which such claims are entitled.

Claims (73)

A method for determining the identity of a nucleotide in a target nucleic acid sequence, comprising:
(a) (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
providing a composition comprising;
(b) forming a multivalent binding complex between two or more nucleotide moieties of a polymer-nucleotide conjugate and two or more copies of the target nucleic acid sequence of the composition of (a). contacting the composition with the polymer nucleotide conjugate under conditions sufficient to ensure that the two or more nucleotide moieties are not incorporated into the two or more primer nucleic acid molecules, and wherein the polymer-nucleotide conjugate is Comprising one or more nucleotide moieties; and
(c) detecting the multivalent binding complex to determine the identity of the nucleotide in the target nucleic acid sequence.
Decision method including. The method of claim 1, wherein the target nucleic acid sequence is DNA. 2. The method of claim 1, wherein the step of detecting the binding complex is performed in the absence of unbound or solution-borne polymer nucleotide conjugate. The method of claim 1, wherein the target nucleic acid sequence has been cloned or amplified, or was produced by cloning or amplification. 2. The method of claim 1, wherein the step of detecting the multivalent binding complex comprises measuring fluorescence. 2. The method of claim 1, wherein the contacting step includes the use of one type of polymer-nucleotide conjugate. 2. The method of claim 1, wherein the contacting step includes the use of two or more types of polymer-nucleotide conjugates. 8. The method of claim 7, wherein each type of the two or more types of polymer-nucleotide conjugates comprises a different type of nucleotide moiety. 9. The method of claim 8, wherein the contacting step includes 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. 2. The method of claim 1, wherein the polymer-nucleotide conjugate comprises a blocked nucleotide moiety. 11. The method of claim 10, wherein the blocked nucleotide is a 3'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkyl hydroxylamine nucleotide. The method of claim 1, wherein the contacting step occurs in the presence of an ion that stabilizes the multivalent bond complex. 2. The method of claim 1, wherein the contacting step occurs in the presence of strontium ions, magnesium ions, calcium ions, or any combination thereof. 2. The method of claim 1, wherein the polymerase molecule is catalytically inert. The method of claim 1, wherein the polymerase molecule has been rendered catalytically inactive by mutation or chemical modification. 2. The method of claim 1, wherein the polymerase molecule is rendered catalytically inactive by the absence of a necessary ion or cofactor. 2. The method of claim 1, wherein the polymerase molecule is catalytically active. The method of claim 1 , wherein the polymer-nucleotide conjugate does not include blocked nucleotide moieties. The method of claim 1 , wherein the multivalent binding complex has a duration of greater than 2 seconds. 2. The method according to claim 1, wherein the method is carried out at a temperature in the range of 25°C to 42°C. 2. The method of claim 1, wherein the polymer-nucleotide conjugate further comprises one or more fluorescent labels, and wherein 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 A decision method having a contrast to noise ratio of greater than 20 in the detection step. 2. The method of claim 1, wherein the composition of (a) is deposited on the surface using a buffer incorporating a polar aprotic solvent. The method of claim 1, 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 the nucleotide moiety is complementary to the next base of the target nucleic acid sequence. A method for determining if not performed under conditions that destabilize the multivalent binding complex. The method of claim 1, wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches, and wherein the two or more nucleotide moieties are attached to the branches. 25. The method of claim 24, wherein the polymer has a star, comb, cross-linked, bottle brush, or dendrimer shape. The method of claim 1, wherein the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tag, and combinations thereof. 2. The method of claim 1, further comprising a dissociation step to destabilize the multivalent binding complex formed between the composition of (a) and the polymer-nucleotide conjugate, wherein the dissociation step allows removal of the polymer-nucleotide conjugate. How to decide what to do. 28. The method of claim 27, further comprising an extension step of incorporating a nucleotide complementary to the next base of the target nucleic acid sequence into the two or more primer nucleic acid molecules. 29. The method of claim 28, wherein the extending step occurs simultaneously with or after the dissociating step. 2. The method of claim 1, wherein the polymer-nucleotide conjugate comprises one or more detectable labels. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete