Polyvalent binding compositions for nucleic acid analysis

Abstract

To provide systems that include multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle, applicable for detecting protein-protein interactions, protein-nucleic acid interactions, nucleic acid hybridization, and the like.SOLUTION: A system comprises: a composition of a nucleotide conjugate comprising a polymer core and a plurality of nucleotide moieties attached thereto; and a solid support having a surface containing a plurality of primed nucleic acid sequences attached thereto, wherein a plurality of primer nucleic acid sequences in the plurality of primed nucleic acid sequences, together with the plurality of nucleotide moieties, remain unextended during the primer extension reaction, and at least two or more nucleotide moieties among the plurality of nucleotide moieties are bound to at least two or more primed nucleic acid sequences among the plurality of primed nucleic acid sequences.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] cross reference This application is a continuation-in-part application of U.S. Patent Application No. 16 / 579,794 filed on 23 September 2019, and claims the interests of U.S. Provisional Application No. 62 / 897,172 filed on 6 September 2019 and U.S. Provisional Application No. 62 / 852,876 filed on 24 May 2019, each of which is incorporated herein by reference as a whole.

[0002] This disclosure relates, overall, to polyvalent binding compositions and their use in the analysis of nucleic acid molecules. In particular, the inventive concept relates to polyvalent binding compositions having multiple copies of nucleotides attached to particles or polymer cores, thereby effectively increasing the local concentration of nucleotides and enhancing the binding signal. Polyvalent binding compositions may be applied, for example, in the fields of sequencing and microarrays of biosensors. [Background technology]

[0003] Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical situations, including diagnostics, prognosis, biotechnology, and forensic biology. Various sequencing methods are being developed, including de novo sequencing methods such as the Maxam-Gilbert method and chain termination method, shotgun sequencing, and bridge PCR, as well as next-generation methods such as Polony sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, HeliScope single-molecule sequencing, and SMRT® sequencing. Despite advances in DNA sequencing, many challenges remain unresolved for cost-effective, high-throughput sequencing. This disclosure provides novel solutions and approaches that address many of the shortcomings of existing technologies. [Overview of the project]

[0004] Disclosed herein is a method for determining nucleotide identity in a target nucleic acid sequence, the method comprising: 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 allow the formation of a polyvalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition of (a), 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 polyvalent binding complex, thereby determining the nucleotide identity in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the step of detecting the polyvalent binding composition is carried out in the absence of an unbound or solution-derived polymer-nucleotide conjugate. In some embodiments, the target nucleic acid sequence is replicated, amplified, or produced by replication or amplification. In some embodiments, one or more detectable labels are fluorescent labels. In some embodiments, the step of detecting the polyvalent complex includes fluorescence measurement. In some embodiments, the contact includes the use of one polymer-nucleotide conjugate. In some embodiments, the contact includes the use of two or more polymer-nucleotide conjugates. In some embodiments, each of the two or more polymer-nucleotide conjugates contains a different species of nucleotide moiety. In some embodiments, the contact includes the use of three polymer-nucleotide conjugates, where each of the three polymer-nucleotide conjugates contains a different species of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate contains 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-alkylhydroxylamine nucleotide. In some embodiments, the contact is carried out in the presence of an ion that stabilizes the polyvalent conjugate composition. In some embodiments, the contact is carried out in the presence of a strontium ion, a magnesium ion, a calcium ion, or any combination thereof. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule is catalytically inactive by mutation or chemical modification. In some embodiments, the polymerase molecule is catalytically inactive by the absence of a required ion or cofactor. In some embodiments, the polymerase molecule is catalytically active. In some embodiments, the polymer-nucleotide conjugate does not contain a blocked nucleotide moiety. In some embodiments, the duration of the polyvalent conjugate is greater than 2 seconds. In some embodiments, the method can be carried out at temperatures in 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 a target nucleic acid sequence are deposited, attached to, or hybridized to a surface, where the fluorescence image of the polyvalent conjugate on the surface has a contrast-to-noise ratio greater than 20 in the detection step. In some embodiments, the composition of (a) is deposited on a surface using a buffer incorporating a polar aprotic solvent. In some embodiments, the contact step is carried out under conditions that stabilize the polyvalent conjugate when the nucleotide portion is complementary to the next base of the target nucleic acid sequence, and destabilize the polyvalent conjugate when the nucleotide portion is not complementary to the next base of the target nucleic acid sequence. In some embodiments, the polymer-nucleotide conjugate comprises a polymer having multiple branches, and the two or more nucleotide portions are attached to the branches. In some embodiments, the polymer configuration is star-shaped, comb-shaped, cross-shaped, bottlebrush-shaped, or dendrimer-shaped.In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the method further comprises a dissociation step that destabilizes the polyvalent binding complex formed between composition (a) and the polymer-nucleotide conjugate, the dissociation step allowing for the removal of the polymer-nucleotide conjugate. In some embodiments, the method further comprises an extension step for incorporating nucleotides 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 is performed simultaneously with or after the dissociation step.

[0005] Disclosed herein is a method for determining nucleotide identity in a target nucleic acid sequence, the method comprising: 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 allow the formation of a polyvalent binding complex between the polymer-nucleotide conjugate and the two or more copies of the target nucleic acid sequence in the composition of (a), wherein the polymer-nucleotide conjugate comprises two or more copies of a reversibly terminated nucleotide moiety and one or more optionally cleavable and detectable labels; and c. detecting the polyvalent binding complex, thereby determining the nucleotide identity in the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is DNA. In some embodiments, the method further includes, after detection of the polyvalent binding complex, contacting the composition of (a) with a polymer-nucleotide conjugate containing a reversibly terminated nucleotide or two or more copies of a reversibly terminated nucleotide. In some embodiments, the target nucleic acid sequence is replicated, amplified, or produced by replication or amplification. In some embodiments, one or more detectable labels are fluorescent labels. In some embodiments, the step of detecting the polyvalent complex includes fluorescence measurement. In some embodiments, the contact includes the use of one polymer-nucleotide conjugate. In some embodiments, the contact includes the use of two or more polymer-nucleotide conjugates. In some embodiments, each of the two or more polymer-nucleotide conjugates contains a different species of nucleotide moiety. In some embodiments, the contact includes the use of three polymer-nucleotide conjugates, where each of the three polymer-nucleotide conjugates contains a different species of nucleotide moiety. In some embodiments, the polymer-nucleotide conjugate contains a blocked nucleotide moiety.In some embodiments, the blocked nucleotide is 3'-O-azidomethyl, 3'-O-methyl, or 3'-O-alkylhydroxylamine. In some embodiments, the contact is carried out in the presence of an ion that stabilizes the polyvalent binding composition. In some embodiments, the polymerase molecule is catalytically inactive. In some embodiments, the polymerase molecule is 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 contain a blocked nucleotide moiety. In some embodiments, the method can be carried out at a temperature in 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, adhered to, or hybridized to a surface, where the fluorescence image of the polyvalent binding complex on the surface has a contrast-to-noise ratio greater than 20 in the detection step.

[0006] Furthermore, disclosed herein is a system comprising: a) one or more computer processors individually or collectively programmed to perform a method, the method comprising: i) contacting a substrate, which includes multiple copies of a target nucleic acid sequence anchored on its surface, with a reagent comprising a polymerase and one or more primer nucleic acid sequences complementary to one or more regions of the target nucleic acid sequence, in order 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 allow the formation of a polyvalent 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 portion and a detectable label; and iii) acquiring and processing an image of the substrate surface to detect the polyvalent binding complex, thereby determining the identity of nucleotides in the target nucleic acid sequence. In some embodiments, the system further comprises a fluid module configured to deliver a series of reagents to the substrate surface in a predetermined order and at predetermined time intervals. In some embodiments, the system further includes an imaging module configured to acquire an image of the substrate surface. In some embodiments, (ii) and (iii) are repeated two or more times to determine the identity of a set of two or more nucleotides in a target nucleic acid sequence. In some embodiments, the sequence of steps further includes a dissociation step that destabilizes the polyvalent binding complex, the dissociation step allowing for the removal of the polymer-nucleotide conjugate. In some embodiments, the sequence of steps further includes an extension step for 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 is performed simultaneously with or after the dissociation step. In some embodiments, the detectable label includes a fluorophore, and the image includes a fluorescence image.In some embodiments, the fluorophore is cyanine dye 3 (Cy3), and an image is acquired under non-signal saturation conditions using an inverted fluorescence microscope equipped with a 20X objective lens, NA=0.75, a dichroic mirror optimized for 532 nm light, a bandpass filter optimized for cyanine dye-3 emission, and a camera, while the surface is immersed in 25 mM ACES, pH 7.4 buffer, the fluorescence image of the polyvalent binding complex on the surface has a contrast-to-noise ratio greater than 20 in the detection step. 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 the base call is characterized by a Q score greater than 25 for at least 80% of the determined nucleotide identities. In some embodiments, the accuracy of the base call is characterized by a Q score greater than 30 for at least 80% of the determined nucleotide identities. In some embodiments, the accuracy of the base call is characterized by a Q score greater than 40 for at least 80% of the determined nucleotide identities.

[0007] Disclosed herein is a composition comprising a) a polymer core and b) two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core, wherein the linker length depends on the nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core. Furthermore, disclosed herein is a composition comprising a) a mixture of polymer-nucleotide conjugates, each of which comprises i) a polymer core and ii) two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core, wherein the linker length depends on the nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties attached to the polymer core, and the mixture comprises polymer-nucleotide conjugates having at least two different attached nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties. In some embodiments, the polymer core comprises a polymer having multiple branches, and two or more nucleotides, nucleotide analogs, nucleosides, or nucleoside analog moieties are attached to the branches. In some embodiments, the polymer configuration is star-shaped, comb-shaped, cross-shaped, bottlebrush-shaped, or dendrimer-shaped. In some embodiments, the polymer-nucleotide conjugate comprises one or more binding groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof. In some embodiments, the 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-alkylhydroxylamine nucleotide. In some embodiments, the polymer-nucleotide conjugate further comprises one or more fluorescent labels.

[0008] In some embodiments, the Disclosure provides a method for determining nucleotide identity in a target nucleic acid, the method comprising, in any order, 1) providing a composition comprising a target nucleic acid comprising two or more repeats of the same sequence, two or more primer nucleic acids complementary to one or more regions of the target nucleic acid, and two or more polymerase molecules; 2) contacting the composition with a polyvalent conjugate or embedded composition comprising a polymer-nucleotide conjugate under conditions sufficient to allow the formation of a conjugate or embedded complex between the polymer-nucleotide conjugate and the composition of step (a), wherein the polymer-nucleotide conjugate comprises two or more copies of a nucleotide and optionally one or more detectable labels; and 3) detecting the conjugate or embedded complex to establish the nucleotide identity in the target nucleic acid polymer. In some further embodiments, the Disclosure provides the method wherein the target nucleic acid is DNA, and / or the target nucleic acid is replicated by any commonly performed method of DNA replication or amplification, such as rolling circle amplification, bridge amplification, helicase-dependent amplification, isothermal bridge amplification, rolling circle multiple substitution amplification (RCA / MDA), and / or recombinase-based replication or amplification methods. In some further embodiments, the Disclosure provides the method wherein the detectable label is a fluorescent label, and / or the detection of the complex comprises fluorescence measurement. In some further embodiments, the Disclosure provides the method wherein the polyvalent conjugate composition comprises one type of polymer-nucleotide conjugate, the polyvalent conjugate composition comprises two or more polymer-nucleotide conjugates, and / or each of the two or more polymer-nucleotide conjugates comprises different species of nucleotides. In some embodiments, the present disclosure provides a method wherein the bound or incorporated complex further comprises a blocked nucleotide, in particular the blocked nucleotide being a 3'-O-azidomethyl nucleotide, a 3'-O alkylhydroxylamino nucleotide, or a 3'-O methyl nucleotide.In some further embodiments, the Disclosure provides the method wherein the contact is carried out in the presence of strontium ions, barium, magnesium ions, and / or calcium ions. In some embodiments, the Disclosure provides the method wherein the polymerase molecule is catalytically inactive, such as by mutation, chemical modification, or absence of a required ion or cofactor. In some embodiments, the Disclosure also provides the method wherein the polymerase molecule is catalytically active, and / or the binding complex does not contain blocked nucleotides. In some embodiments, the Disclosure provides the method wherein the binding complex has a duration of more than 2 seconds, and / or the method may be carried out at a temperature of 15°C or higher, 20°C or higher, 25°C or higher, 35°C or higher, 37°C or higher, 42°C or higher, 55°C or higher, 60°C or higher, 72°C or higher, or within the range defined by any of the above. In some embodiments, the Disclosure provides the method wherein the binding composition is deposited, attached to, or hybridized on a surface having a contrast-to-noise ratio of more than 20 in the detection step. In some embodiments, the Disclosure provides a method wherein the composition is deposited under buffer conditions that incorporate a polar aprotic solvent. In some embodiments, the Disclosure provides a method wherein the contact is carried out under conditions that stabilize the conjugated composition when the nucleotide is complementary to the next base of the target nucleic acid, and destabilize the conjugated composition when the nucleotide is not complementary to the next base of the target nucleic acid. In some embodiments, the Disclosure provides a method wherein the polymer-nucleotide conjugate comprises a polymer having a plurality of branches, and the plurality of copies of the first nucleotide are attached to the branches, in particular, the first polymer having a star-shaped, comb-shaped, cross-linked, bottlebrush-shaped, or dendrimer-shaped configuration. In some embodiments, the Disclosure provides a method wherein the polymer-nucleotide conjugate comprises one or more conjugate groups selected from the group consisting of avidin, biotin, affinity tags, and combinations thereof.In some embodiments, the Disclosure provides the method further comprising a dissociation step for destabilizing the binding complex formed between the composition of (a) and the polymer-nucleotide conjugate and removing the polymer-nucleotide conjugate. In some embodiments, the Disclosure further comprises an extension step of incorporating a nucleotide complementary to the following base of the target nucleic acid into the primer nucleic acid, and optionally, the extension step is performed concurrently with or after the dissociation step.

[0009] In some embodiments, the Disclosure provides a composition comprising a branched polymer having two or more branches and two or more copies of nucleotides, wherein the nucleotides are attached to a first plurality of the branches or arms, and optionally, one or more interaction portions are attached to a second plurality of the branches or arms. In some embodiments, the composition may include one or more labels on the polymer. In some embodiments, the Disclosure provides the composition wherein the nucleosides have a surface density of at least four nucleotides per polymer. In some embodiments, the Disclosure provides the composition comprising, or incorporating, nucleotides or nucleotide analogs modified to prevent incorporation into a nucleic acid chain elongating during a polymerase reaction. In some embodiments, the composition may comprise, or incorporate, nucleotides or nucleotide analogs that are reversibly modified to prevent incorporation into a nucleic acid chain elongating during a polymerase reaction. In some embodiments, the Disclosure provides the composition wherein one or more labels comprise a fluorescent label, a FRET donor, and / or a FRET acceptor. In some embodiments, the composition may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more branches or arms, or 2, 4, 8, 16, 32, or 64 or more branches or arms. In some embodiments, the branches or arms may radiate outward from a central portion. In some embodiments, the composition may include one or more interaction portions, which may include avidin or streptavidin, a biotin portion, an affinity tag, an enzyme, an antibody, a minibody, a receptor, or other proteins, a non-protein tag, a metal affinity tag, or any combination thereof. In some embodiments, the disclosure provides the composition in which the polymer comprises polyethylene glycol, polypropylene glycol, polyvinyl acetate, polylactic acid, or polyglycolic acid.In some embodiments, the Disclosure provides compositions in which a nucleotide or nucleotide analog is attached to a branch or arm by a linker, and in particular, the linker comprises PEG, the PEG linker portion having an average molecular weight of about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 50 kDa, about 100 kDa, about 150 kDa, or about 200 kDa or more. In some embodiments, the Disclosure provides compositions in which the linker comprises PEG, the PEG linker portion having an average molecular weight of about 5 kDa to about 20 kDa. In some embodiments, the Disclosure provides compositions in which at least one nucleotide or nucleotide analog comprises a deoxyribonucleotide, a ribonucleotide, a deoxyribonucleoside, or a ribonucleoside, and / or the nucleotide or nucleotide analog is conjugated to a linker via the 5' end of the nucleotide or nucleotide analog. In some embodiments, the Disclosure provides compositions in which one of the nucleotides or nucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyluridine, and / or cytidine, and the linker length is between 1 nm and 1,000 nm. In some embodiments, the Disclosure provides compositions in which at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit elongation during a polymerase reaction or sequencing reaction, such as a nucleotide lacking a 3' hydroxyl group, a nucleotide modified to include a blocking group at the 3' position, and / or a nucleotide modified with a 3'-O-azide group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3-O-benzyl group.In some embodiments, the present disclosure provides the composition, wherein at least one nucleotide or nucleotide analog is a nucleotide that is not modified at the 3' position.

[0010] In some embodiments, the Disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising the steps of: 1) providing a nucleic acid molecule comprising a template chain and a complementary chain at least partially complementary to the template chain; 2) contacting the nucleic acid molecule with one or more nucleic acid binding compositions according to any embodiment disclosed herein; 3) detecting the binding of the nucleic acid binding composition to the nucleic acid molecule; and 4) determining the identity of the terminal nucleotide incorporated into the complementary chain of the nucleic acid molecule, regardless of any particular order. In some embodiments, the Disclosure provides a method comprising the steps of incorporating the terminal nucleotide into the complementary strand, and repeating the contact, detection, and integration steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the Disclosure provides a method wherein the nucleic acid molecule is anchored to a solid support, in particular the solid support 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 Disclosure further comprises embodiments in which the at least one hydrophilic polymer coating layer comprises PEG, and / or the at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least eight branches. In some embodiments, the Disclosure provides a method in which the plurality of oligonucleotide molecules are at least 500 molecules / mm³ 2 at least 1,000 molecules / mm³ 2 at least 5,000 molecules / mm³ 2at least 10,000 molecules / mm³ 2 at least 20,000 molecules / mm³ 2 at least 50,000 molecules / mm³ 2 at least 100,000 molecules / mm³ 2 , or at least 500,000 molecules / mm³ 2The present invention provides the method wherein the nucleic acid molecules are present at a surface density. In some embodiments, the present invention provides the method wherein the nucleic acid molecules are clonally amplified on a solid support. In some embodiments, the present invention provides the method wherein the clonal amplification includes the use of polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), intercircle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the present invention provides the method wherein one or more nucleic acid binding compositions are labeled with a fluorophore, and the detection step includes the use of fluorescence imaging, in particular the fluorescence imaging includes two-wavelength excitation / four-wavelength emission fluorescence imaging. In some embodiments, the Disclosure provides a method in which four different nucleic acid binding compositions, each containing a different nucleotide or nucleotide analog, are used to determine the identity of terminal nucleotides, the four different nucleic acid binding compositions are labeled with distinct fluorophores, and the detection step includes simultaneous excitation at a wavelength sufficient to excite all four fluorophores and fluorescence emission imaging at a wavelength sufficient to detect each fluorophore. In some embodiments, the Disclosure provides a method in which four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, are used to determine the identity of terminal nucleotides, 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), respectively, and the detection step comprises simultaneous excitation at any two of 532 nm, 568 nm, and 633 nm, and fluorescence emission imaging at approximately 570 nm, 592 nm, 670 nm, and 702 nm, respectively, and / or fluorescence imaging comprises dual-wavelength excitation / dual-wavelength emission fluorescence imaging.In some embodiments, the Disclosure provides a method wherein four different nucleic acid binding compositions, each comprising a different nucleotide or nucleotide analog, are used to determine the identity of a terminal nucleotide, and one, two, three, or four different nucleic acid binding compositions are each labeled with a distinct fluorophore or set of fluorophores, and the detection step comprises simultaneous excitation at a wavelength sufficient to excite one, two, three, or four fluorophores or sets of fluorophores, and fluorescence emission imaging at a wavelength sufficient to detect each fluorophore. In some embodiments, the Disclosure provides a method in which three different nucleic acid binding or integration compositions, each containing a different nucleotide or nucleotide analog, are used to determine the identity of a terminal nucleotide, and one, two, or three different nucleotide acid binding or integration compositions are each labeled with a separate fluorophore or set of fluorophore compositions, and the detection step comprises simultaneous excitation at a wavelength sufficient to excite one, two, or three fluorophore or set of fluorophore, and fluorescence emission imaging at a wavelength sufficient to detect each fluorophore, and the detection of a fourth nucleotide is determined or determinable by reference to a “dark” or unlabeled spot or the location of a target nucleotide. In some embodiments, the Disclosure provides a method in which the polyvalent binding or integration composition may contain three polymer-nucleotide conjugates, and each of the three polymer-nucleotide conjugates contains a different species of nucleotide. In some embodiments, the Disclosure provides a method in which the step of detecting the binding or integration complex is carried out in the absence of unbound or solution-derived polymer-nucleotide conjugates.

[0011] In some embodiments, the present disclosure provides the above method, wherein four different nucleic acid binding compositions each comprising a different nucleotide or nucleotide analog, or three different nucleic acid binding compositions or incorporation compositions, are used to determine the identity of a terminal nucleotide, wherein one of the four or three different nucleotide binding compositions or incorporation compositions is labeled with a first fluorophore, one is labeled with a second fluorophore, one is labeled with both the first and second fluorophores, and one is unlabeled or absent, and the detection step comprises simultaneous excitation at a first excitation wavelength and a second excitation wavelength, and images are acquired at a first fluorescence emission wavelength and a second fluorescence emission wavelength. In some embodiments, the present disclosure provides the above method, 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, the first fluorescence emission wavelength is about 570 nm, and the second fluorescence emission wavelength is about 670 nm. In some embodiments, the present disclosure provides the above method, wherein a detection label may comprise one or more parts of a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications can be performed under a single excitation and imaging step. In some embodiments, the present disclosure provides the above method, wherein a sequencing reaction cycle comprising the steps of contacting, detecting, and incorporating / extending is performed in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the present disclosure provides the above method, wherein the average Q-score of base calling accuracy over a sequencing run is 30 or higher, and / or 40 or higher. In some embodiments, the present disclosure provides the above method, wherein 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 of greater than 30, and / or 40 or higher. In some embodiments, the present disclosure provides the above method, wherein at least 95% of the identified terminal nucleotides have a Q-score of greater than 30.

[0012] In some embodiments, the Disclosure provides a reagent comprising one or more nucleic acid-binding compositions and buffers disclosed herein. For example, in some embodiments, the Disclosure provides a reagent comprising one, two, three, four or more nucleic acid-binding compositions or embedded compositions, each of which comprises a single type of nucleotide. In some embodiments, the reagents of the present disclosure comprise one, two, three, four, or more nucleic acid-binding compositions or incorporated compositions, each of which comprises a single nucleotide or nucleotide analog, the nucleotide or nucleotide analog being one or more from the group consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), and deoxyadenosine monophosphate (dAMP); thymidine triphosphate (TTP), thymidine diphosphate (TDP), thymidine monophosphate (TMP), deoxythymidine triphosphate (dTTP), deoxythymidine diphosphate (dTDP), deoxythymidine monophosphate (dTMP), and uridine triphosphate Each of these may correspond to one or more of the group consisting of (UTP), uridine diphosphate (UDP), uridine monophosphate (UMP), deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), and deoxyuridine monophosphate (dUMP); one or more of the group consisting of cytidine triphosphate (CTP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), deoxycytidine triphosphate (dCTP), deoxycytidine diphosphate (dCDP), and deoxycytidine monophosphate (dCMP); and one or more of the group consisting of guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), deoxyguanosine triphosphate (dGTP), deoxyguanosine diphosphate (dGDP), and deoxyguanosine monophosphate (dGMP).In some other or further examples, the Disclosure provides reagents comprising, or further comprising, one, two, three, four or more nucleic acid-binding compositions or embedded compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide, nucleotide analog may correspond to one or more from the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

[0013] Disclosed herein are kits comprising a nucleic acid-binding composition or incorporated composition of any of the embodiments disclosed herein, and / or a reagent of any of the embodiments disclosed herein, and / or one or more buffers, and instructions for their use.

[0014] Disclosed herein are systems for carrying out the methods of any embodiment disclosed herein, the systems comprising nucleic acid binding compositions or integration compositions of any embodiment disclosed herein, and / or reagents of any embodiment disclosed herein. In some embodiments, the systems are configured for repeated sequential contact between tethered, primed nucleic acid molecules and the nucleic acid binding compositions or integration compositions and / or reagents, and for detection of binding or integration of the disclosed nucleic acid binding compositions or integration compositions to one or more primed nucleic acid molecules.

[0015] In some embodiments, the present disclosure provides a composition comprising a particle (e.g., a nanoparticle or a polymer core), wherein the particle comprises a substrate for binding or incorporation of a plurality of enzymes or proteins, wherein the substrate for binding or incorporation of enzymes or proteins binds to one or more enzymes or proteins to form one or more binding complexes or incorporation complexes (e.g., a multivalent binding complex or an incorporation complex), and said binding or incorporation can be observed or identified by observing the position, presence, or persistence of one or more binding complexes or incorporation complexes. In some embodiments, the particle may comprise a polymer, a branched polymer, a dendrimer, a liposome, a micelle, a nanoparticle, or a quantum dot. In some embodiments, said substrate may comprise a nucleotide, a nucleoside, a nucleotide analog, or a nucleoside analog. In some embodiments, the enzyme or protein binding or incorporation substrate may comprise an agent capable of binding to a polymerase. In some embodiments, the enzyme or protein may comprise a polymerase. In some embodiments, observing the position, presence, or persistence of one or more binding complexes or incorporation complexes may comprise fluorescence detection. In some embodiments, the present disclosure provides a composition comprising a plurality of different particles disclosed herein, wherein each particle comprises a single type of nucleoside or nucleoside analog, and each nucleoside or nucleoside analog is associated with a fluorescent label having detectably different emission wavelengths or excitation wavelengths. In some embodiments, the present disclosure provides a composition further comprising one or more labels, e.g., a fluorescent label, on the particle. In some embodiments, the present disclosure provides the composition, wherein the composition comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 tethered nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs tethered to the particle. In some embodiments, the present disclosure provides that the density of nucleosides or nucleoside analogs is 0.001 to 1,000,000 / μm 2 , 0.01 to 1,000,000 / μm 2 , 0.1 to 1,000,000 / μm2 、1~1,000,000 / μm 2 、10~1,000,000 / μm 2 、100~1,000,000 / μm 2 、1,000~1,000,000 / μm 2 、1,000~100,000 / μm 2 、10,000~100,000 / μm 2 、or 50,000~100,000 / μm 2The present invention provides the composition present in a surface density within a range defined by any two of the aforementioned values. In some embodiments, the present invention provides the composition wherein a nucleoside or nucleoside analog is present within a nucleotide or nucleotide analog. In some embodiments, the present invention provides the composition comprising, or incorporating, a nucleotide or nucleotide analog modified to prevent incorporation into a nucleic acid chain elongating during a polymerase reaction. In some embodiments, the present invention provides the composition comprising, or incorporating, a nucleotide or nucleotide analog reversibly modified to prevent incorporation into a nucleic acid chain elongating during a polymerase reaction. In some embodiments, the present invention provides the composition wherein one or more labels comprise a fluorescent label, a FRET donor, and / or a FRET acceptor. In some embodiments, the present invention provides the composition wherein a substrate (e.g., a nucleotide, a nucleotide analog, a nucleoside, or a nucleoside analog) is attached to particles by a linker. In some embodiments, the present disclosure provides compositions in which at least one nucleotide or nucleotide analog is a nucleotide modified to inhibit elongation during a polymerase reaction or sequencing reaction, such as a nucleotide lacking a 3' hydroxyl group, a nucleotide modified to include a blocking group at the 3' position, a nucleotide modified with a 3'-O-azide group, a 3'-O-azidomethyl group, a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group, and / or a nucleotide not modified at the 3' position.

[0016] In some embodiments, the Disclosure provides a method for determining the sequence of a nucleic acid molecule, comprising, in any order: 1) providing a nucleic acid molecule comprising a template strand and a complementary strand at least partially complementary to the template strand; 2) contacting the nucleic acid molecule with one or more nucleic acid binding compositions or integration compositions according to any embodiment disclosed herein; 3) detecting the binding or integration of the nucleic acid binding composition or integration composition to the nucleic acid molecule; and 4) determining the identity of a terminal nucleotide incorporated into the complementary strand of the nucleic acid molecule. In some embodiments, the method may further include the steps of incorporating the terminal nucleotide into the complementary strand, and repeating the contact, detection, and integration steps for one or more additional iterations, thereby determining the sequence of the template strand of the nucleic acid molecule. In some embodiments, the Disclosure provides the method wherein the nucleic acid molecule is clonely amplified on a solid support. In some embodiments, the Disclosure provides a method for clonal amplification that includes the use of polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand binding (SSB) protein-dependent amplification, or any combination thereof. In some embodiments, the Disclosure provides a method in which a sequencing reaction cycle, including contact, detection, and integration steps, is carried out in less than 30 minutes, less than 20 minutes, or less than 10 minutes. In some embodiments, the Disclosure provides a method in which the mean Q score of base call accuracy over sequencing runs is 30 or greater, or 40 or greater. In some embodiments, the Disclosure provides a method in which 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, or greater than 40. In some embodiments, the present disclosure provides the method wherein at least 95% of the identified terminal nucleotides have a Q score greater than 30.

[0017] In some embodiments, the Disclosure provides a reagent comprising one or more nucleic acid-binding compositions or embedded compositions and buffers disclosed herein. In some embodiments, the Disclosure provides a reagent comprising one, two, three, four or more nucleic acid-binding compositions or embedded compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog comprises a nucleotide, a nucleotide analog, a nucleoside or a nucleoside analog. In some embodiments, the Disclosure provides a method comprising one, two, three, four or more nucleic acid-binding compositions or embedded compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog may correspond to one or more from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP; 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 one or more from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, the Disclosure provides a method comprising one, two, three, four or more nucleic acid-binding compositions or embedded compositions, each of which comprises a single type of nucleotide or nucleotide analog, and the nucleotide or nucleotide analog may correspond to one or more from the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, a, and dGMP.

[0018] In some embodiments, the present disclosure provides a kit comprising any of the compositions disclosed herein and / or any of the reagents disclosed herein, one or more buffers, and instructions for their use.

[0019] In some embodiments, the Disclosure provides a system for carrying out any of the methods disclosed herein, wherein the method may include the use of any of the compositions disclosed herein and / or any of the reagents disclosed herein, one or more buffers, and optionally one or more nucleic acid molecules tethered or attached to a solid support, and the system is configured to be carried out repeatedly for sequential contact between the nucleic acid molecules and the compositions and / or the reagents, and for detection of binding or incorporation of the nucleic acid binding composition or incorporation composition to one or more nucleic acid molecules.

[0020] In some embodiments, the disclosure provides compositions, such as those disclosed herein, for use in increasing the contrast-to-noise ratio (CNR) of surface-bound or associated labeled nucleic acid complexes.

[0021] In some embodiments, the Disclosure provides compositions disclosed herein for use in establishing or maintaining control over the duration of signals from surface-bound or associated labeled nucleic acid complexes.

[0022] In some embodiments, the Disclosure provides compositions disclosed herein for use in establishing or maintaining control over the duration of fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signals from surface-bound or associated labeled nucleic acid complexes.

[0023] In some embodiments, the present disclosure provides compositions disclosed herein for use in increasing the specificity, accuracy, or read length of nucleic acid sequencing and / or genotyping applications.

[0024] In some embodiments, the present disclosure provides compositions disclosed herein for use in increasing specificity, accuracy, or read length in linkage or integration sequencing, synthesis sequencing, single-molecule sequencing, or ensemble sequencing methods.

[0025] In some embodiments, the present disclosure provides reagents disclosed herein for use in increasing the contrast-to-noise ratio (CNR) of a surface-bound or associated labeled nucleic acid complex.

[0026] In some embodiments, the disclosure provides reagents disclosed herein for use in establishing or maintaining control over the duration of signals from surface-bound or associated labeled nucleic acid complexes.

[0027] In some embodiments, the Disclosure provides reagents disclosed herein for use in establishing or maintaining control over the duration of fluorescent, luminescent, electrical, electrochemical, colorimetric, radioactive, magnetic, or electromagnetic signals from surface-bound or associated labeled nucleic acid complexes.

[0028] In some embodiments, the present disclosure provides reagents disclosed herein for use in increasing the specificity, accuracy, or read length of nucleic acid sequencing and / or genotyping applications.

[0029] In some embodiments, the present disclosure provides reagents disclosed herein for use in increasing specificity, accuracy, or read length in conjugate or embedding sequencing, synthesis sequencing, single-molecule sequencing, or ensemble sequencing methods.

[0030] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent that each publication, patent, or patent application is intended to be incorporated by specific and individual reference. In the event of any conflict between the terminology used herein and the terminology used in any cited document, the terminology used herein shall prevail. [Brief explanation of the drawing]

[0031] The patent or application file must include at least one drawing created in color. A copy of the publication of this patent or patent application containing the color drawing will be provided by the relevant office after the required fees have been requested and paid.

[0032] Novel features of the inventive concept disclosed herein are made concrete in the appended claims. A better understanding of the features and advantages of the compositions, methods, and systems of the present invention will be obtained by referring to the following detailed description, which specifies exemplary embodiments in which the principles of the embodiments of the inventive concept are utilized, and to the following appended drawings. [Figure 1A] The process involves utilizing non-limiting examples of polyvalent binding compositions for sequencing target nucleic acids. Figure 1A shows non-limiting example 4 for attaching target nucleic acids to a surface. [Figure 1B] The process involves utilizing a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1B shows the target nucleic acid clonally to form a cluster of amplified target nucleic acid molecules. [Figure 1C] The process involves utilizing a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1C shows a non-limiting example of priming a target nucleic acid to produce a primed target nucleic acid. [Figure 1D] The process involves utilizing a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1D shows a non-limiting example of contacting a primed target nucleic acid with a polyvalent binding composition and polymerase to form a binding complex. [Figure 1E]The process demonstrates the use of a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1E shows a non-limiting example of an image of a binding complex captured on a surface. [Figure 1F] This describes a process utilizing a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1F shows a non-limiting example of extending a primer chain by one nucleotide. [Figure 1G] The process involves utilizing a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1G shows a non-limiting example of another cycle in which the primed target nucleic acid is contacted with the polyvalent binding composition and polymerase to form a binding complex. [Figure 1H] The process demonstrates the use of a non-limiting example of a polyvalent binding composition for sequencing a target nucleic acid. Figure 1H shows a non-limiting example of an image of the binding complex trapped on the surface during a subsequent sequencing cycle. [Figure 2] Figure 2 shows a flowchart outlining the steps for sequencing the target nucleic acid and extending the primer chain by single-nucleotide addition. [Figure 3] Figure 3 shows a flowchart outlining the steps for sequencing the target nucleic acid and extending the primer chain by incorporating the nucleotides onto the particle-nucleotide conjugate. [Figure 4A] A non-limiting example of detecting target nucleic acids using polymer-nucleotide conjugates is shown. Figure 4A shows the process of contacting polymerase and polymer-nucleotide conjugates with several nucleic acid molecules. [Figure 4B] This shows a non-limiting example of detecting target nucleic acids using polymer-nucleotide conjugates. Figure 4B shows the binding complex formed between polymerase, polymer-nucleotide conjugate, and target nucleic acid molecule. [Figure 5A] A schematic diagram shows non-limiting examples of various configurations of polymer-nucleotide conjugates. Figure 5A shows polymer-nucleotide conjugates with various multi-arm configurations. [Figure 5B]Schematic diagrams of non-limiting examples of various polymer-nucleotide conjugate configurations are shown. Figure 5B shows a polymer-nucleotide conjugate with polymer branches radiating from the center. [Figure 5C] Schematic diagrams of non-limiting examples of various polymer-nucleotide conjugate configurations are shown. Figure 5C shows a polymer-nucleotide conjugate with a biotin binding moiety. [Figure 6] Figure 6 shows a generalized graphical representation of the increase in signal intensity observed during the cleaning and removal of bonded, sustained, and multivalent substrates. [Figure 7] Figures 7A–7J show fluorescence images of the sequencing reaction steps using a polyvalent PEG substrate composition. Figure 7A is a red and green fluorescence image of a DNA RCA template (G and A first base) after exposure to 500 nM base-labeled nucleotides (A-Cy3 and G-Cy5) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2. The images were collected after washing with an imaging buffer having the same composition as the exposure buffer but without nucleotides or polymerase. Contrast was scaled to maximize visualization of the faintest signals, but no signals persisted after washing with the imaging buffer (Figure 7A, inset). Figures 7B–E are fluorescence images showing polyvalent PEG-nucleotide (base-labeled) ligands PB1 (Figure 7B), PB2 (Figure 7C), PB3 (Figure 7D), and PB5 (Figure 7E) with an effective nucleotide concentration of 500 nM, after mixing with exposure buffer and imaging with imaging buffer as described above. Figure 7F is a fluorescence image showing 2.5 uM of polyvalent PEG-nucleotide (base-labeled) ligand PB5, after mixing with exposure buffer and imaging with imaging buffer as described above. Figure 7GI is a fluorescence image showing further base discrimination by exposing the polyvalent conjugated composition to inactive mutants of Klenow polymerase (Figure 7G), D882H (Figure 7H), and D882E (Figure 7I). Figure 7J is a fluorescence image showing further base discrimination by exposing the polyvalent conjugated composition to inactive mutants of wild-type Klenow (control) enzyme. [Figure 8A]The effectiveness of a multivalent reporter composition in determining the base sequence of a DNA sequence across five sequencing cycles is demonstrated. Figure 8A shows images of the template and the expected sequence obtained after each sequencing cycle. [Figure 8B] The effectiveness of the multivalent reporter composition in determining the base sequence of a DNA sequence across five sequencing cycles is demonstrated. Figure 8B shows the aligned sequencing results using the images taken in Figure 8A. [Figure 9] AJ in Figure 9 shows fluorescence images of polyvalent polyethylene glycol (PEG) polymer-nucleotide (base-labeled) conjugates with effective nucleotide concentrations of 500 nM and various PEG branch lengths, after contact with a support surface containing a DNA template (containing G or A at the first base and prepared using rolling circle amplification (RCA)) in an exposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr+2. The images were acquired after washing with an imaging buffer having the same composition as the exposure buffer but lacking nucleotides and polymerase. The panel shows images obtained using polyvalent PEG-nucleotide ligands with arm lengths as follows: A in Figure 9: 1-K PEG. B in Figure 9: 2-K PEG. C in Figure 9: 3-K PEG. D in Figure 9: 5-K PEG. E in Figure 9: 10-K PEG. F in Figure 9: 20-K PEG. Figure 9G shows an image obtained using 10K PEG and inactive Klenow polymerase containing mutant D882H. Figure 7H shows an image obtained using 10K PEG and inactive Klenow polymerase containing mutant D882E. Figure 7I shows an image obtained using 10K PEG and inactive Klenow polymerase containing mutant D882A. Figure 7J shows an image obtained using 10K PEG and active wild-type Klenow polymerase. [Figure 10] Figure 10 shows a quantitative representation of fluorescence intensity separated by color values ​​in the image shown in AF of Figure 9, where orange traces correspond to red labels (Cy3 labels; A base) and blue traces correspond to green labels (Cy5 labels; G base). [Figure 11]Figure 11 shows the normalized fluorescence from a multivalent substrate bound to a DNA cluster, as described in AJ of Figure 7, and the substrate complex is formed in the presence (condition B) and absence (condition A) of Triton-X100 (0.016%). [Figure 12A] Plots of normalized fluorescence intensity measured for polyvalent polymer-nucleotide conjugates and free nucleotides are shown. Figure 12A shows two replicas of polyvalent polymer-nucleotide conjugates bound to the conjugated composition versus DNA clusters (conditions A and B), formed using labeled free nucleotides (condition C) after 1 minute. [Figure 12B] Figure 12B shows the normalized fluorescence intensity plots measured for the polyvalent polymer-nucleotide conjugate and free nucleotides. Figure 12B also shows the time course of fluorescence of the polyvalent substrate complex over 60 minutes. [Modes for carrying out the invention]

[0033] I. Definition As used herein, “nucleic acids” (also known as “polynucleotides,” “oligonucleotides,” “ribonucleic acid (RNA),” or “deoxyribonucleic acid (DNA)”) are linear polymers of two or more nucleotides, or their variants or functional fragments, linked by covalently bonded nucleoside bonds. In naturally occurring examples of nucleic acids, the nucleoside bond is a phosphate diester bond. However, other examples may optionally include other nucleoside bonds, such as phosphorothiolate bonds, and may or may not contain phosphate groups. Nucleic acids include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA, DNA / RNA hybrids, peptide-nucleic acid (PNA), and hybrids of PNA with DNA or RNA, and may also include other types of nucleic acid modifications.

[0034] As used herein, “nucleotide” refers to a nucleotide, a nucleoside, or an analog thereof. Nucleotides include both naturally occurring and chemically modified nucleotides and may include, but are not limited to, nucleosides, ribonucleotides, deoxyribonucleotides, protein nucleic acid residues, or derivatives. Examples of nucleotides include adenine, thymine, uracil, cytosine, guanine, or their residues; deoxyadenine, deoxythymine, deoxyuracil, deoxycytosine, deoxyguanine, or their residues; adenine PNA, thymine PNA, uracil PNA, cytosine PNA, guanine PNA, or their residues or equivalents; N-glycosides or C-glycosides of purines or pyrimidine bases (e.g., deoxyribonucleosides containing 2-deoxy-D-ribose, or ribonucleosides containing D-ribose).

[0035] As used herein, "complementary" refers to the topological compatibility or matching of the interacting surfaces of a ligand molecule and its receptor. Therefore, it may be stated that the receptor and its ligand are complementary, and furthermore, that their contact surface properties are complementary to each other.

[0036] As used herein, “branched polymer” refers to a polymer having multiple functional groups that facilitate the conjugation of biologically active molecules such as nucleotides, the functional groups may be attached to the side chains of the polymer or directly to the central core or central main chain of the polymer. A branched polymer may have a linear main chain having one or more functional groups that detach from the main chain for conjugation. A branched polymer may also be a polymer having one or more side chains, where the side chains have sites suitable for conjugation. Examples of functional groups include, but are not limited to, hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide, thiol, alkanic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and toresylate.

[0037] As used herein, “polymerase” refers to an enzyme containing a nucleotide-binding moiety that 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 integration, strand displacement activity, and nucleotide binding or integration and recognition. A polymerase may include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing nucleotide-binding or integration moieties.

[0038] As used herein, “duration” refers to the length of time that a binding complex formed between a target nucleic acid, polymerase, and conjugate or unconjugated nucleotide remains stable without the binding components dissociating from the complex. 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, for example, by observing the signal from the labeled component of the binding complex. For example, a labeled nucleotide or a labeling reagent containing one or more nucleotides may be present in the binding complex, and thus it becomes possible to detect a signal from the label during the duration of the binding complex. One non-limiting example of labeling is fluorescent labeling.

[0039] II. Methods for analyzing target nucleic acids Disclosed herein are polyvalent conjugated or embedded compositions and their use in the analysis of nucleic acid molecules involved in sequencing or other bioassay applications. Increased binding or embedding of nucleotides to enzymes (e.g., polymerases) or enzyme complexes may be influenced by increasing the effective concentration of nucleotides. This increase may be achieved by increasing the concentration of nucleotides in a free solution or by increasing the amount of nucleotides adjacent to the relevant binding or embedding site. The increase may further be achieved by physically restricting the number of nucleotides to a limited volume, which results in a localized increase in concentration, and thus such a structure can bind or embed at the binding or embedding site with a higher apparent binding force than may be observed for unconjugated, untethered, or otherwise unrestricted individual nucleotides. Non-limiting means for making such restrictions are provided by providing polyvalent conjugated or embedded compositions in which multiple nucleotides are conjugated to particles such as polymers, branched polymers, dendrimers, micelles, liposomes, microparticles, nanoparticles, quantum dots, or other suitable particles known in the art.

[0040] The polyvalent conjugate or embedded composition disclosed herein may comprise at least one particle-nucleotide conjugate, the particle-nucleotide conjugate having 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 embedded complex with the polymerase and the target nucleic acid, the binding or embedded complex exhibiting higher stability and longer duration than a binding or embedded complex formed using a single unconjugated or detethered nucleotide. Each nucleotide parenchyma of the polyvalent conjugate composition may bind to the complementary N+1 nucleotide of a primed target nucleic acid molecule, thereby forming a polyvalent conjugate complex comprising two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a polyvalent conjugate composition (e.g., a polymer-nucleotide conjugate). Each nucleotide moiety of a polyvalent binding composition can bind to the complementary N nucleotide of a primed target nucleic acid molecule, thereby forming a polyvalent binding complex containing two or more target nucleic acid molecules, two or more polymerase (or other enzyme) molecules, and a polyvalent binding composition (e.g., polymer-nucleotide conjugate). From this bound complex, the nucleotide can examine the complementary base before incorporating a modified, reversibly blocked nucleotide that extends the replicated strand by one base. Furthermore, one can imagine examining the N nucleotide by the bound complex by advancing with the reversibly terminated nucleotide and probing the subsequent N+1 bases before and after unblocking. Thus, by reading what has been examined twice, error checking can be performed and the overall accuracy of the base call can be improved. A key distinguishing factor from conventional methods is that the binding is used to examine matched bases, whereas the stepping or incorporation step is used only to advance along the extension strand.

[0041] Polyvalent binding compositions or embedding compositions can be used to localize detectable signals to active regions of biochemical interactions, such as sites of protein-nucleic acid interactions, nucleic acid hybridization reactions, or enzymatic reactions such as polymerase reactions. For example, the polyvalent binding compositions or embedding compositions described herein can be used to identify sites of base embedding in nucleic acid chains elongated during polymerase reactions and to provide base identification for sequencing and array-based applications. When nucleotides are complementary to the target nucleic acid, increased binding or embedding between the target nucleic acid and the nucleotides in the polyvalent binding composition or embedding composition provides an enhanced signal that significantly improves the accuracy of base calling and reduces imaging time.

[0042] Furthermore, the use of a polyvalent binding composition allows the sequencing signal from a given sequence to occur within a cluster region containing multiple copies of the target sequence. A sequencing method incorporating multiple copies of the target sequence has the advantage that the signal can be amplified because multiple simultaneous sequencing reactions exist within a defined region, each providing its own signal. The presence of multiple signals within a defined region also mitigates the impact of a single skipped cycle, due to the fact that signals from numerous correct base calls overwhelm signals from a few skipped or incorrect base calls, thus providing a method to reduce phase errors and / or improve the read length of the sequencing reaction.

[0043] The polyvalent conjugation compositions disclosed herein and their use result in one or more of the following: (i) stronger signals for better base calling accuracy compared to conventional nucleic acid amplification and sequencing methods; (ii) better differentiation of sequence-specific signals from background signals; (iii) reduced requirements for the amount of starting material needed; (iv) increased sequencing speed and reduced sequencing time; (v) reduced phase error; and (vi) improved read length during sequencing reactions.

[0044] In some embodiments, the target nucleic acid may refer to a target nucleic acid sample having one or more nucleic acid molecules. In some embodiments, the target nucleic acid may comprise multiple nucleic acid molecules. In some embodiments, the target nucleic acid may comprise two or more nucleic acid molecules. In some embodiments, the target nucleic acid may comprise two or more nucleic acid molecules having the same sequence.

[0045] A. Sequencing of target nucleic acids Figures 1A-1H illustrate one exemplary method in which a polyvalent binding composition is used to sequence a target nucleic acid. As shown in Figure 1A, the target nucleic acid (102) can be anchored to a solid support surface (101). The target nucleic acid can adhere to the surface directly or indirectly. Not shown in Figure 1A, the target nucleic acid (102) can hybridize to adapters attached to the surface by covalent or non-covalent bonds. When one or more adapters are used to attach the target nucleic acid to the surface, the target surface may contain fragments that are complementary to the adapters and therefore hybridize to the adapters. In some examples, one adapter sequence may be anchored to the surface. In some examples, multiple adapter sequences may be anchored to the surface. In some examples, the target nucleic acid (102) can adhere directly to the solid support surface without the use of adapters. The solid support can be a low nonspecific binding surface.

[0046] In Figure 1B, after an initial step of attaching the target nucleic acid to the surface of a solid support (e.g., via hybridization to an adapter), the target nucleic acid is clonally amplified to form a cluster of amplified nucleic acids. If the target nucleic acid is attached to the surface via an adapter, the surface density of the clonally amplified nucleic acid sequence hybridized to the adapter on the support surface may range to the same extent as the surface density of the tethered adapter (or primer). Clonal amplification may be performed using polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, single-strand joining (SSB), protein-dependent amplification, or any combination thereof.

[0047] Figure 1C shows a non-limiting step of annealing a primer (103) to a target nucleic acid (102) to form a primed target nucleic acid (104). Figure 1B shows one primer used in the annealing step alone, but one or more primers can be used depending on the species of target nucleic acid. In some examples, the adapter used to attach the target nucleic acid to the surface has the same sequence as the primer used to prepare the primed target nucleic acid. The primers may include forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcoding sequences, or any combination thereof. In some examples, one primer sequence may be used in the hybridization step. In some examples, multiple different primer sequences may be used in the hybridization step.

[0048] As shown in Figure 1D, the primed target nucleic acid (104) is combined with a polyvalent binding or integration composition and polymerase (106) to form a binding or integration complex. A non-limiting example of the polyvalent binding or integration composition in Figure 1D includes four particle-nucleotide conjugates (105a), (105b), (105c), and (105d). Each particle-nucleotide conjugate has multiple copies of a nucleotide attached to the particle, and the four particle-nucleotide conjugates each cover four types of nucleotides. A particle-nucleotide conjugate having a nucleotide complementary to the following base on the primed target nucleic acid forms a binding or integration complex with the polymerase and the target nucleic acid. In some examples, the polyvalent binding or integration composition may include one, two, or three particle-nucleotide conjugates. In some embodiments, each different type of particle-nucleotide conjugate can be labeled with a separate label. In some embodiments, three of the four nucleotide conjugates may be labeled, and the fourth may be unlabeled or conjugated with an undetectable label. In some embodiments, one, two, three, or four particle-nucleotide conjugates may be labeled with the same label, or each may be labeled with a label corresponding to the identity of the conjugated nucleotide, or remain unlabeled, or be conjugated with an undetectable label, in 3, 2, 1, or 0 particle-nucleotide conjugates. In some embodiments, detection of polymerase complexes incorporating particle-nucleotide conjugates can be performed using four-color detection, where conjugates corresponding to all four nucleotides are present in the sample, and each conjugate has a distinct label corresponding to the nucleotide conjugated to it.In some embodiments, the four particle-nucleotide conjugates may be simultaneously exposed to or in contact with the target nucleic acid, and in some other embodiments, the four particle-nucleotide conjugates may be individually or in groups of two or three, successively exposed to or in contact with the target nucleic acid. In some embodiments, the step of detecting the polymerase complex incorporating the particle-nucleotide conjugates can be carried out using three-color detection, where conjugates corresponding to three of the four nucleotides are present in the sample, three conjugates have distinct labels corresponding to the nucleotides conjugated to them, and one conjugate is unlabeled or conjugated to an undetectable label. In some embodiments, only three types of conjugates are provided, where conjugates corresponding to three of the four nucleotides are present in the sample, three conjugates have distinct labels corresponding to the nucleotides conjugated to them, and one conjugate is absent. In some embodiments, the identity of the nucleotide corresponding to the unlabeled or absent nucleotide conjugate can be determined with respect to the location and / or identity of a known "dark" spot or position of the target nucleic acid that does not exhibit a fluorescent signal. In some embodiments, the present disclosure provides a method for detecting a bound or embedded complex in the absence of unbound or solution-derived polymer-nucleotide conjugates.

[0049] In some embodiments, where three of the four particle-nucleotide conjugates are labeled, or where only three of the four particle-nucleotide conjugates are present, the identity of the nucleotide corresponding to the unlabeled or absent conjugate can be established by monitoring the presence of the unlabeled complex, such as by the absence of a signal or by identifying a “dark” spot or unlabeled region in the sequencing reaction. In some embodiments, detection of the polymerase complex incorporating the particle-nucleotide conjugate can be performed using two-color detection, where conjugates corresponding to two of the four nucleotides are present in the sample, two conjugates have distinct labels corresponding to the nucleotides conjugated to them, and two conjugates are unlabeled or conjugated to an undetectable label. In some embodiments, only two of the four particle-nucleotide conjugates are labeled. In some embodiments, where two of the four particle-nucleotides are labeled, the identity of the nucleotide corresponding to the unlabeled conjugate can be established by monitoring the presence of the unlabeled complex, such as by the absence of a signal or by identifying a “dark” spot or unlabeled region in the sequencing reaction. In some embodiments, where two of the four particle-nucleotides are labeled, the four particle-nucleotide conjugates can be sequentially exposed to or in contact with the target nucleic acid, 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 can be sequentially exposed to or in contact with the target nucleic acid, individually or in groups of two or three, where each contact step indicates 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.

[0050] Figure 1E shows an image taken on the surface after a binding or integration complex has formed between a polymerase, a target nucleic acid, and a particle-nucleotide conjugate having a nucleotide complementary to the next base of the primed target nucleic acid. The image taken includes four binding or integration complexes (107a), (107b), (107c), and (107d) formed on the surface, each binding or integration complex having a different nucleotide that can be distinguished based on a label (e.g., fluorescence color) on the particle-nucleotide conjugate. Using particle-nucleotide conjugates significantly enhances sequencing signals because the binding or integration signal from a particular sequence can be generated within a cluster region containing multiple copies of the target sequence. Figure 1E includes four particle-nucleotide conjugates, each with a different species of nucleotide, but in some ways, one, two, or three particle-nucleotide conjugates, each with a different species of nucleotide and label, can be used. In some embodiments, each different species of particle-nucleotide conjugate may be labeled with the same label, or each with a label corresponding to the identity of its conjugated nucleotide. In some embodiments, three of the four nucleotide conjugates may be labeled, and the fourth may be conjugated to an unlabeled or undetectable label. In some embodiments, particle-nucleotide conjugates 1, 2, 3, or 4 may be labeled with individual labels, while particle-nucleotide conjugates 3, 2, 1, or 0 may be conjugated to an unlabeled or undetectable label, respectively. In some embodiments, the detection step may involve simultaneous and / or sequential excitation of up to four different excitation wavelengths, for example, fluorescence imaging may be performed by detecting single and / or multiple fluorescence emission bands that uniquely classify each of the possible base pairs (A, G, C, or T).In some embodiments, the identity of terminal nucleotides can be determined using four different nucleic acid binding compositions or embedded compositions, each containing a different nucleotide or nucleotide analog, one of the four different nucleic acid binding compositions or embedded compositions being labeled with a first fluorophore, one with a second fluorophore, one labeled with both the first and second fluorophore, and one unlabeled, and the detection step includes simultaneous excitation at a first excitation wavelength and a second excitation wavelength, and the image is acquired at a first fluorescence emission wavelength and a second fluorescence emission wavelength.

[0051] When a polyvalent binding or integration composition is used in place of a single unbound or untethered nucleotide to form a binding or integration complex with polymerase and a primed target nucleic acid, the local concentration of the nucleotide increases many times over, thereby enhancing the signal intensity. The formed binding or integration complex also has a longer duration, which in turn helps to shorten the imaging process. The high signal intensity is due to the high binding or integration affinity of the polymer-nucleotide conjugate (which may contain multiple fluorophores or other labels), thus forming a complex that remains stable throughout the binding or integration and imaging process. The strong binding or integration between polymerase, the primed target chain, and the polymer-nucleotide or nucleotide analog conjugate further means that the thus formed polyvalent binding or integration complex remains stable during the washing process, and the signal intensity remains high even if nucleotide analogs that did not match with the components of the other reaction mixture are washed away. After the imaging process, the binding or integration complex can be destabilized (e.g., by changing the buffer composition), allowing the primed target nucleic acid to be extended by one base.

[0052] The sequencing method may further include a step of incorporating an N+1 or terminal nucleotide into the primed strand, as shown in Figure 1F. In Figure 1F, the primer strand of the primed target nucleic acid (108) may be extended by one base to form the extended nucleic acid (109). The extension step may be performed after or concurrently with the destabilization of the polyvalent binding or integration complex. The primed target nucleic acid (108) can be extended using complementary nucleotides bound to particles in a particle-nucleotide conjugate, or using unconjugated or detethered free nucleotides provided after the removal of the polyvalent binding or integration composition.

[0053] Following the extension step, a contact step, as shown in Figure 1G, can be performed again to form a binding or integration complex and to mimic the next sequencing cycle. The contact, detection, and extension steps can be repeated in one or more cycles, thereby determining the sequence of the target nucleic acid molecule. For example, Figure 1H shows a surface image obtained after performing multiple sequencing cycles, and the image can then be processed to determine the sequence of the target nucleic acid molecule.

[0054] The elongation of a primed target nucleic acid may be prevented or inhibited by the use of blocked nucleotides on the chain or a catalytically inactive polymerase. If a nucleotide in a polymer-nucleotide conjugate has a blocking group that hinders nucleic acid elongation, the incorporation of the nucleotide may be achieved by removing the blocking group from the nucleotide (e.g., by detaching the nucleotide from its polymer, branched polymer, dendrimer, particle, etc.). If the elongation of a primed target nucleic acid is inhibited by the use of a catalytically inactive polymerase, the incorporation of the nucleotide may be achieved by providing a cofactor or activator, such as a metal ion.

[0055] Furthermore, disclosed herein are systems configured to carry out either the disclosed nucleic acid sequencing or nucleic acid analysis methods. These systems may include a fluid flow controller and / or fluid distribution system configured to continuously and repeatedly contact a primed target nucleic acid molecule attached to a solid support with the disclosed polymerase and polyvalent binding composition or incorporated composition and / or reagents. The contact process may be carried out within one or more flow cells. In some examples, the flow cells may be fixed components of the system. In some examples, the flow cells may be removable and / or disposable components of the system.

[0056] The sequencing system may include an imaging module, i.e., one or more light sources, one or more optical components, and one or more image sensors, for imaging and detecting the binding or integration of the disclosed nucleic acid binding or integration composition to a target nucleic acid molecule anchored inside a solid support or flow cell. The disclosed compositions, reagents, and methods may be used for any of a variety of nucleic acid sequencing and analytical applications. Examples include, but are not limited to, DNA sequencing, RNA sequencing, whole-genome sequencing, targeted sequencing, exome sequencing, and genotyping.

[0057] The sequencing system may include a computer-controlled system programmed to carry out the methods of the disclosure. The computer system is programmed or otherwise configured to carry out the methods of the disclosure, including nucleic acid sequencing, commentary on nucleic acid sequencing data and analysis of cellular nucleic acids such as RNA (e.g., mRNA), and characterization of cells from the sequencing data. The computer system may be a user's electronic device or a computer system located remotely from the electronic device. The electronic device may be a mobile electronic device.

[0058] Figure 2 is a flowchart outlining the steps in sequencing a target nucleic acid. (201) describes the step of attaching the 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. Prior to (201), the nucleic acid molecules of the target library may have been prepared to contain fragments complementary to the adapter sequence by ligation or other methods. (202) describes the step of clonal amplification to generate clusters of target nucleic acid molecules on the surface. (203) describes the step of hybridizing sequencing primers to a complementary primer-binding sequence or integration sequence on the target nucleic acid to form a primed target nucleic acid. (204) describes the step of combining the primed target nucleic acid with a polymerase, a polyvalent binding composition or integration composition containing a labeled (e.g., fluorescently labeled) particle-nucleotide conjugate. (204 may further include a step of washing or removing unbound reagents, including polymerase and particle-nucleotide conjugates.)

[0059] Referring again to Figure 2, if the nucleotide on 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 conjugate or integration complex which can be detected by a detection method compatible with labeling the particle-nucleotide conjugate (e.g., fluorescence imaging). (205) may further include a step of measuring the duration of the ternary conjugate or integration complex. In (206), the conjugate or integration complex is destabilized to remove the binding or integration of the particle-nucleotide conjugate and polymerase. Dissociation can be achieved by placing the conjugate or integration complex under conditions that alter the conformation of the polymerase and destabilize the binding or integration (e.g., addition of strontium ions). 206 may further include a step of washing or removing the dissociated particle-nucleotide conjugate and / or polymerase. (207) describes a 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) may be repeated for multiple cycles to determine the sequence of the target nucleic acid.

[0060] Figure 3 is another flowchart outlining the process of sequencing a target nucleic acid, including the steps of cleaving nucleotides from a particle-nucleotide conjugate and incorporating the cleaved nucleotides. (301) describes the step of attaching the 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. Prior to (301), the nucleic acid molecules of the target library may have been prepared to contain fragments complementary to the adapter sequence by ligation or other means. (302) describes a step of clonal amplification to generate clusters of target nucleic acid molecules on a surface. (303) describes hybridizing sequencing primers to complementary primer-binding or incorporation sequences on the target nucleic acid to form a primed target nucleic acid. (304) describes a step of combining the primed target nucleic acid with a polymerase, a polyvalent binding or incorporation composition containing a labeled (e.g., fluorescently labeled) particle-nucleotide conjugate, in which the nucleotides attach to the particle via chemical bonds or interactions that can be cleaved later. (404) may further include a step of washing or removing unbound reagents containing the polymerase and particle-nucleotide conjugate.

[0061] Referring again to Figure 3, if the nucleotide on 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 ternary conjugate or integration complex which can be detected by a detection method compatible with labeling the particle-nucleotide conjugate (e.g., fluorescence imaging). (305) may further include measuring the duration of the ternary conjugate or integration complex. In (306), the polymerase is placed under conditions that make it catalytically active to incorporate the nucleotide. These conditions may include exposing the polymerase to Mg ions or Mn ions in the reaction solution. The nucleotide bound to the polymerase and the primed target nucleic acid is then cleaved from the particle and subsequently incorporated into the primed chain of the primed target nucleic acid. The conjugate or integration complex becomes unstable. (306) may further include the step of washing or removing the dissociated particle-nucleotide conjugate and / or polymerase. After extension, steps (304), (305), and (306) may be repeated in multiple cycles to determine the sequence of the target nucleic acid.

[0062] B. Detection of target nucleic acid molecules Figures 4A–4B illustrate one exemplary method in which a polyvalent binding or embedding composition is used to detect a target nucleic acid. As shown in Figure 4A, a polymer-nucleotide conjugate (401) is placed in contact with polymerase (406), a first nucleic acid molecule (404), and a second nucleic acid molecule (405). The polymer-nucleotide conjugate (401) has multiple polymer branches radiating from the core, some of which attach to the nucleotide or oligonucleotide (402) and some attach to the label (403). If the nucleotide or oligonucleotide (402) on the polymer-nucleotide conjugate (401) is complementary to at least a portion of the first nucleic acid (404), a polyvalent binding or embedding complex is formed as shown in Figure B, and the strong binding or embedding signal helps detect a target nucleic acid having a sequence complementary or partially complementary to the nucleotide or oligonucleotide on the polymer-nucleotide conjugate. In some examples, polymerase, nucleic acid molecules, and at least one polymer-nucleotide conjugate are attached to a solid support.

[0063] The polyvalent binding compositions or embedded compositions described herein may be used in methods for detecting target nucleic acids in a sample. Furthermore, disclosed herein is a system configured to carry out any of the disclosed nucleic acid analysis methods. The system may include a fluid flow controller and / or fluid distribution system configured to continuously and repeatedly contact nucleic acid molecules with the disclosed polymerase and polyvalent binding compositions or embedded compositions and / or reagents. The contacting step may be carried out in one or more flow cells. In some examples, the flow cells may be fixed components of the system. In some examples, the flow cells may be removable and / or disposable components of the system. The system may further include a cartridge containing a sample collection unit and an assay assembly, the sample collection unit configured to collect a sample, and the assay assembly containing at least one reaction site containing a polyvalent binding composition or embedded composition adapted to interact with the analyte, which allows a predetermined portion of the sample to react with an assay reagent contained in the assay assembly to generate a signal indicating the presence of the analyte in the sample, and the signal generated from the analyte to be detected.

[0064] III. Polyvalent bonded compositions or incorporated compositions This disclosure relates to a polyvalent conjugated composition or embedded composition having multiple nucleotides conjugated to particles (e.g., polymers, branched polymers, dendrimers, or equivalent structures). When the polyvalent conjugated composition or embedded composition is brought into contact with polymerase and multiple copies of a primed target nucleic acid, a detectable ternary complex is formed, which in turn can achieve more accurate determination of the bases of the target nucleic acid.

[0065] When a polyvalent conjugation composition or embedded composition is used to replace a single unconjugated or detethered nucleotide and form a complex with polymerase and one or more copies of a target nucleic acid, the local concentration of the nucleotide and the binding force of the complex (if a complex containing two or more target nucleic acid molecules is formed) are increased many times over, which in turn enhances the signal intensity, particularly correct signal versus mismatch. The polyvalent conjugation composition or embedded composition described herein may comprise at least one particle-nucleotide conjugate (each particle-nucleotide conjugate comprising multiple copies of a single nucleotide moiety) for interaction with the target nucleic acid. The polyvalent composition may further comprise two, three, or four different particle-nucleotide conjugates, each having a different nucleotide conjugated to the particle.

[0066] A polyvalent conjugated composition or incorporated composition may contain one, two, three, four, or more species of particle-nucleotide conjugates, where each particle-nucleotide conjugate contains a different species of nucleotide. A first-type particle-nucleotide conjugate may contain nucleotides selected from the group consisting of ATP, ADP, AMP, dATP, dADP, and dAMP. A second-type particle-nucleotide conjugate may contain nucleotides selected from the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third-type particle-nucleotide conjugate may contain nucleotides selected from the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. A fourth-type particle-nucleotide conjugate may contain nucleotides selected from the group consisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments, each particle-nucleotide conjugate contains a single type of nucleotide corresponding to one or more nucleotides selected from the group consisting of ATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP. Each polyvalent conjugation composition or embedded composition may further contain one or more labels corresponding to the specific nucleotide conjugated to each of the conjugates. Non-limiting examples of labels include fluorescent labels, colorimetric labels, electrochemical labels (e.g., glucose or other reducing sugars, or thiols or other redox active moieties), luminescence labels, chemiluminescence labels, spin labels, radioactive labels, stereolabeling, affinity tags, and the like.

[0067] A. Particle-nucleotide conjugates In particle-nucleotide conjugates, multiple copies of the same nucleotide may be covalently or noncovalently bonded to the particle. Examples of particles may include branched polymers; dendrimers; crosslinked polymer particles such as agarose, polyacrylamide, acrylate, methacrylate, cyanoacrylate, and methyl methacrylate particles; glass particles; ceramic particles; metal particles; quantum dots; liposomes; emulsion particles; or other particles known in the art (e.g., nanoparticles, microparticles, etc.). In preferred embodiments, the particles are branched polymers.

[0068] In some examples, particle-nucleotide conjugates (e.g., polymer-nucleotide conjugates) may contain one, two, three, four, five, six, seven, eight, nine, ten, or more copies of nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs tethered to the particle.

[0069] Nucleotides may be linked to particles by a linker, and nucleotides may be attached to one end or position of a polymer. Nucleotides may be conjugated to particles via their 5' ends. In some particle-nucleotide conjugates, one nucleotide is attached to one end or position of a polymer. In some particle-nucleotide conjugates, multiple nucleotides are attached to one end or position of a polymer. The conjugated nucleotide is sterically accessible to one or more proteins, one or more enzymes, and nucleotide binding or integration sites. In some embodiments, nucleotides may be provided separately from nucleotide binding or integration sites such as polymerases. In some embodiments, the linker does not contain photoemitting or photoabsorbing groups.

[0070] The particles may further have binding or embedded parts. In some embodiments, the particles may self-associate without using separate interacting parts. In some embodiments, the particles may self-associate due to buffering or salt conditions, for example, in the case of calcium-mediated interactions of hydroxyapatite particles, lipid or polymer-mediated interactions of micelles or liposomes, or salt-mediated aggregation of metal (such as iron or gold) nanoparticles.

[0071] A particle-nucleotide conjugate may have one or more labels. Examples of labels include, but are not limited to, fluorophores, spin labels, metals or metal ions, colorimetric labels, nanoparticles, PET labels, radioactive labels, or polymers, or other such labels that can make the composition detectable by methods known in the art of detecting macromolecules or molecular interactions. The labels may be attached to the nucleotide (e.g., by attachment to the 5' phosphate moiety of the nucleotide), the particle itself (e.g., a PEG subunit), the ends of the polymer, the central portion, or any other arbitrary position within the polymer-nucleotide conjugate, the other positions being recognized by those skilled in the art as sufficient to render the composition, such as particles, detectable by methods known in the art or described elsewhere herein. In some embodiments, one or more labels are provided to correspond to or distinguish a particular particle-nucleotide conjugate.

[0072] In some embodiments, the label is a fluorophore. Non-limiting examples of the fluorescent portion include fluorescein and derivatives of fluorescein such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidefluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, rhodamine and TRITC, TMR, Rhodamine derivatives such as Lisamin Rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-Rhodamine, TMR-Iodoacetamide, Lisamin Rhodamine B sulfonyl chloride, Lisamin 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 BODIPY Derivatives such as FL C3-SE, 530 / 550 BODIPY 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, Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue Derivatives such as blue hydrazide, Lucifer Yellow and Lucifer Yellow iodoacetamide, derivatives such as Lucifer Yellow CH, cyanine and indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, derivatives such as Cy3 and Cy5, lanthanide chelate compounds,This includes, but is not limited to, other dyes known in the art, such as those described in 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 any combination thereof, or derivatives thereof. Cyanine dyes may exist in either sulfonated or unsulfonated forms and may consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3(it is 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indole, Or it may contain 1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidine-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indole-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indole-5-sulfonate),Cy5(it's 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H -Indole-1-ium, or 1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindoline-2-ylidene)penta-1,3-dien-1-yl) It comprises -3,3-dimethyl-3H-indole-1-ium-5-sulfonate) and Cy7 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indole-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indrolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indole-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indrolium-5-sulfonate), where "Cy" stands for "cyanine" and the first digit indicates the number of carbon atoms between the two indolenin groups. Cy2, which is an oxazole derivative rather than indorenine, and its benzo derivatives Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0073] In some embodiments, the detection label may be a FRET pair, allowing multiple classifications to be performed in a single excitation and imaging step. As used herein, the FRET may include excitation-exchange (Forster) transcription or electron-exchange (Dexter) transcription.

[0074] B. Polymer-nucleotide conjugates One example of a particle-nucleotide conjugate is a polymer-nucleotide conjugate. Several non-limiting examples of polymer-nucleotide conjugates are shown in Figures 5A–5C. For example, Figure 5A shows a polymer-nucleotide conjugate having a "starburst" configuration, which includes a fluorescently labeled streptavidin core and four nucleotides bound to the core via biotinylated linear PEG linkers with molecular weights ranging from 1K to 10K daltons; Figure 5B shows a polymer-nucleotide conjugate having, for example, a 12, 24, 48, or 96-arm dendrimer core and linear PEG linkers with molecular weights ranging from 1K to 10K daltons radiating from the center; and Figure 5C shows an example of a polymer-nucleotide conjugate including a network of streptavidin cores, for example, linked together by branched PEG linkers containing binding or embedding parts such as biotin.

[0075] Examples of suitable linear or branched polymers include linear or branched polyethylene glycol (PEG), linear or branched polypropylene glycol, linear or branched polyvinyl alcohol, linear or branched polylactic acid, linear or branched polyglycolic acid, linear or branched polyglycine, linear or branched polyvinyl acetate, dextran, or other such polymers, or copolymers incorporating two or more of the aforementioned, or other polymers known in the art. In one embodiment, the polymer is PEG. In another embodiment, the polymer may have PEG branches.

[0076] A suitable polymer may feature repeating units incorporating functional groups suitable for derivatization, such as amines, hydroxyls, carbonyls, or allyl groups. The polymer may further have one or more pre-derivative substituents such that one or more specific subunits incorporate a derivatization site or branched site, regardless of whether other subunits incorporate the same site, substituent, or moiety. Pre-derivative substituents may include, or further include, labels such as nucleotides, nucleosides, nucleotide analogs, fluorescent labels, radiolabels, or spin labels, interaction moieties, additional polymer moieties, or any combination thereof.

[0077] In polymer-nucleotide conjugates, the polymer may have multiple branches. Branched polymers can have a variety of configurations, including, but not limited to, starburst ("starburst"), aggregated star ("helter skelter"), bottlebrush, or dendrimer forms. A branched polymer can radiate or incorporate multiple branching points, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, from a central attachment point or central portion. In some embodiments, each subunit of the polymer may optionally constitute a separate branching point.

[0078] The length and size of the branches may vary depending on the type of polymer. For some branched polymers, the branch lengths may be 1–1,000 nm, 1–100 nm, 1–200 nm, 1–300 nm, 1–400 nm, 1–500 nm, 1–600 nm, 1–700 nm, 1–800 nm, or 1–900 nm, or longer, or may fall within or between any of the values ​​disclosed herein.

[0079] In some polymer-nucleotide conjugates, the polymer core may have a size corresponding to an apparent molecular weight of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any two of the values ​​defined above. The apparent molecular weight of the polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, as determined by mass spectrometry, or as determined by other methods known in the art.

[0080] In some branched polymers, the branches may have sizes corresponding to apparent molecular weights of 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15K Da, 20K Da, 30K Da, 50K Da, 80K Da, 100K Da, or any value within the range defined by any two of the above. The apparent molecular weight of a polymer can be calculated from the known molecular weights of a representative number of subunits, as determined by size exclusion chromatography, as determined by mass spectrometry, or as determined by other methods known in the art. A polymer can have multiple branches. The number of branches in a polymer can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or within the range defined by any two of these values.

[0081] For example, in the case of a polymer-nucleotide conjugate containing a branched polymer of a branched PEG having 4, 8, 16, 32, or 64 branches, the polymer-nucleotide conjugate may have nucleotides attached to the ends of the PEG branches such that each end has 0, 1, 2, 3, 4, 5, 6 or more nucleotides attached to it. In one non-limiting example, a branched PEG polymer with 3 to 128 PEG arms may have one or more nucleotides attached to the ends of the polymer branches such that each end has 0, 1, 2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs attached to it. 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.

[0082] In some examples, the length of the linker (e.g., a PEG linker) may be in the range of approximately 1 nm to approximately 1,000 nm. In some examples, the linker length may be at least 1 nm, at least 10 nm, at least 25 nm, at least 50 nm, at least 75 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 700 nm, at least 800 nm, at least 900 nm, or at least 1,000 nm. In some examples, the linker length may extend to a range between any two values ​​in this paragraph. For example, in some examples, the linker length may extend from approximately 75 nm to approximately 400 nm. Those skilled in the art will recognize that in some examples, the linker length may be any value within the range of this paragraph, for example, 834 nm.

[0083] In some cases, the linker length varies depending on the different nucleotides (including deoxyribonucleotides and ribonucleotides), nucleotide analogs (including deoxyribonucleotide analogs and ribonucleotide analogs), nucleosides (including deoxyribonucleosides or ribonucleosides), or nucleoside analogs (including deoxyribonucleoside analogs or ribonucleoside analogs). In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs is, for example, deoxyadenosine, and the linker length is between 1 nm and 1,000 nm. In some cases, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs is, for example, deoxyguanosine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, thymidine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxyuridine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, deoxycytidine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, adenosine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, guanosine, and the linker length is between 1 and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs includes, for example, 5-methyluridine, and the linker length is between 1 and 1,000 nm.In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs contains, for example, uridine, and the linker length is between 1 nm and 1,000 nm. In some examples, one of the nucleotides, nucleotide analogs, nucleosides, or nucleoside analogs contains, for example, cytidine, and the linker length is between 1 nm and 1,000 nm.

[0084] In polymer-nucleotide conjugates, each branch or subset of a polymer has a portion containing a nucleotide (e.g., adenine, thymine, uracil, cytosine, or guanine residue or its derivatives or mimics) attached to it, which can be bound to or incorporated into polymerase, reverse transcriptase, or other nucleotide binding or integration domains. Optionally, this portion may be incorporated into the nucleic acid chain elongated during a polymerase reaction. In some examples, the portion may be blocked so as not to be incorporated into the nucleic acid chain elongated during a polymerase reaction. In some other examples, the portion may be reversibly blocked so as not to be incorporated into the nucleic acid chain elongated during a polymerase reaction until such blocking is removed, after which the portion can be incorporated into the nucleic acid chain elongated during a polymerase reaction.

[0085] Nucleotides can be conjugated to polymer branches via their 5' end. In some examples, nucleotides can be modified to inhibit or prevent their incorporation into the nucleic acid chain elongating during polymerase reactions. For example, nucleotides may include 3'-deoxyribonucleotides, 3'-azidonucleotides, 3'-methylazidonucleotides, or other such nucleotides known or potentially known in the art, so as to prevent their incorporation into the nucleic acid chain elongating during polymerase reactions. In some embodiments, nucleotides may include a 3'-O-azide group, a 3'-O-azidomethyl group, a 3'-phosphorothioate group, a 3'-O-malonyl group, a 3'-O-alkylhydroxylamino group, or a 3'-O-benzyl group. In some embodiments, nucleotides lack a 3'-hydroxyl group.

[0086] The polymer may further have binding or integration sites in each branch or subset of branches. Some examples of binding or integration sites include, but are not limited to, biotin, avidin, streptavidin, polyhistidine domains, complementary pair nucleic acid domains, G-quartet forming nucleic acid domains, calmodulin, maltose-binding proteins, cellulases, maltose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine ​​and its derivatives, antibodies, epitopes, protein A, and protein G. The binding or integration sites may be any interacting molecules or fragments known in the art for facilitating binding or interaction between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interaction domains or fragments.

[0087] In some embodiments, the 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 proteins and maltose; lectins and related polysaccharides; ion-chelate moieties, complementary nucleic acids, triple helix or nucleic acids capable of forming triple helix interactions; nucleic acids capable of forming G-quartets, etc. Those skilled in the art will recognize that many pairs of moieties exist and are commonly used due to their properties of strongly and specifically interacting with one another. It will therefore be readily apparent that such complementary pairs or sets are considered suitable for this purpose when constructing or conceptualizing the compositions of the present disclosure. In some embodiments, the compositions disclosed herein may include compositions in which one element of a complementary interaction moiety attaches to one molecule or polyvalent ligand, and the other elements of the complementary interaction moiety attach to separate molecules or polyvalent ligands. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of a complementary interaction moiety are attached to a single molecule or a multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of a complementary interaction moiety are attached to separate arms or positions on a single molecule or a multivalent ligand. In some embodiments, the compositions disclosed herein may include compositions in which both or all elements of a complementary interaction moiety are attached to the same arm or position on a single molecule or a multivalent ligand. In some embodiments, a composition containing one element of a complementary interaction moiety and a composition containing another element of a complementary interaction moiety may be mixed simultaneously or sequentially. In some embodiments, the interactions between molecules or particles disclosed herein allow for the association or aggregation of multiple molecules or particles, for example, to increase a detectable signal. In some embodiments, a fluorescence signal, a colorimetric quantitative signal, or a radioactive signal is enhanced.In other embodiments, other interacting moieties disclosed herein or known in the art are contemplated. In some embodiments, the compositions provided herein may be provided so as to be mixed simultaneously or sequentially, comprising one or more molecules containing a first interacting moiety, such as one or more imidazole or pyridine moieties, and one or more additional molecules containing a second interacting moiety, such as a histidine residue. In some embodiments, the compositions comprise one, two, three, four, five, six, or more imidazole or pyridine moieties. In some embodiments, the compositions comprise one, two, three, four, five, six, or more histidine residues. In such embodiments, the interactions between the provided molecules or particles may be facilitated by the presence of divalent cations such as nickel, manganese, magnesium, calcium, or strontium. In some embodiments, for example, a (His)3 group may interact with a (His)3 group on another molecule or particle via the coordination of nickel or manganese ions.

[0088] A polyvalent binding composition or integration composition may comprise one or more buffers, salts, ions, or additives. In some embodiments, typical additives may include, but are not limited to, surfactants such as betaine, spermidine, 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® series polymers; arginine, histidine, imidazole, or any combination thereof; or any substance known in the art as a DNA "relaxer" (a compound having the effect of altering the duration of DNA so that sites within the strand have increased accessibility to DNA binding or integration sites, altering the number of conjugates or crosslinks within the polymer, or altering the stereodynamics of the DNA molecule).

[0089] The polyvalent binding composition or incorporated composition may contain zwitterionic compounds as additives. Further representative additives are found in Lorenz, TCJ Vis.Exp.(63), e3998, doi:10.3791 / 3998(2012), which is cited herein by reference in relation to the disclosure of additives for promoting nucleic acid binding or kinetics, or for promoting the manipulation, use, or storage of nucleic acids. In some embodiments, representative cations may include, but are not limited to, sodium, magnesium, strontium, potassium, manganese, calcium, lithium, nickel, cobalt, or other such cations known in the art for promoting nucleic acid interactions such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, and protein binding.

[0090] IV. Binding of target nucleic acids to a polyvalent binding composition or embedded composition When a polyvalent conjugate or embedding composition is used to replace a single unconjugated or detethered 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 force of the complex (if a complex containing two or more target nucleic acid molecules is formed), increases many times over, thereby enhancing the signal intensity, particularly correct signal-to-mismatch. This disclosure intends to contact a polyvalent conjugate or embedding composition with a polymerase and a primed target nucleic acid to determine the formation of a ternary conjugate or embedding complex.

[0091] Figure 6 illustrates the use of the disclosed polymer-nucleotide conjugate to achieve increased signal intensity, persistence, and wash / removal steps during binding. Due to increased local concentrations of nucleotides on the polymer-nucleotide conjugate and / or the formation of non-covalent bonds with two or more primed target nucleic acid molecules, binding of the polymerase, primed target chain, and polymer conjugate nucleotide is more preferable when the nucleotide is complementary to the next base of the target nucleic acid. The formed binding complex has a longer duration, which helps increase the signal and shorten the imaging step. The high signal intensity resulting from the use of the disclosed polymer-nucleotide conjugate remains stable throughout the binding and imaging steps. The strong binding between the polymerase, primed target chain, and polymer conjugate nucleotide or nucleotide analog also means that the thus formed binding complex remains stable during the wash step, once other reaction mixture components and mismatched nucleotide analogs are washed away. After the imaging step, the binding complex may become unstable (e.g., due to changes in the buffer composition), and the primed target nucleic acid may subsequently be elongated by one base. After extension, the binding and imaging steps can be repeated using the disclosed polymer-nucleotide conjugate to determine the identity of the next base.

[0092] As an example, Figure 6 provides a graphical representation of the increase in signal intensity during binding, persistence, and washing / removal of the multivalent substrate described herein, which represents the experimentally observed change in signal intensity. Thus, the compositions and methods of this disclosure provide a robust and controllable means for establishing and maintaining a ternary enzyme complex, thereby providing a significantly improved means for identifying and / or measuring the presence of the complex and controlling its persistence. This provides an important solution to problems such as determining N+1 base identity in nucleic acid sequencing applications.

[0093] Without intending to be bound to any particular theory, the polyvalent binding compositions disclosed herein have been observed to associate with polymerase nucleotide complexes to form ternary complexes at a time-dependent rate, substantially slower than the association rate known to be obtained by nucleotides in free solution. Thus, the on-rate (K) on The on-rate of a single nucleotide or a nucleotide not attached to a multivalent ligand complex is substantially and surprisingly slower. However, importantly, the off-rate (K) of a multivalent ligand complex is significantly slower. off The rate of resorption is substantially slower than that observed for nucleotides in free solution. Therefore, the polyvalent ligand complexes of this disclosure provide a remarkable and beneficial improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (particularly compared to such complexes formed with free nucleotides), enabling, for example, a significant improvement in imaging quality for nucleic acid sequencing applications over currently available methods and reagents. Importantly, this property of the polyvalent binding compositions disclosed herein makes the formation of the visible ternary complex controllable, and as a result, subsequent visualization, modification, or processing steps can be performed essentially regardless of the dissociation of the complex; i.e., the complex can be formed, imaged, modified, or used in other ways as needed, and the complex remains stable until a positive dissociation step is performed, such as exposing the complex to a dissociation buffer.

[0094] In some examples, the duration of a polyvalent binding complex formed using the disclosed particle nucleotide or polymer-nucleotide conjugate may range from about 0.1 seconds to about 600 seconds under non-unstable conditions. In some examples, the duration may be at least 0.1 seconds, at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 20 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 540 seconds, or at least 600 seconds. In some examples, the duration may range between any two values ​​specified in this paragraph. For example, in some examples, the duration may range from about 10 seconds to about 360 seconds. Those skilled in the art will recognize that in some examples the duration may be any value within the range specified in this paragraph, for example, 78 seconds.

[0095] In various embodiments, polymerases suitable for the binding or integration interactions described herein may include any polymerase in its original form or those that may be known in the art. For example, it is known that each organism encodes one or more DNA polymerases within its genome. Examples of suitable polymerases may include, but are not limited to, Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases such as T4, RB69, and phi29 bacteriophage DNA polymerases, and Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III, and Escherichia coli DNA polymerase III alpha and epsilon; reverse transcriptases, such as 9-degree N polymerase, HIV type M or O reverse transcriptase, avian myeloblastosis virus reverse transcriptase, or Moloney's mouse leukemia virus (MMLV) reverse transcriptase, or telomerases. Further non-limiting examples of DNA polymerases may include those from various archaeal genera such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or their variants, including polymerases known in the art such as Vent®, Deep Vent®, Pfu, KOD, Pfx, Therminator®, and Tgo polymerase. In some cases, the polymerase is a Klenow polymerase.

[0096] The ternary complex has a longer duration when the nucleotides on the polymer-nucleotide conjugate are complementary to the target nucleic acid than when they are non-complementary. The ternary complex further has a longer duration when the nucleotides on the polymer-nucleotide conjugate are more complementary to the target nucleic acid than non-conjugated or non-tethered complementary nucleotides. For example, in some embodiments, the ternary complex may have a duration over a time range defined by less than 1 second, greater than 1 second, greater than 2 seconds, greater than 3 seconds, greater than 5 seconds, greater than 10 seconds, greater than 15 seconds, greater than 20 seconds, greater than 30 seconds, greater than 60 seconds, greater than 120 seconds, greater than 360 seconds, greater than 3600 seconds, or greater than or two of these values.

[0097] The duration can be measured by observing the onset and / or duration of the binding complex, for example, by observing the signal from the labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent containing one or more nucleotides may be present in the binding complex, and thus it becomes possible to detect a signal from the label during the duration of the binding complex.

[0098] It has been observed that different ranges of durations can be achieved with different salts or ions, for example, magnesium ions (Mg 2+ This shows that complexes formed in the presence of (Sr) form faster than complexes formed with other ions. Furthermore, for example, strontium ions (Sr) 2+ The complexes formed in the presence of ) were observed to readily form and dissociate completely or substantially completely upon ion recovery or washing with a buffer lacking one or more components of the composition of the polymer and / or one or more nucleotides and / or one or more interacting moieties, for example, a buffer containing a chelating agent that can cause or accelerate the removal of divalent cations from a polyvalent complex containing a reagent. Therefore, in some embodiments, the composition of the present disclosure is Mg 2+ Includes. In some embodiments, the composition of the present disclosure is Ca 2+Includes. In some embodiments, the composition of the present disclosure is Sr 2+ Includes. In some embodiments, the composition of the present disclosure includes cobalt ions (Co 2+ ) include. In some embodiments, the compositions of the Disclosure include MgCl2. In some embodiments, the compositions of the Disclosure include CaCl2. In some embodiments, the compositions of the Disclosure include SrCl2. In some embodiments, the compositions of the Disclosure include CoCl2. In some embodiments, the compositions do not contain magnesium or are substantially magnesium-free. In some embodiments, the compositions do not contain calcium or are substantially calcium-free. In some embodiments, the methods of the Disclosure provide a step of contacting one or more nucleic acids with one or more compositions of the Disclosures Disclosed herein that lack either calcium or magnesium, or both calcium and magnesium.

[0099] The dissociation of the ternary complex can be controlled by changing the buffer conditions. After the imaging step, a buffer with an increased salt content is used to induce the dissociation of the ternary complex so that the labeled polymer-nucleotide conjugate can be washed away, providing a means by which the signal can be attenuated or terminated, such as in the transition between one sequencing cycle and the next. In some embodiments, this dissociation may be affected by washing the complex with a buffer lacking the required metal or cofactor. In some embodiments, the washing buffer may contain one or more compositions for the purpose of maintaining pH adjustment. In some embodiments, the washing buffer may contain one or more monovalent cations, such as sodium. In some embodiments, the washing buffer lacks or substantially lacks divalent cations, such as strontium, calcium, magnesium, or manganese, for example. In some embodiments, the washing buffer further contains chelating agents, such as EDTA, EGTA, nitrilotriacetic acid, polyhistidine, or imidazole. In some embodiments, the washing buffer may maintain the pH of the environment at the same level as in the case of the bound complex. In some embodiments, the washing buffer may raise or lower the pH of the environment relative to the levels observed in the binding complex. In some embodiments, the pH may be within the range of 2–4, 2–7, 5–8, 7–9, 7–10, or less than 2, or greater than 10, or within the range defined by any two of the values ​​provided herein.

[0100] The addition of certain ions may affect the binding of polymerase to a primed target nucleic acid, the formation of a ternary complex, the dissociation of the ternary complex, or the incorporation of one or more nucleotides into the nucleic acid during extension, such as during polymerase reactions. In some embodiments, the relevant anions may include chlorides, acetates, glucons, sulfates, phosphates, and the like. In some embodiments, ions may be incorporated into the compositions of this disclosure by adding one or more acids, bases, or salts such as NiCl2, CoCl2, MgCl2, MnCl2, SrCl2, CaCl2, CaSO4, SrCO3, and BaCl2. Representative salts, ions, solutions, and conditions are found in Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, AR, Ed. (2000), which is incorporated herein by reference in its entirety, particularly with respect to Chapter 17 and the disclosure of relevant salts, ions, salt solutions, and ionic solutions.

[0101] This disclosure envisions a step of contacting a polyvalent conjugated composition or embedded composition comprising at least one particle-nucleotide conjugate with one or more polymerases. The contact step can optionally be carried out 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 contact step comprises contacting the polyvalent conjugated composition or embedded composition with one polymerase. In some embodiments, the contact step comprises contacting the composition comprising one or more nucleotides with a plurality of polymerases. The polymerases may be conjugated to a single nucleic acid molecule.

[0102] The binding of the target nucleic acid to the polyvalent binding composition may be provided in the presence of a catalytically inactivated polymerase. In one embodiment, the polymerase may be catalytically inactivated by mutation. In one embodiment, the polymerase may be catalytically inactivated by chemical modification. In some embodiments, the polymerase may be catalytically inactivated by the absence of a required substrate, ion, or cofactor. In some embodiments, the polymerase enzyme may be catalytically inactivated by the absence of a magnesium ion.

[0103] The binding of the target nucleic acid to the polyvalent binding composition is carried out in the presence of a polymerase in which the binding solution, reaction solution, or buffer lacks magnesium or manganese. Alternatively, the binding of the target nucleic acid to the polyvalent binding composition is carried out in the presence of a polymerase in which the binding solution, reaction solution, or buffer contains calcium or strontium.

[0104] When a catalytically inactive polymerase is used to assist the interaction of nucleic acids with a polyvalent binding composition, the interaction between the composition and the polymerase stabilizes the ternary complex so that the complex is detectable by fluorescence or by other methods disclosed herein or known in the art. Unbound polymer-nucleotide conjugates can be optionally washed away before detection of the ternary binding complex.

[0105] The step of contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein is carried out in a solution containing either calcium or magnesium, or both calcium and magnesium. Alternatively, the step of contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein is carried out in a solution lacking either calcium or magnesium, or both calcium and magnesium, and in another step, regardless of the order of the steps, includes adding either calcium or magnesium, or both calcium and magnesium, to the solution. In some embodiments, the step of contacting one or more nucleic acids with the polymer-nucleotide conjugates disclosed herein is carried out in a solution lacking strontium, and in another step, regardless of the order of the steps, includes adding strontium to the solution.

[0106] V. Use of polyvalent binding compositions or embedded compositions combined with low nonspecific binding surfaces Disclosed herein are solid supports comprising low nonspecific binding surface compositions that enable improved nucleic acid hybridization and amplification performance. Generally, the disclosed supports may comprise a substrate (or support structure), a covalently or non-covalently low-binding chemically modified layer, e.g., a silane layer, a polymer film, and one or more layers of one or more covalently or non-covalently bonded primer sequences that can be used to anchor single-stranded target nucleic acids to the support surface. In some examples, by varying the surface formulation, e.g., the chemical composition of one or more layers, the coupling chemistry used to crosslink one or more layers to and / or to the support surface, and the total number of layers, nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the support surface is minimized or reduced compared to a comparable monolayer. Often, the surface formulation may be varied so that nonspecific hybridization at the support surface is minimized or reduced compared to a comparable monolayer. The surface formulation may be modified so that nonspecific amplification at the support surface is minimized or reduced compared to a comparable monolayer. The surface formulation may also be modified so that the specific amplification rate and / or yield at the support surface is maximized. An amplification level suitable for detection is achieved in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more amplification cycles, depending on the examples disclosed herein.

[0107] Examples of materials for fabricating substrates or support structures 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 combinations thereof. Various compositions of both glass and plastic substrates are intended.

[0108] The substrate or support structure may exhibit any of the various geometric shapes and dimensions known to those skilled in the art, and may contain any of the various materials known to those skilled in the art. For example, in some examples, the substrate or support structure may be locally planar (e.g., including 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 irregular (e.g., including the outer surface of a non-porous bead or particle of an irregular shape). In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid non-porous surface. In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous so that the coating described herein penetrates the porous surface and the nucleic acid hybridization and amplification reactions performed thereon occur within the pores.

[0109] A substrate or support structure comprising one or more chemically modified layers, for example, a layer of a low nonspecific binding polymer, may be independent or integrated into another structure or assembly. For example, in some examples, the substrate or support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may comprise one or more surfaces within a microplate format, for example, the bottom surfaces of wells within a microplate. As described above, in some preferred embodiments, the substrate or support structure comprises an internal surface of a capillary (such as a lumen surface). In an alternative preferred embodiment, the substrate or support structure comprises an internal surface of a capillary (such as a lumen surface) etched into a planar chip.

[0110] As described above, the nonspecific low-binding supports of this disclosure exhibit reduced nonspecific binding of proteins, nucleic acids, and other components of hybridization and / or amplification formulations used in solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given support surface can be evaluated qualitatively or quantitatively. For example, in some cases, exposure of the surface to a fluorescent dye (e.g., cyanine dyes such as Cy3 or Cy5, fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and / or a fluorescently labeled protein (e.g., polymerase) under a standardized set of conditions, followed by a specified washing protocol and fluorescence imaging may be used as a qualitative tool for comparing nonspecific binding on supports containing formulations of various surfaces. In some examples, surface exposure to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerases), such as Cy3 and Cy5, under a standardized set of conditions, followed by a specified washing protocol and fluorescence imaging, may be used as a quantification tool for comparing nonspecific binding on supports, including various surface formulations, provided that the fluorescence signal is linearly (or predictably) related to the number of fluorophores on the support surface (e.g., under conditions where signal saturation and / or self-quenching of fluorophores are not a concern), and care is taken to ensure that fluorescence imaging is performed under conditions where appropriate calibration standards are used. In some examples, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, may be used to evaluate the quantification of the degree to which nonspecific binding is exhibited by the various support surface formulations of this disclosure.

[0111] Some surfaces disclosed herein exhibit a ratio of specific binding to a fluorophore such as Cy3 and non-specific binding that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range specified herein. Some surfaces disclosed herein exhibit a ratio of specific fluorescence to non-specific fluorescence of a fluorophore such as Cy3 and non-specific fluorescence that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range specified herein.

[0112] As described above, in some examples, the degree of nonspecific binding exhibited by the disclosed low-binding supports may be evaluated using a standardized protocol for contacting the surface with labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof) with nucleotides, labeled oligonucleotides, etc., under a standardized set of incubation and rinsing conditions, and then detecting the amount of label remaining on the surface and comparing the resulting signal with a suitable calibration standard. In some examples, the label may include fluorescent labels. In some examples, the label may include radioisotopes. In some examples, the label may include other detectable labels known to those skilled in the art. In some examples, the degree of nonspecific binding exhibited by a given support surface formulation may therefore be evaluated in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit region. In some examples, the low-binding supports of this disclosure have a binding density of 0.001 molecules / μm 2 Less than 0.01 molecules / μm 2 Less than 0.1 molecules / μm 2 Less than 0.25 molecules / μm 2 Less than 0.5 molecules / μm 2 Less than 1 molecule / μm2 Less than 10 molecules / μm 2 Less than 100 molecules / μm 2 Less than 1,000 molecules / μm 2 Nonspecific protein binding of less than 1 / 2000 (or nonspecific binding of other specified molecules (e.g., cyanine dyes such as Cy3 or Cy5, fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein)) may be observed. Those skilled in the art will know that a given support surface of this disclosure may fall within any value in this range, for example, 86 molecules / μm 2 It will be understood that nonspecific binding of less than 0.5 molecules / µm may be observed. For example, several modified surfaces disclosed herein showed 0.5 molecules / µm after 15 minutes of contact with a 1 µM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) buffer, followed by three rinses in deionized water. 2 It exhibits nonspecific binding of proteins less than 0.25 molecules / µm. Several modified surfaces disclosed herein show this binding rate of 0.25 molecules / µm. 2This shows nonspecific binding of less than 1 μM of Cy3 dye molecules. In an independent nonspecific binding assay, 1 μM labeled Cy3SA (ThermoFisher), 1 μM Cy5SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (JenaBiosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (JenaBiosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (JenaBiosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (JenaBiosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (JenaBiosciences) were incubated on a low-binding substrate in a 384-well plate format at 37°C for 15 minutes. Each well was rinsed 2-3 times with 50 μl of deionized RNase / DNase-free water and 2-3 times with 25 mM ACES buffer pH 7.4. The 384-well plates were imaged on a GETyphoon instrument using Cy3, AF555, or Cy5 filter sets (depending on the dye test performed) with 800 PMT sensitivity adjustments and a resolution of 50-100 μm as specified by the manufacturer. For higher resolution imaging, images were acquired using an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) equipped with a total internal reflection fluorescence (TIRF) objective lens (100X, 1.5 NA, Olympus), a CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or Olympus DP80 color and monochrome camera), an illumination source (Olympus 100W Hg lamp, Olympus 75W Xe lamp, Olympus U-HGLGPS fluorescence light source, etc.), and an excitation wavelength of 532 nm or 635 nm.Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), for example, dichroic reflectors / beam splitters at 405, 488, 532, or 633 nm, and bandpass filters were selected at 532LP or 645LP to match the appropriate excitation wavelength. Several modified surfaces disclosed herein exhibit nonspecific binding of dye molecules to less than 0.25 molecules / µm².

[0113] In some examples, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of a fluorophore such as Cy3 to 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 an intermediate value defined by the range specified herein. In some examples, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence signals of a fluorophore such as Cy3 to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range specified herein.

[0114] Low background surfaces consistent with the disclosures herein may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50 specific dye molecules per nonspecifically adsorbed molecule, between specific dye adhesion (e.g., Cy3 adhesion) and nonspecific dye adsorption (e.g., Cy3 dye adsorption). Similarly, low background surfaces consistent with the disclosures herein, on which fluorophores, e.g., Cy3, are attached when exposed to excitation energy, may exhibit a ratio of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1 between specific fluorescence signals (e.g., generated from Cy3-labeled oligonucleotides attached to the surface) and nonspecific adsorbed dye fluorescence signals.

[0115] In some examples, the degree of hydrophilicity (or "wetting" by aqueous solutions) of the disclosed support surface may be evaluated, for example, by measuring the water contact angle with a small water droplet placed on the surface, measured, for example, using an optical tensile meter. In some examples, a static contact angle may be determined. In some examples, a progressive or receding contact angle may be determined. In some examples, the water contact angle of the hydrophilic, low-bonding support surface disclosed herein may be in the range of about 0 to about 30 degrees. In some examples, the water contact angle of the hydrophilic, low-bonding support surface disclosed herein may be 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 examples, the contact angle is 40 degrees or less. Those skilled in the art will understand that a given hydrophilic, low-bonding support surface of this disclosure may exhibit a water contact angle having a value somewhere within this range.

[0116] In some cases, the hydrophilic surfaces disclosed herein facilitate a reduction in the washing time of bioassays, often due to a decrease in the nonspecific binding of biomolecules to low-binding surfaces. In some cases, the appropriate washing step may be performed in less than 60, 50, 40, 30, 20, 15, or 10 seconds. For example, in some cases, the appropriate washing step may be performed in less than 30 seconds.

[0117] Some of the low-binding surfaces of this disclosure exhibit significant improvements in stability or endurance to prolonged exposure to solvents and high temperatures, or to repeated cycles of solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces may be tested by fluorescently labeling functional groups on the surface, or tethered biomolecules on the surface (e.g., oligonucleotide primers), and monitoring the fluorescence signals before, during, and after prolonged exposure to solvents and rising temperatures, or to repeated cycles of solvent exposure or temperature changes. In some examples, the degree of change in fluorescence used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours (or any combination of these percentages measured over these periods). In some examples, the degree of fluorescence change used to assess surface quality may be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of these percentages measured over this range of cycles) over 5, 10, 20, 30, 40, 50, 60, 700, 800, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 cycles of repeated exposure to changes in solvent and / or temperature.

[0118] In some examples, the surfaces disclosed herein may exhibit a high ratio of specific signals to non-specific signals or other backgrounds. For example, when used for nucleic acid amplification, some surfaces may exhibit an amplification signal at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 100 times greater than the signal of adjacent uncollected regions of the surface. Similarly, some surfaces may exhibit an amplification signal at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 100 times greater than the signal of adjacent increased nucleic acid population regions of the surface.

[0119] In some examples, the disclosed low-background surface fluorescence images, when used in nucleic acid hybridization or amplification applications to create clusters of hybridized or cloned nucleic acid molecules (e.g., those directly or indirectly labeled with fluorescent dyes), exhibit contrast-to-noise ratios (CNRs) greater than 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 250.

[0120] One or more primers may be attached to or anchored to the support surface. In some examples, one or more adapters or primers may include spacer sequences, adapter sequences for hybridization to adapter-linked target library nucleic acid sequences, forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcode sequences, or any combination thereof. In some examples, one primer or adapter sequence may be anchored to at least one layer of the surface. In some examples, at least two, three, four, five, six, seven, eight, nine, ten, or more than ten different primers or adapter sequences may be anchored to at least one layer of the surface.

[0121] In some examples, the length of the tethered adapter and / or primer sequence may range from about 10 nucleotides to about 100 nucleotides. In some examples, the length of 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 some examples, the length of 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. Any combination of the lower and upper limits set out in this paragraph may form the range included in this disclosure, for example, in some examples, the length of the tethered adapter and / or primer sequence may range from about 20 nucleotides to about 80 nucleotides. Those skilled in the art will recognize that the length of the tethered adapter and / or primer sequence may have any value within this range, for example, about 24 nucleotides.

[0122] In some examples, the resulting surface density of primers on the low-binding support surface of this disclosure is approximately 100 primer molecules / μm 2 Approximately 100,000 primer molecules / μm 2 It can be in the range of. In some examples, the resulting surface density of primers on the low-binding support surface of this disclosure is about 1000 primer molecules / μm 2 ~Approximately 1,000,000 primer molecules / μm 2 The range may be as follows: In some examples, the surface density of the primer is at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm 2 This is possible. In some examples, the surface density of the primer can be up to 1,000,000, up to 100,000, up to 10,000, or up to 1,000 molecules / μm 2It is possible. Either the lower and upper limits described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the surface density of the primer is approximately 10,000 molecules / μm 2 The range may be approximately 100,000 molecules. Those skilled in the art will know that the surface density of primer molecules can be any value within this range, for example, approximately 455,000 molecules / μm. 2 It will be recognized that these may be present. In some cases, the surface density of the target library nucleic acid sequence initially hybridized to the adapter or primer sequence on the support surface may be less than or equal to that indicated by the surface density of the anchored primer. In some cases, the surface density of the clonely amplified target library nucleic acid sequence hybridized to the adapter or primer sequence on the support surface may be in the same range as that indicated by the surface density of the anchored primer.

[0123] The local density mentioned above does not exclude variations in density across the entire surface, and as a result, the surface may have, for example, a density of 500,000 / μm. 2 It may include a region having one oligodendrome, while also including at least a second region having a substantially different local density.

[0124] VI. Exemplary Alternative Embodiments A disclosed method for determining the sequence of a target nucleic acid includes: a) contacting a double-stranded or partially double-stranded target nucleic acid molecule, comprising a sequenced template strand and an extended primer strand, with one or more disclosed nucleic acid binding compositions; and b) detecting the binding of the nucleic acid binding composition to the nucleic acid molecule, thereby determining the presence of one of the one or more nucleic acid binding compositions on the nucleic acid molecule and the identity of the next nucleotide (i.e., N+1 or terminal nucleotide) incorporated into the complementary strand.

[0125] The sequencing method may further include the step of incorporating an N+1 or terminal nucleotide into a primer strand, and then repeating the steps of contacting, detecting, and incorporating for one or more additional repeats, thereby determining the sequence of the template strand of the nucleic acid molecule. After the step of detecting the ternary complex, the primed strand of the primed target nucleic acid is extended to one base before the next round of analysis is performed. The primed target nucleic acid may be extended using conjugated nucleotides attached to the polymer in the polyvalent binding composition, or using unconjugated or detethered free nucleotides provided after the polyvalent binding composition has been removed.

[0126] The elongation of a primed target nucleic acid may be prevented or inhibited due to blocked nucleotides on the chain or the use of a catalytically inactive polymerase. If a nucleotide in a polymer-nucleotide conjugate has a blocking group that hinders nucleic acid elongation, the incorporation of the nucleotide may be achieved by removing the blocking group from the nucleotide (e.g., by detaching the nucleotide from its polymer, branched polymer, dendrimer, particle, etc.). If the elongation of a primed target nucleic acid is inhibited due to the use of a catalytically inactive polymerase, the incorporation of the nucleotide may be achieved by providing a cofactor or activator, such as a metal ion.

[0127] Detection of the ternary complex is achieved before, simultaneously with, or after the incorporation of nucleotide residues. In some embodiments, the primed target nucleic acid may comprise a target nucleic acid having multiple primed sites for attachment of polymerases and / or nucleic acid binding moieties. In some embodiments, the multiple polymerases may attach to a single target nucleic acid molecule, such as multiple sites within the target nucleic acid molecule. In some embodiments, the multiple polymerases may be bound to a polyvalent binding composition disclosed herein, comprising multiple nucleotides. In some embodiments, the target nucleic acid molecule may be the product of strand substitution synthesis, rolling circle amplification, linking or fusion of multiple copies of a query sequence, or other methods for producing nucleic acid molecules containing multiple copies of the same sequence, known in the Art or disclosed elsewhere herein. Thus, in some embodiments, the multiple polymerases may attach to multiple identical or substantially identical sites within the target nucleic acid containing multiple identical or substantially identical copies of the query sequence. In some embodiments, the multiple polymerases may then engage in interaction with one or more polyvalent binding compositions. However, in preferred embodiments, the number of binding sites in the target nucleic acid is at least two, and the number of nucleotides or substrate portions present on the particle-nucleotide conjugate, such as a polymer-nucleotide conjugate, is also two or more.

[0128] To provide an optimized signal, for example, to provide identification of nucleotides at specific positions in a nucleic acid sequence, it may be advantageous to provide a polyvalent binding composition in combination with other elements. In some embodiments, the compositions disclosed herein are provided in combination with a surface that provides low background binding or low levels of protein binding, particularly a hydrophilic or polymer-coated surface. A typical surface can be found, for example, in U.S. Patent Application No. 16 / 363,842, the contents of which are incorporated herein by reference in whole.

[0129] In some examples, nucleic acid molecules are anchored to the surface of a solid support, for example, via hybridization of a template strand to an adapter nucleic acid sequence or primer nucleic acid sequence anchored to the solid support. In some examples, the solid support comprises glass, fused silica, silicon, or a polymer substrate. In some examples, the solid support comprises a low nonspecific binding coating comprising one or more hydrophilic polymer layers (e.g., PEG layers), where at least one of the hydrophilic polymer layers comprises a branched polymer molecule (e.g., a branched PEG molecule having 4, 8, 16, or 32 branches).

[0130] The solid support has approximately 1,000 primer molecules / μm 2 ~Approximately 1,000,000 primer molecules / μm 2 The oligonucleotide adapter or primer is anchored to at least one hydrophilic polymer layer with a surface density in the range of [range]. In some examples, the surface density of the oligonucleotide primer is at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules / μm 2 In some cases, the surface density of oligonucleotide primers can be up to 1,000,000, 100,000, 10,000, or 1,000 molecules / μm. 2 This may also be the case. Either the lower and upper limits described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the surface density of the primer is approximately 10,000 molecules / μm 2 ~Approx. 100,000 molecules / μm 2 It may be within this range. Those skilled in the art will know that the surface density of primer molecules can be any value within this range, for example, about 455,000 molecules / μm 2 They will recognize that they may have it.

[0131] Those skilled in the art will recognize that in a series of repetitive sequencing reactions, one or more sites may fail to incorporate a nucleotide during a given cycle, and therefore one or more sites may become asynchronous with the majority of the elongated nucleic acid chain. Under conditions where the sequencing signal originates from a reaction occurring with a single copy of the target nucleic acid, these incorporation failures also produce individual errors in the output sequence. An object of this disclosure is to describe a method for reducing this type of error in sequencing reactions. By using a polyvalent substrate that can be incorporated into the elongated chain by increasing the probability of recombination during early dissociation of the ternary polymerase complex, the frequency of “skipped” cycles in which bases are not incorporated can be reduced. Accordingly, in some embodiments, this disclosure intends to use a polyvalent substrate disclosed herein in which the nucleoside moiety is contained within a nucleotide having a free or reversibly modified 5'-phosphate, diphosphate, or triphosphate moiety, and the nucleotide is unstable or linkage-linked to a particle or polymer disclosed herein. In some embodiments, the Disclosure aims to reduce the intrinsic error rate by skipped assembly as a result of using the multivalent substrates disclosed herein.

[0132] The disclosure further envisions a sequencing reaction in which a sequencing signal originating from or related to a given sequence originates from or occurs within a definable region containing multiple copies of a target sequence. Sequencing methods incorporating multiple copies of a target sequence have the advantage of signal amplification because multiple simultaneous sequencing reactions exist within a defined region, each providing its own signal. The presence of multiple signals within a defined region also mitigates the impact of a single skipped cycle, as signals from numerous correct base calls may overwhelm signals from a few skipped or incorrect base calls. The disclosure further envisions including free, unlabeled nucleotides during the extension reaction or in separate parts of the extension cycle to provide incorporation at sites that may have been skipped in previous cycles. For example, unlabeled, blocked nucleotides may be added during or after an incorporation cycle so that they may be incorporated into skipped sites. The unlabeled blocked nucleotides may consist of the same species as the nucleotides attached to the polyvalent binding substrate or the substrate that is present or has been present during a particular cycle, or a mixture of 1, 2, 3, 4 or more species of unlabeled blocked nucleotides.

[0133] When each sequencing cycle proceeds to completion, each reaction within a defined region provides an identical signal. However, as described elsewhere in this specification, in a series of iterative sequencing reactions, occasionally one or more sites fail to incorporate nucleotides during a given cycle, and therefore one or more sites become asynchronous with the majority of the extending nucleic acid chain. This problem, called "phasing," causes degradation of the sequencing signal because the signal is contaminated with spurious signals from sites that skipped one or more cycles. This can lead to errors in base identification. The gradual accumulation of skipped cycles over multiple cycles further reduces the effective read length due to the gradual degradation of the sequencing signal in each cycle. A further object of this disclosure is to provide a method for reducing phasing errors and / or improving read length in sequencing reactions.

[0134] The sequencing method may include contacting a target nucleic acid or a plurality of target nucleic acids, comprising multiple linked or unlinked copies of a target sequence, with a polyvalent binding composition described herein. Contacting the target nucleic acid, or a plurality of target nucleic acids comprising multiple linked or unlinked copies of a target sequence, with one or more particle-nucleotide conjugates may provide a substantially increased local concentration of the correct nucleotides being investigated in a given sequencing cycle, thereby suppressing signals from improper incorporation or phase nucleic acid strands (i.e., elongated nucleic acid strands with one or more skipped cycles).

[0135] A method for obtaining nucleic acid sequence information may involve contacting a target nucleic acid or a plurality of target nucleic acids with a particle-nucleotide conjugate, wherein the target nucleic acid or a plurality of target nucleic acids comprises a plurality of linked or unlinked copies of the target sequence. This method reduces the sequencing error rate, as indicated by a reduction in misidentification of bases, reporting of non-existent bases, or failure to report correct bases. In some embodiments, the reduction in the sequencing error rate may include a reduction of 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the error rate observed using a monovalent ligand comprising a free nucleotide, labeled free nucleotide, protein or peptide-bonded nucleotide, or labeled protein or peptide-bonded nucleotide.

[0136] A method for obtaining nucleic acid sequence information may include contacting a target nucleic acid or a plurality of target nucleic acids with one or more particle nucleotide conjugates, wherein the template nucleic acid or the plurality of target nucleic acids comprises a plurality of linked or unlinked copies of the target sequence. In this method, the average read length increases by 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, or more compared to the average read length observed using a monovalent ligand including free nucleotides, labeled free nucleotides, protein or peptide-bonded nucleotides, or labeled protein or peptide-bonded nucleotides.

[0137] In this method, the average read length increases by 10 nucleotides (NT), 20 NT, 25 NT, 30 NT, 50 NT, 75 NT, 100 NT, 125 NT, 150 NT, 200 NT, 250 NT, 300 NT, 350 NT, 400 NT, 500 NT, or more, compared to the average read length observed using free nucleotides, labeled free nucleotides, protein or peptide-bonded nucleotides, or monovalent ligands containing labeled protein or peptide-bonded nucleotides.

[0138] In some cases, the disclosed compositions and methods may have average read lengths ranging from 100 to 1,000 nucleotides for sequencing applications. In some examples, the average read length may be 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, 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 examples, the average read length may be within a range limited by any two values ​​within this range, for example, between 375 and 825 nucleotides. Those skilled in the art will recognize that in some examples, the average read length may be any value within the range specified in this paragraph, for example, 523 nucleotides.

[0139] Using a polyvalent conjugated composition for sequencing effectively reduces sequencing time. The sequencing reaction cycle, including the contact, detection, and incorporation steps, is carried out for a total time ranging from about 5 minutes to about 60 minutes. In some examples, the sequencing reaction cycle is carried out for 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 examples, the sequencing reaction cycle is carried out for a maximum of 60 minutes, a maximum of 50 minutes, a maximum of 40 minutes, a maximum of 30 minutes, a maximum of 20 minutes, a maximum of 10 minutes, or a maximum of 5 minutes. Any combination of the lower and upper limits described in this paragraph may form the range included in this disclosure, for example, in some examples, the sequencing reaction cycle is carried out for a total time ranging from about 10 minutes to about 30 minutes. Those skilled in the art will recognize that the sequencing cycle time may be any value within this range, for example, about 16 minutes.

[0140] In some examples, the disclosed nucleic acid sequencing compositions and methods will provide an average base call accuracy of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% accurate over the course of the sequencing run. In some examples, the disclosed nucleic acid sequencing compositions and methods will provide an average base call accuracy of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% accurate for every 1,000 bases, 10,000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases being called.

[0141] Using a polyvalent binding composition for sequencing provides more accurate base readout. The disclosed compositions and methods for nucleic acid sequencing will provide an average Q score in the range of about 20 to about 50 with accuracy of base calling over sequencing runs. In some examples, 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 this range, for example, about 32.

[0142] In some examples, the disclosed nucleic acid sequencing compositions and methods provide a Q score greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some examples, the disclosed nucleic acid sequencing compositions and methods provide a Q score greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some examples, the disclosed nucleic acid sequencing compositions and methods provide a Q score greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some examples, the disclosed nucleic acid sequencing compositions and methods provide a Q score greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides. In some examples, the disclosed nucleic acid sequencing compositions and methods provide a Q score greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or N+1) nucleotides.

[0143] The disclosed nonspecific low-binding supports and associated nucleic acid hybridization and amplification methods may be used to analyze nucleic acid molecules derived from any of the various cells, tissues, or samples known to those skilled in the art. For example, nucleic acids may be extracted from cells or tissue samples containing one or more types of cells, derived from eukaryotes (such as animals, plants, fungi, and protists), primordial bacteria, or eubacteria. In some cases, nucleic acids may be extracted from prokaryotes or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Nucleic acids are extracted in diverse ways from, for example, primitive or immortalized rodents, pigs, cats, dogs, cattle, horses, primates, or human cell lines. Nucleic acids may be extracted from any of the following cells, organs, or tissues (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells of the heart, lungs, brain, liver, kidneys, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Nucleic acids may be extracted from normal or healthy cells. Alternatively or in combination, nucleic acids may be extracted from abnormal cells such as cancer cells, or from pathogenic cells that infect the host. Some nucleic acids may be extracted from a distinct subset of cell types, such as immune cells (including T cells, cytotoxic (killer) T cells, helper T cells, αβT cells, γδT cells, T cell progenitor cells, B cells, B cell progenitor cells, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophilic cells, mast cells, monocytes, dendritic cells, and / or macrophages or any combination thereof), undifferentiated human stem cells, differentiated human stem cells, and rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, myeloid cells, progenitor cells, foam cells, mesenchymal cells, or trophoblast cells). The nucleic acids may further include nucleic acids derived from viral samples and subviral pathogens such as viroids and infectious RNA. Nucleic acids may originate from clinical or other samples such as saliva, saliva, ocular fluid, synovial fluid, blood, feces, urine, tissue exudate, sweat, pus, and drainage fluids.Nucleic acids may also be derived from plant or fungal samples such as leaves, cambium, roots, meristematic tissue, pollen, eggs, seeds, spores, inflorescences, and mycelium. Nucleic acids may also be derived from environmental or industrial samples such as water, air, dust, and food. Other cells, tissues, and samples are contemplated and consistent with the disclosures herein.

[0144] Nucleic acid extraction from cells or other biological samples may be carried out using any of the many techniques known to those skilled in the art. For example, a DNA extraction procedure may include (i) taking a cell or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove any surfactants, proteins, salts, or other reagents used during the cell membrane lysis step.

[0145] Various suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep® kit series from Promega (Madison, WI).

[0146] V II. System System Modules: As described above, further disclosed herein are systems configured to perform either the disclosed nucleic acid sequencing or nucleic acid detection and analysis methods. In some examples, the disclosed systems may comprise one or more polyvalent binding compositions described herein, one or more buffers, and / or one or more nucleic acid molecules tethered to a solid support.

[0147] In some examples, the system may further include a fluid flow controller and / or fluid distribution system configured to continuously and repeatedly contact a template nucleic acid molecule hybridized to a nucleic acid molecule (e.g., an adapter or primer) tethered to a solid support with a disclosed polyvalent binding composition and / or reagent. In some examples, the contact may be performed within one or more flow cells. In some examples, the flow cells may be fixed components of the system. In some examples, the flow cells may be removable and / or disposable components of the system.

[0148] In some examples, the system may further include an imaging module, which includes, for example, one or more light sources, one or more optical components (e.g., lenses, mirrors, prisms, optical filters, tinted glass filters, narrowband interference filters, broadband interference filters, dichroic reflectors, diffraction gratings, apertures, optical fibers, or optical waveguides) and one or more image sensors (e.g., charge-coupled device (CCD) sensors or cameras, complementary metal-oxide-semiconductor (CMOS) image sensors or cameras, or negative channel metal-oxide-semiconductor (NMOS) image sensors or cameras) for imaging and detection of the binding of the disclosed polyvalent binding composition to target (or template) nucleic acid molecules anchored inside a solid support or flow cell.

[0149] Processor and Computer System: One or more processors may be used to implement the systems for nucleic acid sequencing or other nucleic acid detection and analysis methods disclosed herein. One or more processors may include hardware processors such as a central processing unit (CPU), graphics processing unit (GPU), general-purpose processing unit, or computing platform. One or more processors may consist of any of the following: a variety of suitable integrated circuits (e.g., application-specific integrated circuits (ASICs) specifically designed to implement deep learning network architectures, or field-programmable gate arrays (FPGAs) to accelerate computation time and / or to facilitate deployment), microprocessors, emerging next-generation microprocessor designs (e.g., memristor-based processors), or logic devices. Although this disclosure is described with reference to processors, other types of integrated circuits and logic devices may also be applicable. The processor may have any suitable data manipulation capabilities. For example, the processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data manipulations. One or more processors may be single-core or multi-core processors, or multiple processors configured for parallel processing.

[0150] One or more processors or computers used to implement the disclosed methods may be part of a larger computer system and / or may be operably coupled to a computer network ("Network") with the assistance of a communication interface to facilitate the transmission and sharing of data. The Network may be a local area network, an intranet and / or extranet, an intranet and / or extranet that is in communication with the Internet, or the Internet. In some cases, the Network may be a telecommunications and / or data network. The Network may, in some cases, include one or more computer servers that enable distributed computing, such as cloud computing. The Network may, in some cases with the assistance of a computer system, implement a peer-to-peer network, which may allow devices coupled to a computer system to act as clients or servers.

[0151] A computer system may also include memory or storage locations (e.g., random access memory, read-only memory, flash memory, Intel® Optane® technology), electronic storage devices (e.g., hard disks), communication interfaces for communicating with one or more other systems (e.g., network adapters), and peripherals, such as caches, other memory, data storage devices, and / or electronic display adapters. The memory, storage devices, interfaces, and peripherals may communicate with one or more processors, such as CPUs, via a communication bus, as seen on a motherboard, for example. The storage devices may be data storage devices (or data repositories) for storing data.

[0152] One or more processors, for example, a CPU, execute a set of machine-readable instructions embedded in a program (or software). These instructions are stored in memory. The instructions target the CPU, which then programs or otherwise configures itself to implement the methods of this disclosure. Examples of operations performed by the CPU include fetching, decoding, executing, and writing back. The CPU may be part of a circuit, such as an integrated circuit. One or more other components of a system may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0153] Storage devices store files such as drivers, libraries, and saved programs. Storage devices also store user data, such as user-specific preferences and user-specific programs. A computer system may include one or more additional data storage devices located outside the computer system, such as those situated on remote servers communicating with the computer system via an intranet or the internet.

[0154] Some aspects of the methods and systems described herein may be executed by machine-executable code stored in an electronic storage location of a computer system, such as memory or an electronic storage device. The machine-executable or machine-readable code may be provided in the form of software. During use, the code is executed by one or more processors. In some embodiments, the code is retrieved from storage devices and stored in memory for immediate use by one or more processors. In some cases, electronic storage devices are excluded, and machine-readable instructions are stored in memory. The code may be pre-compiled and configured for use in a machine with one or more processors adapted to execute the code, or it may be compiled at runtime. The code may be supplied in a programming language that can be selected to enable the code to be executed in a pre-compiled or as-compiled format.

[0155] Various aspects of the technology can often be considered as “products” or “manufactured goods,” such as “computer programs or software products,” in the form of machine (or processor) executable code and / or related data stored on some kind of machine-readable medium, where executable code includes a number of instructions for controlling a computer or computer system in performing one or more methods disclosed herein. Machine executable code may be stored in optical storage devices, including optically readable media such as optical discs, CD-ROMs, DVDs, or Blu-ray discs. Machine executable code may be stored in electronic storage devices such as memory (e.g., read-only memory, random-access memory, flash memory) or on hard disks. “Storage” type media can include any or all of the tangible memory of a computer or processor, or its associated modules, such as various semiconductor memory chips, optical drives, tape drives, disk drives, etc., which can provide non-temporary storage at any time for software coding the methods and algorithms disclosed herein.

[0156] All or part of the software code is communicated from time to time over the Internet or various other telecommunication networks. Such communications enable, for example, the loading of software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media used to transmit software-coded instructions include light waves, radio waves, and electromagnetic waves, such as those used in physical interfaces between local devices, over wired and optical terrestrial communication line networks, and by various atmospheric links. Physical elements involving such waves, such as wired or wireless links and optical links, are also considered media for transmitting software-coded instructions to perform the methods disclosed herein. Unless limited to non-temporary and tangible “storage” media as used herein, terms such as computer or machine-readable media refer to media involved in providing instructions to a processor for execution.

[0157] A computer system may include, for example, an electronic display for providing images captured by a machine vision system, or may be in communication with such a display. Often, the display may provide a user interface (UI). Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0158] System control software: In some examples, the disclosed system may include a computer (or processor) and a computer-readable medium containing code for providing manual, semi-automatic, or fully automatic control of all system functions, such as a fluid flow controller and / or fluid distribution system (or subsystem), a temperature control system (or subsystem), an imaging system (or subsystem), etc., to a user interface. In some examples, the system computer or processor may be an integrated component of the instrument system (e.g., a microprocessor or motherboard embedded in the instrument). In some examples, 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 the instrument control software include, but are not limited to, volumetric fluid flow rate, fluid velocity, timing and duration of sample and reagent addition, and rinsing steps. Examples of temperature control functions that may be provided by the instrument control software include, but are not limited to, specifying temperature setpoints and controlling the timing, duration, and ramp rate of temperature changes. Examples of imaging system control functions that may be provided by the instrument control software include, but are not limited to, autofocus functions, control of exposure time and intensity of illumination or excitation light, control of image acquisition speed, exposure time, and data storage options.

[0159] Image Processing Software: In some examples of the disclosed systems, the system may further include a computer-readable medium containing code for providing image processing and analysis functions. Examples of image processing and analysis functions that may be provided by the software include, but are not limited to, manual, semi-automatic, or fully automatic image exposure adjustment (e.g., white balance, contrast adjustment, signal averaging, and other noise reduction functions), manual, semi-automatic, or fully automatic edge detection and object identification (e.g., for identifying clusters of amplified template nucleic acid molecules on a substrate surface), manual, semi-automatic, or fully automatic signal intensity measurement and / or thresholding in one or more detection channels (e.g., one or more fluorescence emission channels), and manual, semi-automatic, or fully automatic statistical analysis (e.g., for comparing signal intensity to a reference value for base calling purposes).

[0160] In some examples, system software may provide integrated real-time image analysis and instrument control, as a result the sample loading, reagent addition, rinsing, and / or imaging / base calling steps may be extended, modified, or repeated as needed until, for example, the optimal base calling result is obtained. Real-time or post-processed image analysis functions may be implemented using any of the various image processing and analysis algorithms known to those skilled in the art. Examples include, but are not limited to, Canny edge detection methods, Canny-Deriche edge detection methods, first-order gradient edge detection methods (such as the Sobel operator), second-order derivative edge detection methods, phase-coherence edge detection methods, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods), feature and pattern recognition algorithms (e.g., generalized Hough transform, circular Hough transform for detecting arbitrary shapes), and mathematical analysis algorithms (e.g., Fourier transform, fast Fourier transform, wavelet analysis, autocorrelation), or combinations thereof.

[0161] In some examples, system control and image processing / analysis software may be written as separate software modules. In other examples, system control and image processing / analysis software may be incorporated into an integrated software package. [Examples]

[0162] VIII. Examples 1. Preparation of polyvalent binding compositions As shown in Figure 5A, one type of multi-arm substrate was prepared by reacting propargylamine dNTP with biotin-PEG-NHS. This aqueous reaction was completed and purified to obtain a pure biotin-PEG-dNTP species. In another reaction, several different PEG lengths were used, corresponding to average molecular weights ranging from 1 kDa to 20 kDa. The biotin-PEG-dNTP species were mixed with either freshly prepared or commercially available dye-labeled streptavidin (SA) using a Dye:SA ratio of 3–5:1. The mixing of biotin-PEG-dNTP with dye-labeled streptavidin was carried out in the presence of excess biotin-PEG-dNTP to ensure saturation of the biotin-binding site on each streptavidin tetramer. The complete complex was purified from excess biotin-PEG-dNTP by size exclusion chromatography. Each nucleotide species was individually transformed and purified, and then mixed together to prepare a 4-base mixture for sequencing.

[0163] Another species of multi-arm substrate, as shown in Figure 5A, was prepared in a single pot by reacting multi-arm PEG NHS with excess Dye-NH2 and propargylamine dNTPs. Various multi-arm PEG NHS variants were used, ranging from 4 to 16 arms and molecular weights from 5 kDa to 40 kDa. After the reaction, excess small molecule dyes and dNTPs were removed by size exclusion chromatography. Each nucleotide species was independently conjugated and purified, and then mixed together to prepare a 4-base mixture for sequencing.

[0164] Class II substrates, as shown in Figure 5B, were prepared using a single pot reaction to simultaneously conjugate the dye and dNTPs. Alkyne-PEG-NHS was reacted with excess propargylamine dNTPs. The product (alkyne-PEGdNTPs) was then purified and homogenized by chromatography. Multiple PEG lengths were used, with average molecular weights varying between 1 KDa and 20 KDa. Dendrimer cores containing a variable number of separated azide-conjugated sites (12, 24, 48, 96) were used. Conjugation of alkyne dyes and alkyne-PEG-dNTPs to the dendrimer cores occurred in a single pot reaction containing excess dye and dNTP species via copper-mediated click chemistry. After the reaction, excess small molecule dyes and dNTPs were removed by size exclusion chromatography. Each nucleotide species was conjugated and purified independently and then mixed together to create a tetranucleotide mixture for sequencing. Note that this scheme allows for immediate substitution of alternative cores such as dextran, other polymers, and proteins.

[0165] Class III polymer-nucleotide conjugates, as shown in Figure 5C, were constructed by reacting 4- or 8-arm PEG NHS with a saturated mixture of biotin and propargylamine dNTPs. This reaction was then purified by size exclusion chromatography. The result was a multi-arm PEG containing a discrete distribution of biotin and nucleotides. This heterogeneous population was then reacted with dye-labeled streptavidin and purified by size exclusion chromatography. Each nucleotide species was conjugated and purified independently, and then mixed together to create a tetranucleotide mixture for sequencing. Note that the biotin distribution is adjustable by the input ratio of biotin-NH2 to propargylamine dNTPs.

[0166] 2. Detection of the ternary complex The binding reaction using a polyvalent binding composition containing a PEG polymer-nucleotide conjugate was analyzed to detect the possible formation of a ternary complex, and fluorescence images of the various steps are illustrated in Figures 7A to 7J. In Figure 7A, 20 nM Klenow polymerase and 2.5 mM Sr +2 The images show red and green fluorescence images after exposure of the templates (first bases of G and A) for DNA rolling circle application (RCA) to labeled nucleotides (A-Cy3 and G-Cy5) with 500 nM bases in an exposure buffer containing the following: The polyvalent PEG substrate compositions were prepared using varying ratios of 4-arm PEGamine (4ArmPEG-NH), biotin-PEGamine (biotin-PEG-NH), and nucleotide (Nuc), as follows: Samples 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. The images were collected after washing with an imaging buffer of the same composition as the exposure buffer but without nucleotides or polymerase.

[0167] The contrast was scaled to maximize visualization of the darkest signals, but the signals did not persist after washing with imaging buffer (inset in Figure 7). In Figures 7B–7E, the fluorescence images show polyvalent PEG nucleotide (base-labeled) ligands at at least 500 nM after mixing in exposure buffer and imaging in imaging buffer, as described above (Figure 7B: PB1, Figure 7C: PB2, Figure 7D: PB3, Figure 7E: PB5). Figure 7F: The fluorescence image shows polyvalent PEG nucleotide (base-labeled) ligand PB5 at 2.5 μM after mixing in exposure buffer and imaging in imaging buffer. In Figures 7G–7I, the fluorescence images further show base discrimination upon exposure of inactive variants of Krenow polymerase with polyvalent ligands (Figure 7G: D882H, Figure 7H: D882E, Figure 7I: D882A, and wild-type Krenow (control) enzyme is shown in Figure 7J).

[0168] Using polyvalent ligand formulations, base discrimination may be possible by providing polymerase ligand interactions with increased binding affinity. Furthermore, it has been shown that increased concentrations of polyvalent ligands produce higher signals, and that various Krenow mutations that knock out catalytic activity can be used for binding affinity-based sequencing.

[0169] 3. Sequence determination of target nucleic acid molecules using a ternary complex To demonstrate sequencing based on a polyvalent ligand reporter, four known templates were amplified using the RCA method on a low-binding substrate. Continuous cycles were performed using 20 nM Klenow polymerase and 2.5 mM Sr +2 The substrate was exposed to an exposure buffer containing the ligand, washed with imaging buffer, and imaged. After imaging, the substrate was washed with a wash buffer (EDTA and high salt), and blocked nucleotides were added to advance to the next base. The cycle was repeated for 5 cycles. Spots were detected using standard imaging processing and spot detection, and the sequences were called using a two-color scheme of red and green (G-Cy3 and A-Cy5) to identify the template being cycled. As shown in Figures 8A and 8B, the polyvalent ligand can provide base discrimination throughout all 5 sequencing cycles.

[0170] 4. Control of nucleotide dissociation from the ternary complex The ternary complex is prepared and imaged as in Example 2. The complex is imaged over various time periods, e.g., about 60 seconds, to demonstrate the persistence of the ternary complex. After time has elapsed, the complex is washed with the same buffer used for its formation, which lacks any divalent cation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, and 0.016% TritonX100 (without SrOAc), or the complex is washed with the same buffer used for its formation, which contains a chelating agent but lacks any divalent cation, e.g., 100 nm - 100 mM EDTA, 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, and 0.016% TritonX100 (without SrOAc). The fluorescence of the complex is observed over time, allowing for the observation and quantification of the dissociation of the ternary complex. A typical time course for this dissolution is shown in Figure 6.

[0171] 5. Extension of the complementary sequence of the target nucleic acid As in Example 4, after preparing, imaging, and dissociating the ternary complex, a deblocking solution is poured from the 3' end of the DNA strand to be extended into a chamber containing bound DNA molecules, such as an O-azidomethyl group, an O-alkylhydroxylamino group, or an O-amino group, in sufficient quantities to remove the blocking portion. Subsequently or simultaneously, an extension solution is poured into the chamber containing the bound DNA molecules. The extension solution contains a buffer, a divalent cation sufficient to support polymerase activity, active polymerase, and appropriate amounts of all four nucleotides, the nucleotides being blocked, such as by the incorporation of a 3'-O-azidomethyl group, a 3'-O-alkylhydroxylamino group, or an O-amino group, so that further extension cannot be supported after a single nucleotide is added to the DNA strand to be extended. The strand to be extended is thus extended by one and a few bases, and the catalytically inactive polymerase and the binding of the polyvalent binding substrate can be used to call the next base in the cycle.

[0172] Alternatively, nucleotides attached to the polyvalent substrate may adhere via unstable binding, and consequently, the buffer can flow into a chamber containing the bound DNA molecule with sufficient divalent cations or other cofactors to provide a catalytically active polymerase. Before, after, or simultaneously with this, conditions can be provided to cleave a base from the polyvalent substrate so that the polyvalent substrate can be incorporated into the elongation chain. This cleavage and incorporation results in labeling of the polyvalent substrate and dissociation of the polymer backbone by just one base while the elongating DNA chain is stretched. Washing is performed to remove the used polymer backbone, and a new polyvalent substrate flows into the chamber containing the bound DNA molecule, making it possible to call a new base as in Example 1.

[0173] 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 exposed to a single sequencing cycle and imaged as described in Example 1. As shown in Figures 9A–J, increasing the length of the PEG branch increased the signal to a length corresponding to a clear average PEG MW of 5K Da (Figures 9A–D). The use of longer PEG arms resulted in a decrease in the fluorescence signals of both Cy3-A and Cy5-G (Figures 9E–9G). Quantitative measures of signal intensity are shown graphically in Figure 10.

[0174] 7. Enhancement of the binding of polyvalent substrates by the addition of surfactants. Polyvalent substrates were prepared and assembled into binding complexes in and without the presence of a surfactant. One set used 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, and 0% TritonX100 (Condition A), while the other set used 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, and 0.016% TritonX100. Figure 11 shows the normalized fluorescence of these polyvalent substrates bound to DNA clusters, clearly demonstrating enhanced fluorescence intensity in the substrate complex formed in the presence of Triton-X100 (0.016%) (Condition B).

[0175] 8. Evaluation of the time course of multivalent substrate bonding A multivalent substrate was prepared as in Example 2 and assembled into a binding complex. A complex was also formed using free labeled nucleotides under the same buffer conditions. The complex was imaged over a 60-minute period to characterize its duration. Figures 12A and 12B show representative results. The multivalent binding complex remained stable over timescales greater than 60 minutes (Figure 12B), while the labeled free nucleotides dissociated in less than 1 minute (Figure 12A).

[0176] VIII. Conclusion This disclosure provides greatly improved methods and compositions for DNA sequencing and biosensor applications. It should be understood that the above features are illustrative and not limiting. Many embodiments will be obvious to those skilled in the art in considering the above description. For example, while the concept of the present invention is described primarily in relation to the use of polymer-nucleotide conjugates, it will be readily apparent to those skilled in the art that other types of particle-nucleotide conjugates can be used. For example, in some embodiments, it may be desirable to use particle nucleotide conjugates comprising quantum dots, liposomes, or emulsion particles. Alternatively, conjugation may be achieved by non-covalent bonds such as hydrogen bonds or other interactions. Thus, the scope of the concept of the present invention should not be determined in relation to the above description, but rather in relation to such claims, along with a full range of equivalents to which appended claims are given.

Claims

1. A system for sequencing, detecting and / or analyzing a target nucleic acid sequence, wherein the system is: A polymer-nucleotide conjugate composition comprising a polymer core, multiple nucleotide moieties, one or more detectable labels, and polymerase, A solid support having a surface to which a plurality of primed target nucleic acid sequences are attached, wherein the plurality of primed target nucleic acid sequences include sequencing primers that hybridize to complementary primer-binding sequences or embedded sequences to form the plurality of primed target nucleic acid sequences, the sequencing primers include nucleotides that are blocked so as not to support further extension, and the polymer-nucleotide conjugate composition is conjugated to at least two of the plurality of primed target nucleic acid sequences by two or more nucleotide moieties of the plurality of nucleotide moieties, the solid support, A system that includes this.

2. The aforementioned system, Fluid flow controller, Fluid distribution system, or The combination of the fluid flow controller and the fluid distribution system further includes, Here, the fluid flow controller or the fluid distribution system is configured to continuously and repeatedly guide the polymer-nucleotide conjugate composition to the plurality of primed target nucleic acid sequences. The system according to claim 1.

3. The system according to claim 1, wherein the solid support is a flow cell.

4. The system according to claim 3, wherein the surface is one or more internal surfaces of the flow cell.

5. The system according to claim 4, wherein the at least two primed target nucleic acid sequences hybridize to at least a portion of the at least two primer nucleic acid sequences anchored to the solid support.

6. The aforementioned system, An imaging module operably coupled to the flow cell, One or more computer processors programmed to execute a series of machine-readable instructions, wherein the series of machine-readable instructions are (a) A step of bringing the polymer-nucleotide conjugate composition into contact with the surface under conditions sufficient to form a polyvalent binding complex between the polymer-nucleotide conjugate and two or more copies of the primed target nucleic acid sequence. (b) A step of imaging the surface with the imaging module in order to detect the multivalent binding complex, and (c) One or more computer processors comprising the step of determining at least two nucleotides of the at least two primed target nucleic acid sequences, The system according to claim 4, further comprising:

7. The imaging module, which is operably coupled to the flow cell, Light source and Optical components and Image sensor and, The system according to claim 6, including the system described in claim 6.

8. The system according to claim 1, wherein the one or more detectable labels include fluorophores.

9. The system according to claim 1, wherein the primed target nucleic acid sequences of the plurality of primed target nucleic acid sequences each include a blocked nucleotide at its 3' end, wherein the blocked nucleotide includes a blocking group.

10. The system according to claim 9, wherein the blocking group comprises a 3'-O-azidomethyl nucleotide, a 3'-O-methyl nucleotide, a 3'-O-alkylhydroxylamino group, a 3'-phosphorothioate group, a 3'-O-malonyl group, or a 3'-O-benzyl group.

11. The system according to claim 1, wherein the plurality of primed target nucleic acid sequences include concatenation or fusion of a plurality of copies of a query sequence.

12. The system according to claim 1, further comprising a composition of a second polymer-nucleotide conjugate comprising a second polymer core and a second plurality of nucleotide moieties attached thereto, wherein the second plurality of nucleotide moieties comprises nucleic acid bases of a different type from the nucleic acid bases of the plurality of nucleotide moieties.

13. The system according to claim 1, wherein the plurality of nucleotide portions are covalently bonded to the polymer core.

14. The system according to claim 1, wherein the polymer core comprises a branched polymer, a dendrimer, a cross-type polymer, or a combination thereof.

15. The system according to claim 1, wherein the polymer-nucleotide conjugate composition further comprises a linker that binds the plurality of nucleotide portions to the polymer core, wherein the linker has a molecular weight of about 5,000 daltons (Da) to about 20,000 daltons (Da).

16. The system according to claim 1, wherein the polymer-nucleotide conjugate composition further comprises one or more binding groups selected from the group including avidin, biotin, affinity tags, and combinations thereof.

17. The polymer-nucleotide conjugate composition, bound to at least two primed target nucleic acid sequences, forms a polyvalent binding complex. The system according to claim 1, wherein the polyvalent binding complex further comprises two or more polymerase molecules such that the two or more nucleotide moieties are not incorporated into the at least two primed target nucleic acid sequences from the polymer-nucleotide conjugate composition.

18. The system according to claim 1, wherein the polymer-nucleotide conjugate composition bound to at least two primed target nucleic acid sequences forms a polyvalent binding complex having a duration greater than 2 seconds.

19. The polymer-nucleotide conjugate composition, bound to at least two primed target nucleic acid sequences, forms a polyvalent binding complex. The system according to claim 1, wherein the fluorescence image of the polyvalent binding complex on the surface has a contrast-to-noise ratio of more than 20.

Images