Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes

Abstract

Methods, compositions, kits and devices comprising a fluid comprising a ternary complex and Li + , wherein the ternary complex comprises a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid molecule. + As a substitute or supplement for the above, the fluid may contain betaine or metal ions that inhibit polymerase catalysis, such as Ca 2+ In addition to Li + In addition, the fluid may also contain polyethyleneimine (PEI) with or without betaine.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based upon and claims the benefit of U.S. Provisional Application No. 62 / 662,888, filed on April 26, 2018, which is incorporated herein by reference in its entirety. Background of the Invention

[0004] The present disclosure generally relates to the capture, detection and sequencing of nucleic acids. More specifically, the present disclosure relates to, for example, Sequencing By Binding TM (SBB TM ) method, each ternary complex includes a triggering template nucleic acid, a polymerase and a cognate nucleotide.

[0005] SBB TM The technology utilizes the transient binding of a polymerase and a homologous nucleotide to a primed template nucleic acid as a means of identifying the template base at the end of the primer. A series of steps of extending the primer and detecting the next template base allows the sequence of the template to be determined. For example, exemplary SBBs are disclosed in co-owned U.S. Patent Application Publications 2017 / 0022553 A1 and 2018 / 0044727 A1 and U.S. Patent Application Serial Nos. 15 / 873,343 (published as US 2018 / 0208983 A1) and 15 / 851,383 (published as US 2018 / 0187245 A1). TM Technology, each of which is incorporated herein by reference.

[0006] SBB TM The technical difficulty is that the ternary complex is an equilibrium binding product. The equilibrium binding product coexists with the unbound binding partner in the solution. Removing the unbound binding partner from the equilibrium reaction will cause the binding product to dissociate. TM When labeled nucleotides are used in the method, the unbound labeled nucleotides provide the desired function of maintaining equilibrium, which in turn maintains the ternary complex. However, the unbound labeled nucleotides undesirably generate background signals that obscure the detection of the ternary complex. Similar difficulties arise when labeled polymerases are used instead of labeled nucleotides.

[0007] There is a need for methods of maintaining detectable levels of ternary complexes while reducing the concentration of labeled background components. The present invention satisfies this need and also provides related advantages. Summary of the invention

[0008] The present disclosure provides methods, compositions, kits and devices including a fluid comprising a ternary complex and Li +, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid molecule. + Alternatively or in addition, the fluid may contain betaine and / or metal ions such as Ca that inhibit polymerase catalysis. 2+ In addition to Li + In addition, the fluid may also contain polyethyleneimine (PEI) with or without betaine.

[0009] Also provided is a method for detecting a primed template nucleic acid, comprising the following steps: (a) providing a ternary complex and Li + wherein the ternary complex comprises a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid molecule; and (b) when the ternary complex is in a fluid containing Li + Optionally, the method further comprises step (c): identifying the next correct base of the triggered template nucleic acid molecule according to the result of step (d). + As a substitute or supplement, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain PEI with or without betaine.

[0010] The method for detecting a primed template nucleic acid may include the following steps: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of the same type of polymerase present in the ternary complex and an excess of the same type of nucleotide present in the ternary complex; (b) using a mixture containing Li + The fluid replaces the excess polymerase and excess nucleotides; and (c) when the ternary complex is reacted with a liquid containing Li + Optionally, the method further comprises step (d): identifying the next correct base of the triggered template nucleic acid molecule according to the result of step (c). + As a substitute or supplement, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain PEI with or without betaine.

[0011] In some embodiments, the method of the present disclosure may include the step of extending the primer. For example, the method of detecting the primed template nucleic acid may include the following steps: (a) providing a ternary complex and Li +wherein the ternary complex comprises a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid molecule; (b) when the ternary complex is in a fluid containing Li + (c) identifying the next correct base of the primed template nucleic acid molecule according to the result of step (b); and (d) extending the primer of the primed template nucleic acid. Optionally, the method may further include step (e): repeating steps (a) to (d) using the primed template nucleic acid with the extended primer instead of the primed template nucleic acid. + As a substitute or supplement, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain PEI with or without betaine.

[0012] In another example of a method comprising a primer extension step, the steps of the method may include: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of the same type of polymerase present in the ternary complex and an excess of the same type of nucleotide present in the ternary complex; (b) + The fluid replaces the excess polymerase and excess nucleotides; (c) when the ternary complex is mixed with a liquid containing Li + (d) identifying the next correct base of the primed template nucleic acid molecule according to the result of step (c); and (e) extending the primer of the primed template nucleic acid. Optionally, the method may further include step (f): repeating steps (a) to (e) using the primed template nucleic acid with the extended primer instead of the primed template nucleic acid. + As a substitute or supplement, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain PEI with or without betaine.

[0013] In yet another example of a method comprising a primer extension step, the steps of the method may include: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of nucleotides of the same type present in the ternary complex; (b) + (c) When the ternary complex is mixed with a liquid containing Li +(d) identifying the next correct base of the primed template nucleic acid molecule according to the result of step (c); and (e) extending the primer of the primed template nucleic acid. Optionally, the method may further include step (f): repeating steps (a) to (e) using the primed template nucleic acid with the extended primer instead of the primed template nucleic acid. + Alternatively or in addition, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain PEI with or without betaine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A Graphs showing the "on" and "off" signal strengths for various examination conditions.

[0015] Figure 1B A graph showing purity under various examination conditions.

[0016] Figure 2 Graphs showing the "on" and "off" signal strengths for various examination conditions.

[0017] Figure 3 A graph showing purity under different detection conditions is shown.

[0018] Figure 4A Shown is a graph of the "on" and "off" signal intensities for four different base types over 20 sequencing cycles performed in the absence of LiCl.

[0019] Figure 4B Shown is a graph of the "on" and "off" signal intensities for four different base types over 20 sequencing cycles performed in the presence of 5 mM LiCl.

[0020] Figure 4C Shown are graphs of the "on" and "off" signal intensities for four different base types over 20 sequencing cycles performed in the presence of 50 mM LiCl.

[0021] Figure 5A Shown is a graph of the purity of four different base types over 20 sequencing cycles performed in the absence of LiCl.

[0022] Figure 5B Shown is a graph of the purity of four different base types over 20 sequencing cycles performed in the presence of 5 mM LiCl.

[0023] Figure 5CShown is a graph of the purity of four different base types over 20 sequencing cycles performed in the presence of 50 mM LiCl.

[0024] Fig. 6A Shown is a graph of the signal-to-noise ratio over 20 sequencing cycles performed in the absence of LiCl.

[0025] Figure 6B Shown is a graph of the signal-to-noise ratio over 20 sequencing cycles performed in the presence of 5 mM LiCl.

[0026] Figure 6C Shown is a graph of the signal-to-noise ratio over 20 sequencing cycles performed in the presence of 50 mM LiCl.

[0027] Fig. 7A Shown is a graph of the average error rate over 20 sequencing cycles performed in the absence of LiCl.

[0028] Figure 7B Shown is a graph of the average error rate for 20 sequencing cycles performed in the presence of 5 mM LiCl.

[0029] Figure 7C Shown is a graph of the average error rate for 20 sequencing cycles performed in the presence of 50 mM LiCl.

[0030] Figure 8 Shown is the percent loss of signal intensity after 60 seconds for the ternary complex in the presence of different combinations of lithium, calcium or betaine.

[0031] Fig. 9 Shown is a graph of purity values ​​for sequencing cycles run in the presence of different combinations of lithium, calcium or betaine.

[0032] Fig.10 Shown are the results of stability testing of the ternary complex in the presence and absence of PEI.

[0033] Fig.11 Shown are the results of stability testing of the ternary complex in the presence of different concentrations of PEI. DETAILED DESCRIPTION

[0034] Described herein is a method for allowing rapid and efficient identification of homologous nucleotides in members of a population of primed template nucleic acid molecules. As described below, this can be achieved in different ways.

[0035] Typically, a preformed ternary complex with a detectable marker (e.g., a labeled nucleotide) can be separated from a non-complexed polymerase and / or nucleotide (e.g., a non-complexed labeled nucleotide or a non-complexed labeled polymerase) and then imaged in the presence of a stabilizing solution for nucleotide identification. Stabilizing solutions advantageously allow ternary complex detection over an extended period of time. It should be understood that there are many ways to form a ternary complex, and the manner in which the ternary complex is formed does not generally affect the effectiveness of the stabilization methods described herein.

[0036] Embodiments of the methods described herein can be used to form a reversible complex (e.g., a ternary complex) by contacting a polymerase, nucleotides, and a primed template nucleic acid under conditions that allow the three components to form a complex while excluding extension at the 3' end of the primer. Extension can be excluded by a variety of means described herein, including but not limited to the presence of a terminator portion on the primer, the presence of a polymerase inhibitor such as an inhibitory metal ion, the absence of a catalytic metal ion, and / or the use of a polymerase variant in which primer extension activity is inhibited (e.g., due to a mutation in the polymerase catalytic domain). If desired, the primed template nucleic acid can be fixed to a solid support. The extent of ternary complex formation reflects the equilibrium binding conditions caused by the presence of different binding components (i.e., polymerase and homologous nucleotides) at their relevant concentrations. Although the net effect of equilibrium is that the formed complex appears to be stable in this binding step, a single complex is actually in a flow state. In fact, the components of the complex can be in a state where they continue to associate and dissociate with the closed primed template nucleic acid molecule at equilibrium, but there is no net change in the concentration of the free component and the bound component.

[0037] Optionally, once the ternary complex is formed, it can be contacted with a stabilizing solution and then detected. Stabilizing solutions can be used to change the chemical environment containing the ternary complex. This means that the ternary complex formed under one condition can be detected under different conditions. Although it is not necessarily desired to be limited by the proposed mechanism, the stabilizing solution can slow down the dissociation of the ternary complex, thereby allowing the detection of the ternary complex within an extended time frame, the dissociation of the ternary complex would originally occur in the absence of one or more binding components (e.g., nucleotides and / or polymerase). Stabilizing solutions can have other effects that improve the ability to detect or manipulate the ternary complex. For example, some stabilizing solutions can inhibit the formation of binary complexes between polymerase and the triggered template nucleic acid (i.e., there is no homologous nucleotide) and / or otherwise increase the ratio of the ternary complex to the binary complex.

[0038] As disclosed herein, the ternary complex, which includes the primed template nucleic acid, the cognate nucleotide (optionally including a detectable label), and the polymerase (optionally including a detectable label), can be detected during or after a wash step that separates the ternary complex from the non-complexed polymerase and non-complexed nucleotides. The wash step can be an imaging wash step using an aqueous stabilizing fluid that includes a stabilizer that retains the complex (e.g., relative to a fluid lacking the stabilizer). Exemplary stabilizers include lithium (Li + ), betaine and / or inhibitory metal ions such as Ca 2+ Particularly useful stabilizers include Li+ in combination with polyethyleneimine (PEI) and optionally further in combination with betaine. It should be understood that Li + , betaine and other metal ions (such as Ca 2+ ) need not be used as a stabilizer to be useful in the methods or compositions described herein. Thus, stabilization of the ternary complex is an optional use of these and other agents described herein.

[0039] The presence of a stabilizer can allow the removal of excessive labeled components (e.g., labeled nucleotides or labeled polymerases) from the reaction vessel while maintaining the ternary complex in the container for detection or other purposes. Therefore, the ternary complex fixed in a container such as a flow cell can be washed with a stabilizing solution and detected when in contact with the stabilizing solution. An advantage of using a stabilizing solution for washing is that the ternary complex can be substantially detected in the absence of excessive non-complex binding components (e.g., labeled nucleotides and / or labeled polymerases), which will undesirably increase the background signal. Therefore, detecting the ternary complex during a stable washing step can involve detecting the complex under varying conditions (e.g., conditions different from the equilibrium caused by the formation of the ternary complex). During the washing step, the net forward reaction that previously caused the formation of the ternary complex no longer occurs or is at least significantly slowed down.

[0040] By passing a stabilizing solution without nucleotides and polymerase through the flow cell to wash the ternary complex, uncomplexed labeled nucleotides and polymerase can be removed and non-specific background signals (e.g., fluorescent background) can be reduced while maintaining the detectability of pre-formed or existing ternary complexes. For example, after contacting with the stabilizing solution for at least 30 seconds, 1 minute, 5 minutes, 10 minutes, or longer, the ternary complex contained in the flow cell can be detected. Alternatively or additionally, it may be desirable to detect a stable ternary complex after contacting with the stabilizing solution for at most 10 minutes, 5 minutes, 1 minute, 30 seconds, or less.

[0041] The compositions and methods used in several embodiments described herein utilize the binding specificity of a ternary complex, which includes a polymerase, a template nucleic acid and a homologous nucleotide for initiation. This specificity can be used to identify the next correct nucleotide of the template nucleic acid for initiation by identifying the nucleotide present in the ternary complex. By this method, the primer can be optionally blocked at its 3'-terminal extension under equilibrium binding conditions in the same reaction mixture, and then the formation of the ternary complex is detected, while excluding the formation of phosphodiester bonds, without the need for intervening reagents (intervening reagents) exchange or washing steps. Alternatively, when excessive nucleotides and polymerases have been removed from the system, the formation of the ternary complex can be detected in the imaging washing step. During the formation and detection of the ternary complex, the optional presence of catalytic metal ions can simulate more natural ternary complex conditions, thus providing an additional benefit that is superior to the method of omitting or replacing catalytic ions. The result of aggregation is the increase in the speed of single nucleotide identification (e.g., in a genotyping program or a single sequencing cycle) and sequence identification (e.g., in the cyclic process of repeated cycles using homologous nucleotide identification and primer extension).

[0042] This disclosure illustrates and describes Sequencing By Binding TM Several aspects of ternary complex stabilization in the context of the technology. It should be understood that the compositions and methods described herein need not be limited to nucleic acid sequencing. For example, the present disclosure provides methods for interrogating a single nucleotide site in a triggered template nucleic acid. Interrogation of a single nucleotide site can be used, for example, in a genotyping method to detect variants (e.g., single nucleotide polymorphisms or SNPs) at a single site. Typically, a genotyping method is performed using a template nucleic acid with a known genetic locus, but allelic variation at the locus is to be determined. Alternatively, identification of a single nucleotide site can be used to assess the properties of a target polymerase, such as the specificity of the polymerase binding to the correct nucleotide. Methods for interrogating only a single nucleotide site in a template nucleic acid can use a single cycle of the Sequencing By Binding described herein. TM Optionally, single nucleotide positions can be interrogated using the methods or reagents disclosed herein in conjunction with the methods or reagents described in commonly owned U.S. Patent No. 9,932,631 and U.S. Provisional Application Serial No. 62 / 448,630, each of which is incorporated herein by reference.

[0043] Another exemplary application of the compositions and methods set forth herein is the capture of allele variants based on polymerase.Capture methods utilize polymerase to be available to form a stable ternary complex with the template and the next correct nucleotide of initiation.For example, a stable ternary complex can be formed between polymerase, target allele and the homologous nucleotides of the allele.Polymerase specificity allows the target allele to be separated from other nucleic acids, including, for example, other alleles that differ from the target allele in a single nucleotide.For example, a ternary complex can be formed between the template and the homologous nucleotides of the SNP allele of the initiation of polymerase, encoding target single nucleotide polymorphism (SNP) allele.Compared with the non-target SNP allele in the same locus, the ternary complex capture will cause the selective capture of the SNP allele, because when forming the ternary complex with the polymerase, the homologous nucleotides have selectivity to the target SNP. The use of stabilizers can be used to improve these capture methods and the methods set forth in U.S. Patent Application Serial No. 15 / 701,358 (now published as U.S. Patent Application Publication No. 2018 / 0208922A1), which is incorporated herein by reference.

[0044] Unless otherwise specified, the terms used herein shall be understood to adopt their ordinary meanings in the relevant art. Several terms used herein and their meanings are as follows.

[0045] As used herein, the term "array" refers to a population of molecules attached to one or more solid carriers so that a molecule at a feature can be distinguished from the molecule at other feature locations. The array may include different molecules, each of which is located at different addressable features on a solid carrier. Alternatively, the array may include a single solid carrier, and each solid carrier plays the role of carrying the characteristic of different molecules, wherein different molecules can be identified according to the position of the solid carrier on the surface to which the solid carrier is attached, or identified according to the position of the solid carrier in a liquid such as a fluid stream. The molecule of the array may be, for example, nucleotide, nucleic acid primer, nucleic acid template, primer nucleic acid template or nuclease, such as polymerase, ligase, exonuclease or a combination thereof.

[0046] As used herein, the term "betaine" means a zwitterionic molecule in a charge-separated form having an onium atom that does not contain a hydrogen atom and is not adjacent to an anionic atom. The anionic atom may be a carboxylic acid group. Ammonium betaine has a cationic functional group that includes a quaternary ammonium. A particularly useful ammonium betaine is N,N,N-trimethylglycine (TMG). Phosphonium betaine has a cationic functional group that includes a phosphonium cation.

[0047] As used herein, the term "binary complex" refers to the intermolecular association between a polymerase and a primed template nucleic acid, excluding nucleotide molecules, such as the next correct nucleotide of the primed template nucleic acid.

[0048] As used herein, the term "blocking portion", when used to refer to a nucleotide, means a portion of a nucleotide that inhibits or prevents the 3' oxygen of a nucleotide from forming a covalent bond with the next correct nucleotide during the nucleic acid polymerization process. The blocking portion of a "reversible terminator" nucleotide can be removed from a nucleotide analog, or otherwise modified to allow the 3'-oxygen of a nucleotide to be covalently linked to the next correct nucleotide. This process is referred to as "deblocking" of a nucleotide analog. Such a blocking portion is referred to herein as a "reversible terminator portion" U.S. Patent Nos. 7,427,673, 7,414,116, 7,057,026, 7,544,794, or 8,034,923; or PCT Publication WO 91 / 06678 or WO 07 / 123744 (each of which is incorporated herein by reference) describes an exemplary reversible terminator portion. The nucleotide having a blocking moiety or a reversible terminator moiety can be located at the 3' end of a nucleic acid, such as a primer, or can be a monomer that is not covalently attached to the nucleic acid.

[0049] As used herein, the term "catalytic metal ion" refers to a metal ion that promotes the formation of a phosphodiester bond between the 3'-OH of a nucleic acid (e.g., a primer) and the phosphate of an introduced nucleotide by a polymerase. A "divalent catalytic metal cation" is a catalytic metal ion with a divalence. The catalytic metal ion can be present at a concentration (as long as no phosphodiester bond formation occurs, it is referred to as a non-catalytic concentration of the metal ion) that stabilizes the formation of a complex between the polymerase, the nucleotide, and the initiated template nucleic acid. The catalytic concentration of the metal ion refers to the amount of the metal ion that is sufficient to cause the polymerase to catalyze the reaction between the 3'-OH group of a nucleic acid (e.g., a primer) and the phosphate group of an introduced nucleotide.

[0050] The term "comprising" is open-ended herein and includes not only the listed elements but also any additional elements.

[0051] As used herein, the term "cycle" or "round" when used in reference to a sequencing procedure refers to the portion of a sequencing run that is repeated to indicate the presence of a nucleotide. Typically, a cycle or round includes several steps, such as delivering reagents, washing away unreacted reagents, and detecting a signal indicating a change in response to the added reagents.

[0052] As used herein, the term "diffusion exchange," when used to refer to members of a binding complex, refers to the ability of the members to move through a fluid to associate or dissociate with each other. Diffusion exchange occurs when there are no barriers preventing the members from interacting to form a complex. However, diffusion exchange is considered to exist as long as access is not absolutely prevented, even if diffusion is delayed, reduced, or altered.

[0053] As used herein, the term "each," when used to refer to a collection of items, is intended to identify a single item in the collection, but does not necessarily refer to each item in the collection. Exceptions may occur if explicit disclosure or context clearly dictates otherwise.

[0054] As used herein, "equilibrium" refers to a state of equilibrium resulting from the equal action of opposing forces. For example, when the rate of formation of the ternary complex is balanced with its rate of dissolution, the ternary complex formed between the initiated template nucleic acid, polymerase, and homologous nucleotides is in equilibrium with the unbound polymerase and homologous nucleotides. Under such conditions, the reversible binding reaction no longer changes the net ratio of product to reactant. If the rate of the forward reaction (e.g., ternary complex formation) is balanced by the rate of the reverse reaction (e.g., ternary complex dissociation), there is no net change.

[0055] As used herein, the term "excess", when used to refer to components capable of forming a complex in a binding reaction, refers to components that are not in a bound state. Taking a reaction to form a ternary complex as an example, the free polymerase or nucleotide in the solution in the reaction vessel together with the ternary complex is an excess of polymerase and nucleotide.

[0056] As used herein, the term "exogenous", when used to refer to a portion of a molecule, means a chemical portion that is not present in the natural analog of the molecule. For example, an exogenous label for a nucleotide is a label that is not present on a naturally occurring nucleotide. Similarly, an exogenous label present on a polymerase is not present on the polymerase in its natural environment.

[0057] As used herein, the term "extension", when used in reference to a nucleic acid, means the process of adding at least one nucleotide to the 3' end of a nucleic acid. The term "polymerase extension", when used in reference to a nucleic acid, refers to a polymerase-catalyzed process of adding at least one nucleotide to the 3' end of a nucleic acid. Nucleotides or oligonucleotides added to a nucleic acid by extension are considered to be incorporated into the nucleic acid. Thus, the term "incorporation" can be used to refer to the process of attaching a nucleotide or oligonucleotide to the 3' end of a nucleic acid by forming a phosphodiester bond.

[0058] As used herein, the term "extendable", when used to refer to a nucleotide, means that the nucleotide has an oxygen or hydroxyl moiety at the 3' position and is capable of forming a covalent bond with the next correct nucleotide if and when incorporated into a nucleic acid. The extendable nucleotide may be located at the 3' position of the primer, or it may be a monomeric nucleotide. The extendable nucleotide lacks a blocking moiety, such as a reversible terminator moiety.

[0059] As used herein, the term "feature", when used to refer to an array, means a position in an array in which a specific molecule is present. A feature may contain only a single molecule, or it may contain a population of several molecules of the same kind (i.e., an ensemble of molecules). Alternatively, a feature may include a population of molecules of different kinds (e.g., a ternary complex population with different template sequences). The features of an array are typically discrete. Discrete features may be continuous, or they may be spaced apart from one another. An array useful herein may have, for example, features spaced less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron or 0.5 micron. Alternatively or additionally, an array may have features spaced greater than 0.5 microns, 1 micron, 5 microns, 10 microns, 50 microns or 100 microns. The area of ​​each feature may be less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less.

[0060] As used herein, the term "label" refers to a molecule or part thereof that provides a detectable feature. The detectable feature can be, for example, a light signal, such as absorptivity of radiation, luminescence or fluorescence emission, luminescence or fluorescence lifetime, luminescence or fluorescence polarization, etc.; Rayleigh and / or Mie scattering; binding affinity to a ligand or receptor; magnetism; electrical properties; charge; mass; radioactivity, etc. Exemplary labels include, but are not limited to, fluorophores, luminophores, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioisotopes, mass labels, charge labels, spin labels, receptors, ligands, etc.

[0061] As used herein, the term "next correct nucleotide" refers to the type of nucleotides that will be combined and / or incorporated at the 3' end of a primer to supplement the base in the template strand hybridized with the primer. The base in the template strand is referred to as the "next base", and just in the template with the 5' of the base that hybridizes with the 3' end of the primer. The next correct nucleotide may be referred to as the "homologue" of the next base, and vice versa. Homologous nucleotides that specifically interact with each other in a ternary complex or a double-stranded nucleic acid are referred to as "pairing" each other. Nucleotides with bases that are not complementary to the next template base are referred to as "incorrect", "mismatched" or "non-homologous" nucleotides.

[0062] As used herein, the term "inhibitory metal ion" refers to a metal ion that, when present, inhibits the formation of phosphodiester bonds required for chemical incorporation of nucleotides into primers. The inhibitory metal ion can interact with the polymerase, for example, by competitive binding compared to the catalytic metal ion. A "divalent inhibitory metal ion" is an inhibitory metal ion having a valence of 2. Examples of divalent inhibitory metal ions include, but are not limited to, Ca 2+ 、Zn 2+ 、Co 2+ 、Ni2+ and Sr 2+ . Trivalent Eu 3+ and Tb 3+ The ion is an inhibitory metal ion having a valency of three.

[0063] As used herein, the term "nucleotide" may be used to refer to a natural nucleotide or an analog thereof. Examples include, but are not limited to, nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs) or non-natural analogs thereof, such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).

[0064] As used herein, the term "polyethyleneimine" or "PEI" refers to a 2 CH 2 ] n Linear polyethyleneimine contains all secondary amines (i.e. [NHCH 2 CH 2 ] n ), in contrast to branched PEI containing primary, secondary and / or tertiary amino groups. The polymer may be in polycationic form. Polyethylene imine is also known in the art as poly(iminoethylene), polyaziridine or poly[imino(1,2-ethanediyl)].

[0065] As used herein, the term "polymerase" can be used to refer to nucleic acid synthases, including but not limited to DNA polymerases, RNA polymerases, reverse transcriptases, primases and transferases. Typically, a polymerase has one or more active sites on which nucleotide binding and / or nucleotide polymerization can occur. A polymerase can catalyze the polymerization of nucleotides to the 3' end of the first strand of a double-stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of the next correct nucleotide to the 3' oxygen group of the first strand of a double-stranded nucleic acid molecule through a phosphodiester bond, thereby covalently incorporating nucleotides into the first strand of a double-stranded nucleic acid molecule. Optionally, under one or more conditions used in the method set forth herein, a polymerase does not need to be able to incorporate nucleotides. For example, a mutant polymerase may be able to form a ternary complex, but cannot catalyze nucleotide incorporation.

[0066] As used herein, the term "primed template nucleic acid" refers to a nucleic acid hybrid having a double-stranded region such that one of the strands has a 3'-end that can be extended by a polymerase. The two strands may be part of a continuous nucleic acid molecule (e.g., a hairpin structure), or the two strands may be separable molecules that are not covalently linked to each other.

[0067] As used herein, the term "primer" refers to a nucleic acid having a sequence that binds to a nucleic acid at or near a template sequence. In general, a primer binds in a configuration that allows template replication (e.g., by polymerase extension of the primer). A primer can be a first portion of a nucleic acid molecule that binds to a second portion of a nucleic acid molecule, the first portion being a primer sequence, and the second portion being a primer binding sequence (e.g., a hairpin primer). Alternatively, a primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having a template sequence (e.g., a dissociable primer). A primer can be composed of DNA, RNA, or its analogs.

[0068] As used herein, a "reaction vessel" is a container that isolates one reagent or reaction (e.g., a binding reaction; an incorporation reaction; etc.) from another reagent or reaction, or provides a space in which a reaction can occur. Non-limiting examples of reaction vessels that can be used in conjunction with the disclosed technology include: flow cells, wells of multiwell plates; microscope slides; open tubes (e.g., capillaries); closed tubes (e.g., microcentrifuge tubes, test tubes, or Eppendorf Tubes TM ); etc. The features to be monitored during the binding and / or incorporation reaction can be contained in the reaction vessel.

[0069] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate can be nonporous or porous. The substrate can optionally be capable of absorbing liquids (e.g., due to porosity), but is generally sufficiently rigid so that the substrate does not substantially expand when absorbing liquids and does not substantially shrink when the liquids are removed by drying. Nonporous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic resins, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM , cycloolefin, polyimide, etc.), nylon, ceramics, resin, Silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glasses, optical fiber strands and polymers.

[0070] As used herein, the term "ternary complex" refers to the intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide. Typically, the polymerase promotes the interaction between the next correct nucleotide and the template strand of the initiated nucleic acid. The next correct nucleotide can interact with the template strand through Walson-Crick hydrogen bonding. The term "stable ternary complex" refers to a ternary complex that promotes or prolongs the existence of or has suppressed its destruction. In general, the stabilization of the ternary complex prevents the nucleotide component of the ternary complex from being covalently incorporated into the initiated nucleic acid component of the ternary complex.

[0071] As used herein, the term "type" or "kind" is used to identify molecules with the same chemical structure. For example, a mixture of nucleotides may include several dCTP molecules. The dCTP molecules will be understood to be nucleotides of the same type (or kind) as each other, but are nucleotides of different types (or kinds) compared to dATP, dGTP, dTTP, etc. Similarly, single DNA molecules with the same nucleotide sequence are DNA of the same type (or kind), while DNA molecules with different sequences are DNA of different types (or kinds). The term "type" or "kind" may also identify parts with the same chemical structure. For example, the cytosine bases in the template nucleic acid will be understood to be bases of the same type (or kind) as each other, regardless of their position in the template sequence.

[0072] The embodiments set forth below and exemplified in the claims can be understood in light of the above definitions.

[0073] The present disclosure provides a method for detecting a primed template nucleic acid. The method may include the following steps: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of the same type of polymerase present in the ternary complex and an excess of the same type of nucleotide present in the ternary complex; (b) using a mixture containing Li + The fluid replaces the excess polymerase and excess nucleotides, and (c) when the ternary complex is combined with a Li + Optionally, the method further comprises step (d): identifying the next correct base of the triggered template nucleic acid molecule according to the result of step (c). + As a substitute or supplement, the fluid may contain betaine and / or inhibitory metal ions such as Ca 2+ In addition to Li + In addition, the fluid may contain polyethyleneimine (PEI) with or without betaine.

[0074] High concentrations of detectably labeled components can be used to drive the formation of transient or reversible ternary complexes to be detected. Unfortunately, non-complexing agents that have detectable labels and are retained in the presence of a particular complex can produce signals that interfere with or mask the desired detection. This is particularly problematic when the signals generated by the detectable label are substantially similar, regardless of whether the labeled component (e.g., a polymerase or nucleotide) is free in solution or contained in a complex (e.g., a ternary complex).

[0075] In certain embodiments, the concentration of nucleotides in the binding reaction mixture substantially exceeds the concentration of the polymerase, and therefore, procedures for detecting ternary complexes using labeled nucleotides may be particularly susceptible to high backgrounds that may obscure the detection of the ternary complex. In addition, the dynamic nature of the ternary complex (e.g., as the ternary complex flows, forms, and dissociates, and exchanges components in its chemical environment) may complicate the detection of the ternary complex product, requiring conventional water washing steps to remove non-complexed reagents from the system. This is because the reversible complex to be detected may be unstable over the period of time used to examine or monitor the ternary complex.

[0076] When using components including detectable labels, there are two technical problems that can affect the detection of multi-component complexes. First, the signal derived from the unbound components of the label can unexpectedly obscure the detection of a particular complex. Second, conventional washing to remove one or more components from the system can promote the dissociation of the reversible complex to be detected. Each of these problems can be a burden when collecting sequencing data.

[0077] The importance of maintaining the stability of bound complexes while detecting the complexes can be appreciated in the context of array-based applications where multiple images are collected along the surface of the array. For example, a flow cell can include an array having a surface area that is larger than a single field of view of an optical imaging system. Thus, the optical system can collect images of different portions of the array by a scanning or stepping process. If the transient complex to be monitored is unstable, lower quality data may be collected for late images compared to early images. As described herein, this problem can be overcome by stabilizing the complex under conditions that allow the collection of data with a high signal-to-background ratio.

[0078] A particularly useful reagent for use in the methods or compositions of the present disclosure (e.g., to stabilize the ternary complex) is lithium. Like other alkali metals, lithium has a single valence electron that is easily given up to form a cation (Li + ). The lithium may be provided to the reaction in the form of a salt, such as LiCl. When contacted with the ternary complex, the concentration of lithium is at least 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM or more. Alternatively or additionally, the lithium may be present at a concentration of up to 250 mM, 100 mM, 50 mM, 25 mM, 10 mM, 5 mM or less.

[0079] Another useful agent for use in the methods or compositions of the present disclosure (e.g., for stabilizing the ternary complex) is betaine. When contacted with the ternary complex, the concentration of betaine is at least 1 mM, 10 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, 3 M, 3.5 M or more. Alternatively or additionally, betaine may be present at a concentration of up to 3.5 M, 2 M, 1 M, 500 mM, 100 mM, 50 mM, 10 mM, 1 mM or less. Betaine may be present with Li + Use in combination, or in Li + Used in the absence of to produce a stabilizing effect.

[0080] Polyethyleneimine (PEI) can be used in the methods or compositions of the present disclosure, for example, to stabilize the ternary complex. When contacted with the ternary complex, PEI can be present at a concentration of at least 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 1% or 5% (w / v). Alternatively or additionally, PEI can be present at a concentration of up to 5%, 1%, 0.1%, 0.05%, 0.01%, 0.001% or 0.0001% (w / v). PEI can be present with Li + Used in combination to stabilize the ternary complex. + When used together, betaine may optionally be present, or betaine may not be present.

[0081] Inhibitory metal ions may also be used in the methods or compositions of the present disclosure, for example as stabilizers. A particularly useful inhibitory metal ion is Ca 2+ When contacted with the ternary complex, the inhibitory metal ion is present at a concentration of at least 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM or more. Alternatively or additionally, the inhibitory metal ion may be present at a concentration of at most 100 mM, 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM or less. Ca 2+ Can be used alone or with Li + , betaine and PEI are used in combination with one or more of them.

[0082] Solutions for stabilizing the ternary complex, such as solutions containing lithium, betaine, PEI, and / or inhibitory metal ions, can be buffered at a desired pH. For example, the pH of the solution can be at least 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. Alternatively or additionally, the buffer can be selected to maintain a pH of at most 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.5, or 7.0. Useful buffering agents include those described in the examples below or those having an appropriate pKa for the desired pH range, including, for example, MOPS (pKa 7.2), BES (pKa 7.2), TES (pKa 7.5), triethanolamine (pKa 7.8), EPPS or HEPPS (pKA 8.0), TRIS (pKa 8.1), Tricine (pKa 8.1), Glycylglycine (pKa 8.3), Bicine (pKa 8.3), TAPPS (pKa 8.4), Morpholine (pKa 8.5), N-methyldiethanolamine (pKa 8.5), 2-amino-2-methyl-1,3-propanediol (pKa 8.8), diethanolamine (pKa 8.9), or AMPSO (pKa 9.1). The buffer can be present at an effective concentration to stabilize the ternary complex, for example, between about 25 mM and 250 mM, between about 25 mM and 100 mM, between about 40 mM and 80 mM, or in other ranges.

[0083] Solutions used to stabilize the ternary complex, such as solutions containing lithium, betaine, PEI, and / or inhibitory metal ions, may include salts such as those described in the Examples section below. Particularly useful salts include, but are not limited to, NaCl, KCl, potassium acetate, ammonium acetate, potassium glutamate, NH 4 Cl or (NH 4 HSO 4 The salt may be present at an effective concentration to stabilize the ternary complex, including, for example, at least 10 mM, 25 mM, 50 mM, 100 mM, 250 mM or more. Alternatively or additionally, the salt concentration may be at most 250 mM, 100 mM, 50 mM, 25 mM or 10 mM.

[0084] Solutions for stabilizing ternary complexes, such as solutions containing lithium, betaine, PEI and / or inhibitory metal ions, may contain other components, such as those that provide the desired viscosity or molecular crowding. Exemplary components include, for example, polysaccharides such as sucrose, Ficoll or dextran; proteins such as lysozyme, albumin or casein; or polymers such as polyvinyl alcohol, polyethylene glycol (PEG 2050, PEG 4600, PEG 6000, PEG 8000, PEG 20000, etc. Viscosity or molecular crowding agents may be present in a concentration of at least about 0.5%, 1%, 3%, 5%, 10% or more. Alternatively or additionally, the concentration may be up to 10%, 5%, 3%, 2%, 1% or 0.5%.

[0085] Other useful components included in the solution for stabilizing the ternary complex include anti-fading agents or photoprotectants, such as DABCO (1,4-diazabicyclo [2.2.2] octane), ascorbate, gallic acid or its derivatives. Other useful anti-fading agents and photoprotectants include, for example, those described in U.S. Patents Nos. 7,993,895, 9,115,353, and 10,036,011 (each of which is incorporated herein by reference). Such agents are particularly useful when present in a solution detected by optical methods such as luminescence and fluorescence. The method disclosed herein may include one or more steps for forming and detecting a ternary complex. Embodiments of the method utilize the specificity that a polymerase can utilize to form a stable ternary complex with a triggered template nucleic acid and the next correct nucleotide. The next correct nucleotide can be non-covalently bound to a stable ternary complex, interacting only with the other members of the complex through non-covalent interactions. Useful methods and compositions for forming stable ternary complexes are described in more detail below and in co-owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or No. 2018 / 0044727 A1; U.S. Patent Application Publication No. 2018 / 0187245 A1 (which claims priority to U.S. Patent Application Serial No. 62 / 440,624) or U.S. Patent Application Publication No. 2018 / 0208983 A1 (which claims priority to 62 / 450,397), each of which is incorporated herein by reference. Typically, the formation and detection of the ternary complex is separated from the step of extending the primer, for example, due to reagent exchange between the steps. However, in some embodiments, the binding, detection and extension steps can be performed in the same mixture.

[0086] Although some catalytic metal ions (e.g., Mg 2+) in the absence of a polymerase, the primed template nucleic acid and the next correct nucleotide can form a ternary complex, but in the absence of a catalytic metal ion, chemical addition of nucleotides is inhibited. Low or insufficient levels of catalytic metal ions can result in non-covalent chelation of the next correct nucleotide in a stable ternary complex. Other methods disclosed herein can also be used to generate stable ternary complexes.

[0087] Optionally, a stable ternary complex can be formed when the primer of the triggered template nucleic acid comprises a blocking portion (e.g., a reversible terminator portion) that prevents the enzymatic incorporation of the introduced nucleotide into the primer. The interaction can occur in the presence of a stabilizer, so that the polymerase-nucleic acid interaction is stabilized in the presence of the next correct nucleotide. The primer of the triggered template nucleic acid can optionally be an extendable primer, or a primer that is blocked from extension at its 3' end (e.g., blocking can be achieved by the presence of a reversible terminator portion at the 3' end of the primer). When the base of the homologous nucleotide is complementary to the next base of the triggered template nucleic acid, the triggered template nucleic acid, the polymerase, and the homologous nucleotide can form a stable ternary complex.

[0088] As described above, conditions that are conducive to or stable ternary complexes can be provided by the presence of a blocking group (e.g., a reversible terminator portion on the 3' nucleotide of a primer) or the absence of a catalytic metal ion, which blocks the enzymatic incorporation of introduced nucleotides into the primer. Other useful conditions include the presence of a ternary complex stabilizer such as an inhibitory metal ion (e.g., a divalent or trivalent inhibitory metal ion) that inhibits the incorporation or polymerization of nucleotides catalyzed by a polymerase. Inhibitory metal ions include, but are not limited to, calcium, strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, and terbium ions. Optionally, the presence of one or more monovalent cations and / or glutamate anions provides conditions that are unfavorable for binary complexes (i.e., complexes between polymerase and the initiated nucleic acid, but lacking homologous nucleotides) or that destabilize the binary complex. As another example of a stable condition, a polymerase engineered to have a reduced catalytic activity or reduced tendency to form a binary complex can be used. Exemplary engineered polymerases are described in U.S. Patent Application Publication No. 2017 / 0314072 A1 or 2018 / 0155698 A1, each of which is incorporated herein by reference.

[0089] The ternary complex stabilizing conditions can be used in the presence of different nucleotides, for example, by destroying the stability of the binary complex, to enhance the affinity difference of the polymerase to the template nucleic acid initiated. Optionally, in the presence of different nucleotides, the conditions cause the polymerase to have different affinities for the template nucleic acid initiated. For example, the conditions include but are not limited to high salt and glutamate ions. For example, the salt can be dissolved in an aqueous solution to produce a monovalent cation, such as a monovalent metal cation (e.g., sodium ion or potassium ion). Optionally, a salt providing a monovalent cation (e.g., a monovalent metal cation) further provides a glutamate ion. Optionally, a glutamate ion source can be potassium glutamate. In some cases, the concentration of potassium glutamate that can be used to change the polymerase affinity of the template nucleic acid initiated is extended from 10mM to 1.6M potassium glutamate, or any amount between 10mM and 1.6M. As described above, high salt refers to a salt concentration of 50mM to 1.5M salt.

[0090] It should be understood that the options for stabilizing the ternary complex described herein need not be mutually exclusive, but can be used in various combinations. For example, the ternary complex can be stabilized by a combination of one or more means, including but not limited to the presence of polymerase domain crosslinks; polymerase to nucleic acid crosslinks; polymerase mutations that stabilize the ternary complex; allosteric inhibition by small molecules; the presence of Li + , PEI, betaine, uncompetitive inhibitor, competitive inhibitor or noncompetitive inhibitor; the absence of catalytic metal ions; the presence of a blocking portion on the primer; or other means described herein.

[0091] The nucleic acid used in the method or composition herein can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, copy DNA (cDNA) etc. RNA can also be used, such as mRNA, ribosomal RNA, tRNA etc. Nucleic acid analogs can also be used as templates in this article. Therefore, the template nucleic acid used herein can be derived from a biological source, a synthetic source or an amplified product. The primer used herein can be DNA, RNA or its analog.

[0092] Particularly useful nucleic acid templates are genomic fragments that include a sequence identical to a portion of a genome. A population of genomic fragments may include at least 5%, 10%, 20%, 30%, or 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a genome. A genomic fragment may have, for example, a sequence substantially identical to at least about 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more nucleotides of a genome. Alternatively or additionally, a genomic fragment may have a sequence substantially identical to no more than 1 x 10 5 pcs, 1x 10 4 pcs, 1x 10 3 The genomic fragment can be a DNA, RNA or an analog thereof.

[0093] Exemplary organisms from which the nucleic acid can be derived include, for example, those from a mammal, such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or non-human primate; a plant, such as Arabidopsis, corn, sorghum, oats, wheat, rice, canola or soybean; an algae, such as Chlamydomonas reinhardtii; a nematode, such as Caenorhabditis elegans; an insect, such as Drosophila melanogaster, a mosquito, a fruit fly, a bee or a spider; a fish, such as a zebrafish; a reptile; an amphibian, such as a frog or an African clawed frog; a dictyostelium discoideum; a fungus, such as pneumocystis carinii, Takifugu rubripes, a yeast, Saccharomyces cerevisiae or Schizosaccharomyces pombe; or a Plasmodium falciparum. Nucleic acids can also be derived from prokaryotes, such as bacteria: Escherichia coli, staphylococci, or mycoplasma pneumoniae; archae; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Nucleic acids can be derived from homogenous cultures or populations of the above organisms, or alternatively from a collection of several different organisms, for example, in a community or ecosystem. Nucleic acids can be isolated using methods known in the art, including, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998) (each of which is incorporated herein by reference).

[0094] Template nucleic acid can be obtained by preparation methods such as genome separation, genome fragmentation, gene cloning and / or amplification. Template can be obtained by amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), etc. Exemplary methods for separating, amplifying and fragmenting nucleic acids to generate templates for array analysis are described in U.S. Patent No. 6,355,431 or No. 9,045,796 (each of which is incorporated herein by reference). Amplification can also be carried out using Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998) (each of which is incorporated herein by reference).

[0095] Any of a variety of polymerases may be used in the methods described herein. Unless otherwise indicated, references to specific polymerases such as those illustrated in this disclosure will be understood to include functional variants thereof. Particularly useful functions of polymerases include forming ternary complexes or catalyzing the polymerization of nucleic acid chains using existing nucleic acids as templates. Specific polymerase activities or properties described herein, for example, forming a polymerase produced by a specific reagent such as Li + , PEI, betaine and / or inhibitory metal ions such as Ca 2+ Stable ternary complexes may be shared by polymerases that have been grouped into known classifications. One particularly useful classification is based on structural homology, such as classifying polymerases into families identified as A, B, C, D, X, Y, and RT. DNA polymerases in Family A include, for example, T7 DNA polymerase, eukaryotic mitochondrial DNA polymerase γ, Escherichia coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I. DNA polymerases in Family B include, for example, eukaryotic DNA polymerases α, δ, and ε; DNA polymerase ζ; T4 DNA polymerase, Phi29 DNA polymerase, and RB69 bacteriophage DNA polymerase. Family C includes, for example, Escherichia coli DNA polymerase III α subunit. Family B archaeal DNA polymerases include, for example, Vent, Deep Vent, Pfu, and 9°N (e.g., Therminator from New England BioLabs Inc.; Ipswich, MA). TMDNA polymerase) polymerase. Family D includes, for example, polymerases from the Euryarchaeota subdomain of Archaea. DNA polymerases in Family X include, for example, eukaryotic polymerases Polβ, polσ, Polλ, and Polμ, and Saccharomyces cerevisiae Pol4. DNA polymerases in Family Y include, for example, Polη, Polι, Polκ, Escherichia coli Pol IV (DINB), and Escherichia coli Pol V (UmuD'2C). The RT (reverse transcriptase family) of DNA polymerases includes, for example, retroviral reverse transcriptases and eukaryotic telomerases. Exemplary RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase; eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0096] The above classification is provided for the purpose of illustration. It should be understood that variations in the classification system are possible. For example, in at least one classification system, Family C polymerase is classified as a subclass of Family X. In addition, polymerases may also be classified according to other characteristics (whether functional or structural), which may or may not overlap with the structural characteristics illustrated above. Some exemplary characteristics will be described in more detail below.

[0097] Polymerases that can be used in the methods or compositions described herein include naturally occurring polymerases and modified variants thereof, including but not limited to mutants, recombinants, fusions, genetically modified bodies, chemically modified bodies, synthetics and the like. Useful polymerases for ternary complex formation and detection are not limited to polymerases with the ability to catalyze polymerization reactions. Optionally, useful polymerases will have the ability to catalyze polymerization reactions under at least one condition that is not used in the formation or inspection process of a stable ternary complex. Optionally, the polymerases participating in the stable ternary complex have improved properties, for example, enhanced binding affinity to nucleic acids, reduced binding affinity to nucleic acids, enhanced binding affinity to nucleotides, reduced binding affinity to nucleotides, enhanced specificity for the next correct nucleotide, reduced specificity for the next correct nucleotide, reduced catalytic rate, catalytic inactivation, etc. Mutant polymerases include, for example, polymerases in which one or more amino acids are replaced by other amino acids or insertion or deletion of one or more amino acids. Exemplary polymerases that can be used to form stable ternary complexes include, for example, wild-type and mutant polymerases described in U.S. Patent Application Serial No. 15 / 866,353 (now published as U.S. Patent Application Publication No. 2018 / 0155698 A1) or U.S. Patent Application Publication No. 2017 / 0314072 A1), each of which is incorporated herein by reference.

[0098] In some embodiments, a polymerase containing an exogenous labeling portion (e.g., an exogenous fluorophore) that can be used to detect a polymerase can be useful. Optionally, a labeling portion can be attached after the polymerase has been at least partially purified using a protein separation technique. For example, the free sulfhydryl or free amine portion of the polymerase can be used to chemically connect the exogenous labeling portion to the polymerase. This can involve chemical bonding with the free amino group of the N-terminus and the polymerase through the side chain of a cysteine ​​residue or through the N-terminus. The exogenous labeling portion can also be attached to the polymerase by protein fusion. Exemplary labeling portions that can be attached by protein fusion include, for example, green fluorescent protein (GFP), phycobiliprotein (e.g., phycocyanin and phycoerythrin) or wavelength shift variants of GFP or phycobiliprotein. In some embodiments, the exogenous label on the polymerase can function as a member of a FRET pair. Another member of a FRET pair can be an exogenous label attached to a nucleotide that is combined with the polymerase in a stable ternary complex. Therefore, a stable ternary complex can be detected or identified by FRET.

[0099] Alternatively, the polymerase involved in the stable ternary complex need not be attached to an exogenous label. For example, the polymerase need not be covalently attached to an exogenous label. Instead, the polymerase can be free of any label until it binds to a labeled nucleotide and / or a labeled nucleic acid (e.g., a labeled primer and / or a labeled template).

[0100] The ternary complex prepared or used according to the present disclosure may optionally include one or more exogenous markers.Before forming the ternary complex, the marker can be attached to the component of the ternary complex (for example, attached to a polymerase, a template nucleic acid, a primer and / or a homologous nucleotide).Exemplary attachment includes covalent attachment or non-covalent attachment, such as those described in this article, in the references cited herein or known in the art.In some embodiments, the component of the mark is delivered in solution to a solid support attached to an unlabeled component, thereby recruiting the marker to the solid support by forming a stable ternary complex.Therefore, the component attached to the carrier can be detected or identified based on the observation of the recruited marker.Whether in the solution phase or on a solid support, the exogenous marker can be used to detect a stable ternary complex or its single component during the inspection step.Exogenous markers can remain attached to the component after the component is separated from other components of the stable ternary complex formed. Exemplary labels, methods for attaching markers, and methods for using labeled components are described in more detail below or in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or 2018 / 0044727 A1, or U.S. Patent Application Serial No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1), No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1), or U.S. Patent Application Publication No. 2018 / 0208983 A1 (which claims priority to Nos. 62 / 450,397 and 62 / 506,759), each of which is incorporated herein by reference.

[0101] As described above, different activities of polymerases can be utilized in the methods described herein. Polymerases can be used, for example, in one or both of the inspection steps, or, as described in further detail below, in the extension step. Different activities can be generated by differences in structure (e.g., by natural activity, mutation, or chemical modification). However, polymerases can be obtained from various known sources and applied according to the teachings and recognized polymerase activities described herein. Useful DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and bacteriophage DNA polymerases. Bacterial DNA polymerases include Escherichia coli (E. coli) DNA polymerases I, II and III, IV and V, Klenow fragments of E. coli DNA polymerases, Clostridium stercorarium (Cst) DNA polymerases, Clostridium thermocellum (Cth) DNA polymerases, and Sulfolobus solfataricus (Sso) DNA polymerases. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and к, as well as Rev1 polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNA polymerases include thermostable and / or thermophilic DNA polymerases, such as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D (Pyrococcus sp.GB-D) polymerase, Thermotogamaritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase, Sulfolobus acidocaldarius DNA polymerase, Thermococcus sp. go N-7 DNA polymerase, Pyrodictium occultum DNA polymerase, Methanococcus vorgonarius (Tgo) DNA polymerase voltae DNA polymerase, Methanococcusthermoautotrophicum DNA polymerase, Methanococcus jannaschii DNA polymerase, Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus horikoshii DNA polymerase, Pyrococcus islandicum DNA polymerase, Thermococcus fumicolans DNA polymerase, Aeropyrum pernix DNA polymerase, and heterodimeric DNA polymerase DP1 / DP2. Engineered and modified polymerases can also be used in conjunction with the disclosed technology. For example, a modified form of the extremely thermophilic marine archaeon Thermococcus species 9°N can be used (e.g., Therminator. TM DNA polymerase). Other useful DNA polymerases, including 3PDX polymerase, are disclosed in US Pat. No. 8,703,461 (the disclosure of which is incorporated herein by reference).

[0102] Useful RNA polymerases include, but are not limited to, viral RNA polymerases, such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase; eukaryotic RNA polymerases, such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0103] Another useful type of polymerase is a reverse transcriptase. Exemplary reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase that maintains eukaryotic chromosome telomeres.

[0104] For some embodiments, polymerases with intrinsic 3'-5' proofreading exonuclease activity can be useful. Polymerases substantially lacking 3'-5' proofreading exonuclease activity are also useful in some embodiments, for example, in most genotyping and sequencing embodiments. The absence of exonuclease activity can be a wild-type property or can be a property conferred by a variant or engineered polymerase structure. For example, an exo-minus Klenow fragment is a mutant form of a Klenow fragment that lacks 3'-5' proofreading exonuclease activity. Klenow fragments and exo-minus variants thereof can be used in the methods or compositions set forth herein.

[0105] Stable ternary complexes can include natural nucleotides, nucleotide analogs or modified nucleotides as needed to suit the specific application or configuration of the method. Optionally, the nucleotide analog has a nitrogenous base, a pentose and a phosphate group, wherein any part of the nucleotide can be modified, removed and / or replaced compared to the natural nucleotide. Nucleotide analogs can be non-incorporable nucleotides (i.e., nucleotides that cannot react with the 3' oxygen of the primer to form a covalently linked nucleotide). Such non-incorporable nucleotides include, for example, monophosphate and diphosphate nucleotides. In another example, the nucleotide may include one or more modifications to the triphosphate group so that the nucleotide cannot be incorporated. Examples of non-incorporable nucleotides can be found in U.S. Patent No. 7,482,120 (which is incorporated herein by reference). In some embodiments, non-incorporable nucleotides can be subsequently modified to be incorporable. Non-incorporable nucleotide analogs include, but are not limited to, α-phosphate modified nucleotides, α-β nucleotide analogs, β-phosphate modified nucleotides, β-γ nucleotide analogs, γ-phosphate modified nucleotides or caged nucleotides. Additional examples of nucleotide analogs are described in US Pat. No. 8,071,755, which is incorporated herein by reference.

[0106] In the present invention, for example, nucleotide analogs for participating in a stable ternary complex may include a terminator that reversibly prevents subsequent nucleotide incorporation at the 3' end of the primer after the analog has been incorporated into the primer. For example, US7,544,794 and US8,034,923 (the disclosures of which are incorporated herein by reference) describe a method in which the 3'-OH group is replaced by a 3'-ONH 2 Partially substituted reversible terminators. Another type of reversible terminator is connected to the nitrogenous base of the nucleotides described in, for example, US8,808,989 (its disclosure is incorporated herein by reference). Other reversible terminators that can be used similarly in conjunction with the methods described herein include those terminators described in the references cited elsewhere herein or US7,956,171, US8,071,755 and US9,399,798 (the disclosures of these United States Patents are incorporated herein by reference). In certain embodiments, the reverse terminator portion can be removed from the primer in a process referred to as "unblocking", thereby allowing subsequent nucleotides to be incorporated. In the context of reversible terminators, compositions and methods for unblocking are described in the references cited herein.

[0107] Alternatively, nucleotide analogs irreversibly prevent nucleotides from incorporating into the 3'-end of the primer into which they have been incorporated. Irreversible nucleotide analogs include 2',3'-dideoxynucleotides (ddNTPs, such as ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs that would otherwise participate in polymerase-mediated primer extension. Thus, the 3' position has a hydrogen moiety instead of the natural hydroxyl moiety. Irreversibly terminated nucleotides are particularly useful for genotyping applications or other applications where primer extension is not required or sequential detection along a template nucleic acid is not desired.

[0108] In certain embodiments, the nucleotide analogs used herein, for example, to participate in a stable ternary complex do not include a blocking group (e.g., a reversible terminator) that prevents subsequent incorporation of a nucleotide at the 3'-end of the primer after the analog has been incorporated into the primer. This may be the case whether or not the extension step is performed using one or more nucleotides having a blocking group (e.g., a reversible terminator).

[0109] In some embodiments, the nucleotides for participating in the formation of a stable ternary complex may include exogenous markers herein, for example. For example, the exogenously labeled nucleotides may include a reversible or irreversible terminator portion, the exogenously labeled nucleotides may be non-incorporable, the exogenously labeled nucleotides may lack a terminator portion, the exogenously labeled nucleotides may be incorporable, or the exogenously labeled nucleotides may be incorporable and non-incorporable. When used to form a stable ternary complex with an unlabeled polymerase, the exogenously labeled nucleotides may be particularly useful. Alternatively, the exogenous marker on the nucleotide may provide a partner in a fluorescence resonance energy transfer (FRET) pair, and the exogenous marker on the polymerase may provide a second partner to the pair. Therefore, FRET detection may be used to identify a stable ternary complex comprising two partners.

[0110] Alternatively, for example, nucleotides used to participate in the formation of a ternary complex herein may lack an exogenous label (i.e., the nucleotide may be "non-labeled"). For example, non-labeled nucleotides may include a reversible or irreversible terminator portion, non-labeled nucleotides may be non-incorporable, non-labeled nucleotides may lack a terminator portion, non-labeled nucleotides may be incorporable, or non-labeled nucleotides may be incorporable and non-terminated. Non-labeled nucleotides may be useful when a label on a polymerase is used to detect a stable ternary complex or when label-free detection is used. Non-labeled nucleotides may also be used in the extension step of the method described herein. It should be understood that the absence of a part or function of a nucleotide means that the nucleotide does not have such a function or part. However, it should also be understood that one or more functions or parts originally known in the art regarding a nucleotide or its analogs described herein or regarding a nucleotide or its analogs may be explicitly omitted in the method or composition described herein.

[0111] Optionally, during or after the formation of a stable ternary complex, nucleotides (e.g., natural nucleotides or nucleotide analogs) are present in the mixture. For example, at least 1, 2, 3, 4 or more nucleotide types may be present. Alternatively or additionally, at most 4, 3, 2 or 1 nucleotide types may be present. Similarly, the one or more nucleotide types present may be complementary to at least 1, 2, 3 or 4 base types in the template nucleic acid. Alternatively or additionally, the one or more nucleotide types present may be complementary to at most 4, 3, 2 or 1 base types in the template nucleic acid. Different base types may be identified by the presence of different exogenous markers on different nucleotides. Alternatively, two or more nucleotide types may have indistinguishable exogenous markers. In the latter form, different nucleotides can still be distinguished due to being delivered separately into the reaction vessels or due to the encoding and decoding schemes described in, for example, U.S. Patent Application Serial No. 15 / 922,787 (published as U.S. Patent Application Publication No. 2018 / 0305749 A1) or U.S. Patent No. 9,951,385 (each of which is incorporated herein by reference).

[0112] Any nucleotide modification that stabilizes the polymerase in the ternary complex can be used in the methods disclosed herein. The nucleotide can be permanently or transiently bound to the polymerase. Optionally, the nucleotide analog is fused to the polymerase, for example, via a covalent linker. Optionally, a plurality of nucleotide analogs are fused to a plurality of polymerases, wherein each nucleotide analog is fused to a different polymerase. Optionally, the nucleotide present in the stable ternary complex is not a means by which the ternary complex is stabilized. Thus, any of a variety of other ternary complex stabilization methods can be combined in a reaction utilizing a nucleotide analog.

[0113] In certain embodiments, the primer strand of the primed template nucleic acid present in the stable ternary complex is not chemically altered by the presence of a polymerase during one or more steps of the methods described herein. For example, the primer does not need to be extended by forming a new phosphodiester bond, nor does it need to be shortened by nucleolytic degradation during the step of forming the stable ternary complex and during the step of detecting the stable ternary complex.

[0114] The specific embodiment of the method set forth herein includes the step of forming a mixture comprising several components. The components of the mixture can be delivered in the container in any desired order, or they can be delivered simultaneously. In addition, some components can be mixed with each other to form the first mixture, and then the first mixture is contacted with other components to form a more complicated mixture. Taking the step of forming a mixture comprising the template nucleic acid, polymerase and multiple different nucleotide types of initiation as an example, it should be understood that the different nucleotide types in the multiple nucleotide types can be contacted with each other, then contacted with the template nucleic acid of initiation. Alternatively, two or more nucleotide types can be delivered to the template nucleic acid and / or the polymerase of initiation respectively. Like this, the first nucleotide type can contact with the template nucleic acid of initiation before contacting with the second nucleotide type. Alternatively or additionally, the first nucleotide type can contact with the polymerase before contacting with the second nucleotide type.

[0115] In certain embodiments, polymerase and nucleotide are soluble in the fluid for stabilizing the ternary complex. For example, the stabilizing solution can advantageously be an aqueous fluid. The stabilizing solution may not contain a solvent that does not solubilize polymerase and / or nucleotide. For example, the ternary composite stabilizing solution may not contain an alcohol or oil that does not solubilize polymerase and / or nucleotide. Exemplary fluids that do not solubilize polymerase and / or nucleotide are described in U.S. Patent Application Serial No. 16 / 164,417 (which requires priority to U.S. Patent Application Serial No. 62 / 574,308) (each of which is incorporated herein by reference). In some embodiments, the ternary composite stabilizing solution can be used as a washing fluid to remove non-complexed nucleotides or polymerase from a fixed ternary composite.

[0116] Prior to contacting with the ternary complex, the ternary complex stabilizing solution may not contain one or more components of the ternary complex binding reaction. + , PEI, betaine or inhibitory metal cations (e.g. Ca 2+ The stabilizing fluid of the present invention may not contain polymerase or nucleotides. Thus, the stabilizing fluid may act as a wash to remove excess polymerase or nucleotides from the binding reaction previously used to form the ternary complex. In certain embodiments, the concentration of the ternary complex in the stabilizing fluid is higher than the concentration of free nucleotides and / or polymerase in the fluid. The free nucleotides and polymerase in such a fluid may be of the same type as those present in the ternary complex. It should be understood that during the formation of the ternary complex, Li + , PEI, betaine, inhibitory metal cations (e.g. Ca 2+ ) or other ternary complex stabilizers. Alternatively, the ternary complex stabilizer can be introduced into the already formed ternary complex.

[0117] The ternary complex can be detected by the presence of an exogenous label on one or more components of the complex. Examples of useful exogenous labels include, but are not limited to, any of a radiolabel moiety, a luminophore moiety, a fluorophore moiety, a quantum dot moiety, a chromophore moiety, an enzyme moiety, an electromagnetic spin label moiety, a nanoparticle light scattering moiety, and various other signal generating moieties known in the art. Suitable enzyme moieties include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Exemplary fluorophore moieties include, but are not limited to, rhodols, resorufins, coumarins, xanthenes, acridines, fluoresceins, rhodamines, erythrins, anthocyanins, o-phthalaldehydes, naphthylamines, fluorescamines, benzoxadiazoles, stilbenes, pyrenes, indoles, borapolyazaindacenes, quinazolinones, eosin, erythrosine, malachite green, CY dyes (GE Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives), DYOMICS and DYLIGHT dyes (Dyomics), including DY-547, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777, and DY-782; Lucifer Yellow; CASCADE BLUE, TEXAS RED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes, including BODIPY 630 / 650 and BODIPY 650 / 670; ATTO dyes (Atto-Tec), including ATTO390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610, ATTO 635; ALEXA FLUOR, including ALEXAFLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750 and ALEXA FLUOR 680 (Molecular Probes); DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any derivative thereof) (Molecular Probes); QUASAR dyes (Biosearch);IRDYES dyes (LiCor), including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester), and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (GlenResearch), including EPOCH REDMONDRED, EPOCH YAKIMA YELLOW, EPOCH GIG HARBOR GREEN; Tokyo green (M. Kamiya et al., 2005 Angew.Chem.Int.Ed.44:5439-5441); and CF dyes, including CF 647 and CF 555 (Biotium), and other dyes known in the art, such as in; Principles of Fluorescence Spectroscopy , Joseph R. Lakowicz (ed.), Plenum Pub Corp, 2nd edition (July 1999) and Richard P. Hoagland Molecular Probes Handbook Those dyes described in the 6th edition of

[0118] The second label can be used in the method of the present disclosure. The second label is a binding moiety that can specifically bind to the partner moiety. For example, the ligand moiety can be connected to a polymerase, a nucleic acid or a nucleotide to allow detection by specific affinity for the labeled receptor. Exemplary binding moiety pairs that can be used include, but are not limited to, antigens and immunoglobulins or active fragments thereof, such as FAbs; immunoglobulins and immunoglobulins (or active fragments thereof, respectively); avidin and biotin, or analogs thereof that are specific to avidin; streptavidin and biotin, or analogs thereof that are specific to streptavidin; or carbohydrates and lectins.

[0119] In some embodiments, the second label can be a chemically modifiable moiety. In this embodiment, a label with a reactive functional group can be incorporated into a stable ternary complex. Subsequently, the functional group can covalently react with the primary labeling moiety. Suitable functional groups include, but are not limited to, amino, carboxyl, maleimido, oxo, groups involved in click reactions, and sulfhydryl.

[0120] The label can be attached to nucleotide, polymerase or other molecules through a joint. The joint present in the nucleotide or polymerase can be, but not necessarily cleavable. For example, the joint can be stable for the conditions used in the method set forth herein, so that the covalent structure of the joint is not changed in any specific step or all steps of the method set forth herein. The joint present in the nucleotide analog can be at least chemically stable as one or more other parts in the analog. For example, in any specific step of the method set forth herein, or in all steps, the joint can be chemically stable as a nitrogenous base, a sugar and / or a phosphoric acid moiety.

[0121] In alternative embodiments, the ternary complex may lack an exogenous label. For example, the stable ternary complex and all components participating in the stable ternary complex (e.g., polymerase, template nucleic acid, primer, and / or cognate nucleotide) may lack one, several, or all exogenous labels described herein or described in the references cited and incorporated herein. In such embodiments, the ternary complex may be detected based on the intrinsic properties of the stable ternary complex, such as mass, charge, intrinsic optical properties, etc. Exemplary methods for detecting unlabeled ternary complexes are described in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1, PCT Application Serial No. PCT / US16 / 68916 (published as WO 2017 / 117243 A1), or U.S. Patent Application Serial No. 62 / 375,379 (now published as U.S. Patent Application Publication No. 2018 / 0044727 A1 or 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044715 A1), each of which is incorporated herein by reference.

[0122] Typically, detection can be achieved in the inspection step by sensing the intrinsic properties of the ternary complex or the labeling portion attached thereto. Exemplary properties that detection can be based on include, but are not limited to, mass, conductivity, energy absorption rate, luminescence (e.g., fluorescence), etc. Detection of luminescence can be performed using methods known in the art related to nucleic acid arrays. Luminophores can be detected based on any of a variety of luminescent properties, including, for example, emission wavelength, excitation wavelength, fluorescence resonance energy transfer (FRET) intensity, quenching, anisotropy, or lifetime. Other detection techniques that can be used in the methods described herein include, for example, mass spectrometry for sensing mass; surface plasmon resonance for sensing binding to a surface; absorbance at wavelengths of energy absorbed by a marker for sensing; calorimetry for sensing temperature changes due to the presence of a marker; conductivity or impedance for sensing electrical properties of a marker, or other known analytical techniques. Examples of reagents and conditions that can be used to generate, manipulate, and detect stable ternary complexes include, for example, those described in commonly owned U.S. Patent Application Publication No. 2017 / 0022553A1; PCT Application Serial No. PCT / US16 / 68916 (published as WO 2017 / 117243A1), or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044715 A1), 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1); 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1); U.S. Patent Application Publication No. 2018 / 0208983 A1 (which claims priority to Nos. 62 / 450,397 and 62 / 506,759), each of which is incorporated herein by reference.

[0123] The detection of the ternary complex can be carried out in different types of reaction mixtures, but is usually carried out under conditions of stable ternary complexes. Conditions can also be selected to reduce the concentration of the type of label (e.g., labeled nucleotide) to below the level for forming the ternary complex. Optionally, the inspection step or substep includes detecting the interaction of the polymerase and the homologous nucleotide with the template nucleic acid initiated in the absence of non-complex labeled nucleotides and polymerase. Optionally, the inspection step or substep includes detecting the labeled nucleotides in the stable ternary complex after removing the unbound nucleotides from contact with the ternary complex using a stabilizing solution. The formation of the ternary complex can be detected or monitored by detecting or monitoring a marker attached to the nucleotides participating in the ternary complex. Optionally, the absence of the formation of the ternary complex is detected or monitored. Optionally, the dissociation of the ternary complex is monitored.

[0124] The step of detecting the ternary complex may be performed in a washing step using a ternary complex stabilizing solution. The stabilizing solution may contain Li + , PEI, betaine or inhibitory metal cations (e.g. Ca 2+ ). Optionally, during the detection step, the stabilizing fluid remains stationary (i.e., does not move or flow). However, the fluid may flow, for example, through a flow cell containing the ternary complex to be detected. Advantageously, detecting the ternary complex during the wash step may reduce background signals associated with unbound nucleotides or polymerases that may have a detectable label. Likewise, by this method, ternary complex detection may be performed in a reaction mixture that is different from the reaction mixture that provides one or more labeled nucleotides to the primed template nucleic acid molecule, which may optionally be capped at its 3'-end with a reversible terminator moiety.

[0125] The inspection and detection of stable ternary complexes can be accomplished in different ways. For example, monitoring can include measuring the association kinetics of the interaction between two or more components of the complex. Monitoring the interaction can include measuring the equilibrium binding signal or equilibrium binding constant. Therefore, for example, monitoring can include measuring the equilibrium binding signal or equilibrium binding constant in the presence of one or more labeled nucleotides. Monitoring the interaction can include, for example, measuring the dissociation kinetics of nucleotides from the template nucleic acid of initiation in the presence of any one of the four nucleotides. Optionally, in the presence of nucleotide molecules, monitoring the interaction of polymerase with the template nucleic acid molecule of initiation includes measuring the dissociation kinetics of the closed complex. The technology for measuring association, equilibrium and dissociation kinetics is known and can be easily modified by those skilled in the art for use in the method set forth herein. See, e.g., Markiewicz et al., Nucleic Acids Research 40(16):7975-84 (2012); Xia et al., J. Am. Chem. Soc. 135(1):193-202 (2013); Brown et al., J. Nucleic Acids, Article ID 871939, 11 pages (2010); Washington et al., Mol. Cell. Biol. 24(2):936-43 (2004); Walsh and Beuning, J. Nucleic Acids, Article ID 530963, 17 pages (2012) and Roettger et al., Biochemistry 47(37):9718-9727 (2008), which are incorporated herein by reference. It should be understood that detection techniques can accumulate signals over relatively short durations, which is generally understood as single time point acquisition. Alternatively, the signal can be monitored continuously over time, which is a typical time-based acquisition. A series of time points can also be acquired in a periodic manner to obtain a time-based acquisition.

[0126] Some embodiments of the methods described herein utilize two or more distinguishable signals to distinguish stable ternary complexes from each other and / or to distinguish one base type from another base type in a template nucleic acid. For example, two or more luminophores can be distinguished from each other based on unique optical properties (such as unique excitation wavelengths or unique emission wavelengths). In a particular embodiment, the method can distinguish different stable ternary complexes based on differences in luminous intensity. For example, a first ternary complex can be detected under conditions where the first ternary complex emits a lower intensity than a second ternary complex. This intensity scaling (sometimes referred to as "grayscale scaling") can utilize any distinguishable intensity difference. Exemplary differences include a specific stable ternary complex having an intensity of 10%, 25%, 33%, 50%, 66% or 75% compared to the intensity of another stable ternary complex to be detected.

[0127] Intensity differences can be produced by using different luminophores, each of which has different extinction coefficients (i.e., resulting in different excitation characteristics) and / or different luminescence quantum yields (i.e., resulting in different emission characteristics). Alternatively, the same luminophore type can be used, but it can be present in different amounts. For example, all members of the first ternary complex population can be labeled with a specific luminophore, while only half of the members of the second population are labeled with the luminophore. In this example, the second population is expected to produce half the signal of the first population. The second population can be produced, for example, by using a mixture of labeled nucleotides and unlabeled nucleotides (in contrast to the first population of mainly labeled nucleotides). Similarly, the second population can be produced, for example, by using a mixture of labeled polymerase and unlabeled polymerase (in contrast to the first population of mainly labeled polymerase). In an alternative labeling scheme, the first ternary complex population can include polymerase molecules with a variety of markers that produce specific luminescent signals, and the second ternary complex population can include polymerase molecules that each only have one of the markers that produce luminescent signals.

[0128] In some embodiments, the checking step is performed in a manner to infer the identity of at least one nucleotide type, for example, as described in co-owned U.S. Patent No. 9,951,385 or U.S. Patent Application Serial No. 15 / 922,787 (published as U.S. Patent Application Publication No. 2018 / 0305749 A1), each of which is incorporated herein by reference. For example, the checking step may include the following steps: (a) providing a fluid comprising a primed template nucleic acid, a polymerase, and nucleotide homologs of the first, second, and third base types in the template under ternary complex stabilizing conditions; (b) optionally removing unbound polymerase and unbound nucleotides from the fluid; (c) checking the fluid to determine whether a ternary complex is formed, wherein the fluid comprises Li +, PEI, betaine and / or inhibitory metal cations (e.g. Ca 2+ ); and (d) identifying the next correct nucleotide of the triggered template nucleic acid molecule, wherein if a ternary complex is detected in step (c), the next correct nucleotide is identified as a homolog of the first, second or third base type, and wherein based on the absence of a ternary complex in step (c), the next correct nucleotide is inferred to be a nucleotide homolog of the fourth base type.

[0129] As an alternative or in addition to using interpolation, the checking step can use a decoding or disambiguation scheme to identify one or more nucleotide types, for example, as described in co-owned U.S. Patent No. 9,951,385 or U.S. Patent Application Serial No. 15 / 922,787 (published as U.S. Patent Application Publication No. 2018 / 0305749 A1) (each of which is incorporated herein by reference). For example, the inspection can be performed by the following steps: (a) contacting a primed template nucleic acid with a polymerase and a first nucleotide mixture under ternary complex stabilization conditions, wherein the first mixture comprises nucleotide homologs of a first base type and nucleotide homologs of a second base type; (b) contacting a primed template nucleic acid with a polymerase and a second nucleotide mixture under ternary complex stabilization conditions, wherein the second mixture comprises nucleotide homologs of the first base type and nucleotide homologs of a third base type; (c) inspecting the products of steps (a) and (b) for signals generated by a ternary complex, the ternary complex comprising the primed template nucleic acid, the polymerase, and the next correct nucleotide, wherein the signal collected for the product of step (a) is ambiguous for the first and second base types, and wherein the signal collected for the product of step (b) is ambiguous for the first and third base types, wherein the signal collected for the product of step (b) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (a) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (b) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (c) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (d) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (e) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (f) is ambiguous for the first and third base types, and wherein the signal collected for the product of step (g ...h) is ambiguous for the first and third base types, and wherein the signal collected for the + , PEI, betaine and / or inhibitory metal cations (e.g. Ca 2+ ) in the presence of a nucleic acid; (d) disambiguating the signal collected in step (c) to identify the base type that binds to the next correct nucleotide. Optionally, to achieve disambiguation, (i) a first base type is associated with the presence of a signal of the product of step (a) and the presence of a signal of the product of step (b), (ii) a second base type is associated with the presence of a signal of the product of step (a) and the absence of a signal of the product of step (b), and (iii) a third base type is associated with the absence of a signal of the product of step (a) and the presence of a signal of the product of step (b). Optionally, prior to step (c), unbound polymerase and / or unbound nucleotides are removed from the products of steps (a) and (b).

[0130] The disclosed technology provides advantages that are superior to various other technologies used in the DNA sequencing field. For example, it is not necessary to have a marker (for example, a FRET partner) on the template nucleic acid of a polymerase or initiation. In fact, in certain embodiments, the polymerase is not labeled, or does not produce any signal for identifying homologous or non-homologous nucleotides in a ternary complex. In the disclosed method, signal energy (for example, FRET) does not need to be transferred between a polymerase and the labeled nucleotide to make the ternary complex detectable. The marker or dye of the detectable nucleotide used in the method herein need not be an intercalating dye (for example, not an intercalating dye disclosed in US8,399,196), which changes its signal generation properties (for example, fluorescent output) when participating in the ternary complex. Similarly, the marker or dye present on the labeled nucleotide need not be a conformation-sensitive dye, which can change spectral characteristics when it is a homologous nucleotide present in a ternary complex.

[0131] In some embodiments, the method of the present disclosure may include the step of extending the primer. For example, the method of detecting the primed template nucleic acid may include the following steps: (a) providing a mixture comprising a ternary complex, the ternary complex comprising the primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture also includes an excess of the same type of polymerase present in the ternary complex and an excess of the same type of nucleotide present in the ternary complex; (b) using a Li + , PEI, betaine and / or inhibitory metal cations (e.g. Ca 2+ ) fluid; (c) when the ternary complex contacts the fluid containing the stabilizer, detecting the ternary complex; (d) identifying the next correct base of the primed template nucleic acid molecule according to the result of step (c); and (e) extending the primer of the primed template nucleic acid. Optionally, the method may further include step (f): repeating steps (a) to (e) using the primed template nucleic acid with the extended primer instead of the primed template nucleic acid.

[0132] In yet another example of a method comprising a primer extension step, the steps of the method may include: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of nucleotides of the same type present in the ternary complex; (b) + , PEI, betaine and / or inhibitory metal cations (e.g. Ca 2+ ) replaces the excess nucleotides, (c) when the ternary complex is reacted with a Li +, PEI, betaine and / or inhibitory metal cations (e.g. Ca 2 + ), (d) identifying the next correct base of the primed template nucleic acid molecule according to the result of step (c); and (e) extending the primer of the primed template nucleic acid. Optionally, the method may further include step (f): repeating steps (a) to (e) using the primed template nucleic acid with the extended primer instead of the primed template nucleic acid.

[0133] The primer extension step can be carried out by contacting the initiated template nucleic acid with an extension reaction mixture. Typically, the fluid present in the previous inspection step is removed and replaced with the extension reaction mixture. Alternatively, the extension reaction mixture can be formed by adding one or more reagents to the fluid present in the inspection step. Optionally, the extension reaction mixture includes a nucleotide composition different from that of the inspection step. For example, the inspection step may include one or more nucleotide types that are not present in the extension reaction, and vice versa. By way of more specific examples, at least one type of nucleotide can be omitted in the extension step, and at least four types of nucleotides can be used in the inspection step. Optionally, one or more nucleotide types are added to the detection mixture used for the primer extension step.

[0134] Nucleotides present in the inspection step may result in unwanted nucleotide incorporation if carried into the extension step. Therefore, a washing step may be used to remove nucleotides prior to the primer extension step. Optionally, free nucleotides may be removed by enzymes such as phosphatases, chemical modifications, or physical separation techniques.

[0135] The primer extension step can use any of a variety of polymerases, including, for example, the polymerases described above in the context of forming a ternary complex. However, the polymerase used for extension has catalytic activity and is used under conditions that do not exclude catalysis. It is not necessary to attach the polymerase used for the extension step to an exogenous marker (e.g., covalently or otherwise). Alternatively, the polymerase used for primer extension can include an exogenous marker, for example, a marker used in a previous inspection step. The formation of the ternary complex and the extension step can use the same type of polymerase, although the conditions are different. Alternatively, these two steps can be performed by different types of polymerases.

[0136] The nucleotide of reversible termination is added to the 3 ' end of the primer to provide the means to prevent adding more than one nucleotide in the primer in the extension step, and further prevent the undesirable extension of the primer in the subsequent inspection step. Therefore, the position adjacent to the nucleotide of the specific type in the template can be checked. In such embodiments, a stable ternary complex can be formed at this position, and it is checked to detect the next correct nucleotide of the template hybridized with the reversible terminated primer of extension. The method can be repeated in a stepwise manner by removing or modifying the reversible terminator part from the primer extended, reversibly terminated to produce an extendable primer.

[0137] Typically, the reversibly terminated nucleotides of primers added in the method set forth herein do not have exogenous markers. This is because the primers extended do not need to be detected in the method set forth herein. However, if desired, the reversibly terminated nucleotides of one or more types used in the method set forth herein can be detected, for example, by the exogenous markers attached to the nucleotides. Exemplary reversible terminators, methods for incorporating them into primers, and methods for modifying primers to further extend (commonly referred to as "unblocking") are described in U.S. Patent Nos. 7,544,794, 7,956,171, 8,034,923, 8,071,755, 8,808,989, or 9,399,798. Additional examples are described in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,211,414, U.S. Pat. No. 7,315,019, U.S. Pat. No. 7,405,281, and US 2008 / 0108082 (each of which is incorporated herein by reference). Additional examples of reagents and conditions that can be used for the polymerase-based primer extension step include, for example, those described in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), No. 15 / 581,383 (published as U.S. Patent Application Publication No. 2018 / 0208922 A1, or U.S. Patent Application Publication No. 2018 / 0208983 A1 (which claims priority to Nos. 62 / 450,397 and 62 / 506,759)), each of which is incorporated herein by reference.

[0138] The method of the present disclosure may include a step of contacting the initiated template nucleic acid with a polymerase and a nucleotide mixture under conditions that produce an extended primer hybrid. One or more nucleotides in the mixture may be reversibly terminated. For example, at least 1, 2, 3, 4 or more nucleotide types may be reversibly terminated in the mixture. Alternatively or additionally, up to 4, 3, 2 or 1 nucleotide types may be reversibly terminated in the mixture. Similarly, one or more nucleotide types reversibly terminated in the mixture may be complementary to at least 1, 2, 3 or 4 base types in the template nucleic acid. Alternatively or additionally, the nucleotide types reversibly terminated in the mixture may be complementary to up to 4, 3, 2 or 1 base types in the template nucleic acid. Reversibly terminated and non-terminated nucleotides may be present in the extension reaction simultaneously. For example, some or all nucleotide types may be delivered simultaneously in a single extension reaction. Alternatively, different nucleotide types may be delivered continuously (individually or in the form of subsets) so that they are combined in a single extension reaction. Use of a mixture of terminating and extendable nucleotides can allow determination of low-resolution sequences and / or extension of read lengths, as described, for example, in U.S. patent application Ser. No. 16 / 265,942 (which claims priority to U.S. patent application Ser. No. 62 / 626,836), each of which is incorporated herein by reference.

[0139] In a specific embodiment, the reagent used in the primer extension step is removed from contact with the template nucleic acid that initiates, before the step of forming a stable ternary complex with the template nucleic acid that initiates. For example, when one or more types of nucleotides in the mixture interfere with the formation or detection of the ternary complex in the subsequent inspection step, it may be necessary to remove the nucleotide mixture used in the extension step. Similarly, it may be necessary to remove the polymerase or cofactor used in the primer extension step to prevent unwanted catalytic activity in the detection step. A washing step may be performed after the removal, in which an inert fluid is used to remove the template nucleic acid that initiates the residual components of the extension mixture.

[0140] The washing step can be performed between any of the various steps described herein. For example, the washing step can be used to separate the separated template nucleic acid from other reagents that are in contact with the template nucleic acid under ternary complex stabilization conditions. This washing can remove one or more reagents so that they do not interfere with the inspection of the mixture, or do not contaminate the second mixture formed on the substrate (or in the container) that was previously in contact with the first mixture. For example, the template nucleic acid that is initiated can be contacted with a polymerase and at least one type of nucleotide to form a first mixture under ternary complex stabilization conditions, and the first mixture can be inspected. Optionally, washing can be performed before inspection to remove reagents that do not participate in the formation of a stable ternary complex. Alternatively or additionally, washing can be performed after the inspection step to remove one or more components of the first mixture from the template nucleic acid that is initiated. Then, the template nucleic acid that is initiated can be contacted with a polymerase and at least one other nucleotide under ternary complex stabilization conditions to form a second mixture, and the ternary complex formation of the second mixture can be inspected. As previously described, optional washing can be performed before the second inspection to remove reagents that do not participate in the formation of a stable ternary complex. The wash in contact with the ternary complex may include a stabilizing liquid, such as Li + , PEI, betaine, inhibitory metal ions (such as Ca 2+ ) or those of other ternary complex stabilizers described herein.

[0141] The method disclosed herein may include multiple repetitions of the steps set forth herein. This repetition may provide the sequence of the template nucleic acid or the signature of the template nucleic acid. The inspection and extension steps may be repeated multiple times, and the optional steps of deblocking or washing away unwanted reactants or products to the primers between different steps may also be repeated multiple times. Therefore, the template nucleic acid that is triggered may be subjected to at least 2, 5, 10, 25, 50, 100 or more steps of the method set forth herein. Not all steps need to be repeated, and the repeated steps do not need to be performed in the same order in each repetition. For example, real-time analysis (i.e., in parallel with the fluid and detection steps of the sequencing method) may be used to identify the next correct nucleotide at each position of the template. However, real-time analysis is not necessary, but after some or all fluids and detection steps are completed, the next correct nucleotide may be identified.

[0142] The stable ternary complex, or the component that can form (i.e. participate in forming) the stable ternary complex can be attached to the solid support. The solid support can be made of any of the various materials used for analytical biochemistry. Suitable materials can include glass, polymeric materials, silicon, quartz (fused silica), borofloat glass, silicon dioxide, silicon-based materials, carbon, metal, optical fiber or optical fiber bundle, sapphire or plastic material. Specific materials can be selected according to the properties required for a specific application. For example, materials that are transparent to the required radiation wavelength are useful for analytical techniques that utilize radiation of the wavelength. On the contrary, it may be necessary to select materials that do not penetrate a certain wavelength of radiation (e.g., opaque, absorptive or reflective). Other properties of the available materials are inertness or reactivity to certain reagents used in downstream processes such as those described herein, or ease of operation, or low manufacturing cost.

[0143] Particularly useful solid carriers are particles, such as beads or microspheres. Bead populations can be used for attachment of stable ternary complex populations or components (e.g., polymerases, templates, primers, or nucleotides) that can form complexes. In some embodiments, it may be useful to use a configuration in which each bead has a single type of stable ternary complex or a single type of component that can form a complex. For example, a single bead can be attached to a single type of ternary complex, a single type of template allele, a single type of template locus, a single type of allele-specific primer, a single type of locus-specific primer, or a single type of nucleotide. Alternatively, different types of components do not need to be separated from each bead. In this way, a single bead can carry a variety of different types of ternary complexes, template nucleic acids, primers, template nucleic acids and / or nucleotides that are triggered. The composition of beads can vary, for example, depending on the format, chemistry, and / or attachment method to be used. Exemplary bead compositions include solid carriers for protein and nucleic acid capture methods and chemical functionality conferred thereon. Such compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex, or cross-linked dextran, such as Sepharose. TM , cellulose, nylon, cross-linked micelles and Teflon TM , and other materials described in the "Microsphere Detection Guide" by Bangs Laboratories, Fishers Ind (which is incorporated herein by reference).

[0144] The geometry of the particles, beads or microspheres may also correspond to a variety of different forms and shapes. For example, they may be symmetrically shaped (e.g., spherical or cylindrical) or irregularly shaped (e.g., controlled pore glass). In addition, the beads may be porous, thereby increasing the surface area available for capturing the ternary complex or its components. Exemplary size ranges of beads used herein may vary from nanometers to millimeters or from about 10 nm to 1 mm.

[0145] In certain embodiments, the beads may be arranged or otherwise spatially separated. Exemplary bead-based arrays that may be used include, but are not limited to, the BeadChip available from Illumina, Inc. (San Diego, CA). TM Arrays or arrays such as those described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570 or 7,622,294 or PCT Publication No. WO 00 / 63437 (each of which is incorporated by reference). The beads can be located at discrete locations on the solid support, such as holes, whereby each location accommodates a single bead. Alternatively, the discrete locations where the beads are located can each include a plurality of beads, as described in, for example, U.S. Patent Application Publication Nos. 2004 / 0263923 A1, 2004 / 0233485 A1, 2004 / 0132205 A1 or 2004 / 0125424 A1 (each of which is incorporated by reference herein).

[0146] As will be appreciated from the bead array embodiments described above, the methods of the present disclosure can be performed in a multiplex format, thereby detecting multiple different types of nucleic acids in parallel in the methods described herein. Although different types of nucleic acids can also be processed sequentially using one or more steps of the methods described herein, parallel processing can save costs, save time, and provide consistency of conditions. The compositions or methods of the present disclosure may include at least 2, 10, 100, 1 x 10 3 1x10 4 1x 10 5 1x 10 6 1x 10 9 Alternatively or additionally, the compositions or methods of the present disclosure may include up to 1 x 10 9 1x 10 6 1x 10 5 1x 10 4 1x 10 3Thus, the various reagents or products (e.g., primed template nucleic acids or stable ternary complexes) useful in the devices or methods described herein can be multiplexed to have different types or species within these ranges.

[0147] Additional examples of commercially available arrays that can be used include, for example, the Affymetrix GeneChip TM Arrays. Spotted arrays may also be used according to some embodiments. A typical spotted array is the CodeLink Array available from Amersham Biosciences. TM Another useful array is one that uses an inkjet printing method, such as SurePrint, available from Agilent Technologies. TM Arrays manufactured by technology.

[0148] Other useful arrays include those used for nucleic acid sequencing applications. For example, arrays for fixing the amplicons (commonly referred to as clusters) of genomic fragments can be particularly useful. Examples of nucleic acid sequencing arrays that can be used herein include those described in the following documents: Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO 91 / 06678, WO 04 / 018497 or WO 07 / 123744, U.S. Patent No. 7,057,026, No. 7,211,414, No. 7,315,019, No. 7,329,492 or No. 7,405,281 or U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0149] Nucleic acid can be attached to a carrier in a manner that provides detection at the single molecule level or the ensemble level. For example, a variety of different nucleic acids can be attached to a solid carrier in such a manner that a single stable ternary complex formed on a nuclear nucleic acid molecule on the carrier can be distinguished from all adjacent ternary complexes formed on the nucleic acid molecule of the carrier. Like this, one or more different templates can be attached to a solid carrier in such a form that the format physically isolates and detects each single molecule template in a manner that single molecules are resolved from all other molecules on the solid carrier.

[0150] Alternatively, the method disclosed herein can be performed for one or more nucleic acid ensembles, which are nucleotide populations with a common template sequence. Clustering methods can be used to attach one or more ensembles to a solid support. In this way, an array can have multiple ensembles, each of which is referred to as a cluster or array feature in this format. Clusters can be formed using methods known in the art such as bridge amplification or emulsion PCR. Useful bridge amplification methods are described in, for example, U.S. Patent No. 5,641,658 or No. 7,115,400, or U.S. Patent Publication No. 2002 / 0055100 A1, No. 2004 / 0002090 A1, No. 2004 / 0096853 A1, No. 2007 / 0128624 A1, or No. 2008 / 0009420 A1. Emulsion PCR methods include, for example, methods described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, or U.S. Patent Publication No. 2005 / 0130173 A1 or No. 2005 / 0064460 A1, each of which is incorporated herein by reference in its entirety. Another useful method for amplifying nucleic acids on a surface to form clusters is rolling circle amplification (RCA), for example, as described in Lizardi et al., Nat. Genet. 19:225-232 (1998) or US 2007 / 0099208 A1 (each of which is incorporated herein by reference).

[0151] In a specific embodiment, a stable ternary complex, a polymerase, a nucleic acid or a nucleotide is attached to a solid support on a flow cell surface or in a flow cell. The flow cell allows for convenient fluid manipulation by moving a solution in and out of the fluid chamber of the ternary complex of the contact binding support. The flow cell also provides detection of fluid manipulation components. For example, a detector can be positioned to detect a signal from a solid support, such as a signal from a marker that is recruited to a solid support due to formation of a stable ternary complex. Exemplary flow cells that can be used are described in, for example, U.S. Patent Application Publication No. 2010 / 0111768A1, WO 05 / 065814 or U.S. Patent Application Publication No. 2012 / 0270305 A1 (each of which is incorporated herein by reference).

[0152] The present disclosure provides systems for detecting nucleic acids, for example, using the methods described herein. For example, the system can be configured for reactions involving checking the interaction between a polymerase and a triggered template nucleic acid in the presence of nucleotides to identify one or more bases in a template nucleic acid sequence. Optionally, the system includes components and reagents for performing one or more steps described herein, including but not limited to: forming at least one stable ternary complex between a triggered template nucleic acid, a polymerase, and the next correct nucleotide; contacting the ternary complex with a stabilizing fluid; detecting one or more stable ternary complexes; extending the primer of each triggered template nucleic acid; and / or identifying nucleotides, nucleotide sequences, or a series of base multiplexes present in the template.

[0153] The system of the present disclosure may include a container or solid support for performing a nucleic acid detection method. For example, the system may include an array, a flow cell, a multi-well plate or other convenient device. The container or solid support may be removable, thereby allowing it to be placed in the system or removed from the system. In this way, the system can be configured to sequentially process multiple containers or solid supports. The system may include a fluid system having a container for accommodating one or more reagents described herein (e.g., a polymerase, a primer, a template nucleic acid, one or more nucleotides, nucleotides for primer extension, a deblocking reagent, a stabilizing solution or a mixture of such components for forming a ternary complex). The fluid system may be configured to deliver the reagent to the container or solid support, for example, via a channel or a droplet transfer device (e.g., an electrowetting device). Any of a variety of detection devices may be configured to detect a container or solid support that interacts with a reagent. Examples include luminescence detectors, surface plasmon resonance detectors, and other detectors known in the art. Exemplary systems having fluid and detection components that can be readily modified for use in the systems of the present invention include, but are not limited to, those described in U.S. patent application serial numbers 62 / 481,289 or and 15 / 922,661 (published as U.S. patent application publication number 2018 / 0280975 A1), U.S. Patent Nos. 8,241,573, 7,329,860, or 8,039,817, or U.S. patent application publication numbers 2009 / 0272914 A1 or 2012 / 0270305 A1 (each of which is incorporated herein by reference).

[0154] Optionally, the system of the present disclosure also includes a computer processing unit (CPU) configured as an operating system component. The same or different CPUs can interact with the system to obtain, store and process signals (e.g., signals detected in the method set forth herein). In a specific embodiment, the CPU can be used to determine the identity of the nucleotides at a specific position present in the template nucleic acid according to the signal. In some cases, the CPU will identify the nucleotide sequence of the template according to the detected signal.

[0155] Useful CPUs may include one or more of a personal computer system, a server computer system, a thin client, a fat client, a handheld or laptop device, a multiprocessor system, a microprocessor-based system, a set-top box, a programmable consumer electronics product, a network PC, a small computer system, a large computer system, a smart phone, and a distributed cloud computing environment including any of the above systems or devices. The CPU may include one or more processors or processing units, a memory architecture that may include RAM and non-volatile memory. The memory architecture may also include removable / non-removable, volatile / non-volatile computer system storage media. In addition, the memory architecture may include one or more readers for reading from and writing to non-removable, non-volatile magnetic media, such as a hard drive, a disk drive for reading from and writing to a removable, non-volatile disk, and / or an optical drive for reading from or writing to a removable, non-volatile optical disk, such as a CD-ROM or DVD-ROM. The CPU may also include various computer system readable media. Such media may be any available media accessible to a cloud computing environment, such as volatile and non-volatile media, as well as removable and non-removable media.

[0156] The memory architecture may include at least one program product having at least one program module implemented as executable instructions, which is configured to perform one or more steps of the method set forth herein. For example, the executable instructions may include an operating system, one or more application programs, other program modules, and program data. Typically, program modules may include routines, programs, objects, components, logic, data structures, etc., which perform specific tasks set forth herein.

[0157] The components of the CPU may be coupled via an internal bus that may be implemented as any one or more of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an accelerated graphics port, and a processor or local bus using any of a variety of bus architectures. By way of example and not limitation, such architectures include an Industry Standard Architecture (ISA) bus, a Micro Channel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, a Video Electronics Standards Association (VESA) local bus, and a Peripheral Component Interconnect (PCI) bus.

[0158] CPU can optionally communicate with one or more external devices such as keyboard, pointing device (pointing device) (such as mouse), display (such as graphical user interface (GUI)) or other devices that are convenient to interact with nucleic acid detection system. Similarly, CPU can communicate with other devices (for example, through network card, modem, etc.). This communication can be carried out through I / O interface. In addition, the CPU of system herein can communicate with one or more networks such as local area network (LAN), wide area network (WAN) and / or public network (for example, Internet) through suitable network adapter.

[0159] The present invention also provides a composition of matter comprising one or more of the components described herein, for example, in the context of a method for stabilizing a ternary complex. In a particular embodiment, the composition comprises a fluid comprising a ternary complex and Li + , wherein the ternary complex includes the initiated template nucleic acid, the polymerase, and the nucleotide homolog of the next correct base of the initiated template nucleic acid molecule. The fluid may also contain betaine, PEI or an inhibitory metal ion (e.g., Ca 2+ ) in one or more. Optionally, the ternary complex is fixed on a solid support, for example, by attaching the template nucleic acid to the solid support. The material for the solid support and the attachment device can be any of a variety of materials described herein or known in the art. For example, the solid support may include an array of features, and each feature may include a fixed ternary complex. The features may have a size, spacing, density or other features consistent with the disclosure described above. One or more components of the ternary complex, such as a polymerase and / or nucleotides, may contain an exogenous marker, such as a luminophore or another marker described herein. The fluid may also contain unbound nucleotide homologues of at least 1, 2, 3 or 4 bases suspected to be present in the template nucleic acid. Unbound nucleotide homologues may have different exogenous markers, which may distinguish one nucleotide type from another nucleotide type. Alternatively or additionally, the fluid may include an unbound polymerase, which optionally contains an exogenous marker. In a specific embodiment, the template nucleic acid that is triggered includes a closed primer (e.g., a reversibly terminated primer). The concentration of the ternary complex in the fluid can be greater than the concentration of unbound nucleotides and / or greater than the concentration of unbound polymerase in the fluid. The fluid can be an aqueous fluid. The fluid can be free of alcohol, oil, or other solvents that do not dissolve nucleotides or polymerase, including, for example, the non-miscible fluids set forth in U.S. Patent Application Serial No. 16 / 164,417 (which claims priority to U.S. Patent Application Serial No. 62 / 574,308) (each of which is incorporated herein by reference).

[0160] The present disclosure also provides kits that can be used, for example, to characterize nucleic acids. The kits may include reagents for performing one or more of the methods described herein. For example, the kit may include reagents that produce a stable ternary complex when mixed with one or more primed template nucleic acids. More specifically, the kit may include Li used in the methods described herein. + , PEI, betaine or inhibitory metal ions (such as Ca 2+ ), including, for example, the method described in the Examples section below. In addition, the kit may include at least one nucleotide type and a polymerase capable of forming a stable ternary complex. The polymerase for the extension step may also be included in the kit. Nucleotides, polymerases, or both may include exogenous markers, for example, as described herein in the context of the various methods.

[0161] Thus, any component or article for practicing the methods described herein can be effectively packaged as a kit. For example, a kit can be packaged to include some, many, or all of the components or articles for practicing the methods described herein. Exemplary components include, for example, labeled nucleotides (e.g., extendable labeled nucleotides); polymerases (labeled or unlabeled); nucleotides with terminator moieties (e.g., unlabeled, reversibly terminated nucleotides); a stabilizing solution, such as a solution containing Li + , PEI, betaine and / or inhibitory metal cations (such as Ca 2+ ) fluids; deblocking reagents as described herein and in the references cited herein, etc. Any such reagents may include, for example, some, many or all of the fluids, components and / or products used to implement one or more subsequent steps for analyzing the triggered template nucleic acid. The kit need not include primers or template nucleic acids. Instead, the user of the kit can provide the triggered template nucleic acid in combination with the kit components. Similarly, the kit may exclude one or more components described herein, and optionally, such excluded components may be provided by the end user.

[0162] One or more auxiliary reagents may also be included in the kit. Such auxiliary reagents may include any of the reagents exemplified above and / or other types of reagents that can be used to implement the methods described herein. The kit may also include instructions. The instructions may include, for example, procedures for preparing any components used in the methods described herein, performing one or more steps of any embodiment of the methods described herein, and / or instructions for performing any subsequent analysis steps using the triggered template nucleic acid.

[0163] In certain embodiments, the test kit includes a box having a container for accommodating a reagent, and also having a fluid component for transferring the reagent from the container to a detection instrument. For example, the fluid assembly can be configured to transfer the reagent to a flow cell, in which a stable ternary complex is detected. The exemplary fluid box that may be included in the test kit (or system) of the present disclosure is described in U.S. Patent Application Serial No. 15 / 922,661 (published as U.S. Patent Application Publication No. 2018 / 0280975 A1, which claims 62 / 481,289 priority) (each of which is incorporated herein by reference).

[0164] Embodiment 1

[0165] Stabilization of the ternary complex using aqueous stabilizing solutions of solubilized polymerase and nucleotides

[0166] This example demonstrates the examination of a blocked primed template nucleic acid in a manner that evaluates the binding of each of four different labeled nucleotides separately. An aqueous solution containing an agent capable of stabilizing a ternary complex is used to: (1) deliver an examination solution containing a polymerase, labeled nucleotides, and magnesium cations; (2) separate the immobilized ternary complex from contact with unbound polymerase and labeled nucleotides; and (3) facilitate ternary complex detection over an extended period of time during an imaging wash step. The different nucleotides used in the procedure are labeled with the same type of fluorescent detectable marker, and each type of nucleotide is delivered and examined separately. Alternatively, the nucleotides may each have a different detectable marker, and several different labeled nucleotide types may have been contacted with the blocked primed template nucleic acid simultaneously.

[0167] The flow cell containing the triggered template nucleic acid was prepared as follows. Template nucleic acid chains synthesized in 12 PCR reactions using 5'-biotinylated primers were prepared and then individually bound to magnetic beads coated with streptavidin. This resulted in a population with 12 bead types, each of which contained a homogenous collection of template chains. The beads used in the procedure have been functionalized with 1mM NHS-PEG4-TCO in phosphate buffered saline (PBS). Next, the beads with fixed template chains were flowed over the surface of the aminosilane flow cell functionalized with tetrazine. The mixture was incubated for 1 hour to allow the modified beads to be covalently attached to the functionalized surface in the flow cell.

[0168] Next, the sequencing primer is flowed into the flow cell and hybridized to the immobilized template strand. In the aggregated bead set, there is a primed template nucleic acid with each of the four bases (i.e., A, C, G, or T) as the next template nucleotide. A reversible terminator nucleotide is then incorporated into the 3'-end of the hybridized sequencing primer to produce a closed set of primed template nucleic acid molecules. This is accomplished using a pH buffered incorporation mixture containing 10 U / ml of Therminator TM polymerase and 200 nM of unlabeled reversibly terminated nucleotide analogs of dATP, dGTP, dCTP, and dTTP. The reversible terminator nucleotides used in this illustrative procedure include 3'-ONH 2 The reversible terminator portion can be obtained by reacting with a NaNO 2 The terminator nucleotides are removed by contact with an acetate buffer solution. A description of such reversible terminator nucleotides can be found in U.S. Pat. No. 7,544,794, the disclosure of which is incorporated herein by reference.

[0169] Next, multiple inspection conditions are screened using fixed beads attached to the closed, triggered template nucleic acid molecules. In the ternary complex formation and detection steps, the reversible terminator portion on the 3' nucleotide of the primer strand excludes nucleotide incorporation. This allows multiple detections of the same base position to determine which conditions support the specific formation and detection of the ternary complex. Four different solutions are introduced into the flow cell to allow the ternary complex to form, respectively, the solution comprising a polymerase and a single nucleotide labeled with a Cy5 moiety on its base (i.e., Cy5-dATP, Cy5-dGTP, Cy5-dCTP or Cy5-dTTP, as described in U.S. Patent Application Serial No. 15 / 873,343 (which is incorporated herein by reference)).

[0170] After delivering each solution containing nucleotides and polymerase to allow ternary complex formation, the flow cell was washed with imaging solution to remove non-complexed nucleotides and polymerase. Ternary complexes were detected during the imaging wash step. The solution (EXAM solution) that provides polymerase and labeled nucleotides to form ternary complexes on the immobilized blocked primed template nucleic acid and the imaging solution (IMG solution) both contained 20 mM Tricine buffer (pH 8.42), 50 mM KCl, 0.1% Tween-80, 10 mM (NH 4 ) 2 SO 4 and 3% sucrose. The solution used to form the ternary complex also contained 20 U / ml of Therminator TM DNA polymerase (New England Biolabs; Ipswich, MA), 1 mM MgCl2 and Cy5-dNTPs (400 nM each for Cy5-dATP, Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP). The polymerase, MgCl 2 Each of the two solutions also contained LiCl and / or betaine as stabilizing additives, wherein the solutions used in the same procedure contained similar concentrations.

[0171] After imaging the ternary complex in the imaging wash step, the polymerase and labeled nucleotides were stripped from the ternary complex by washing the flow cell with a buffered guanidine thiocyanate solution containing EDTA. The flow cell was then prepared for the next base check by washing with a pre-spiking buffer (20 mM Tricine (pH 8.42), 50 mM KCl, 0.1% Tween-80, and 0.1% hydroxylamine). In addition to the imaging solution, which did not contain polymerase, MgCl 2 EXAM and IMG solutions contained the same core components except for the 5 or 50 mM LiCl solution with or without 1 M betaine (N,N,N-trimethylglycine). Table 1 summarizes the concentrations of LiCl and betaine used in the experiments. Figure 1A and Figure 1B The results of these procedures are shown in .

[0172] Table 1

[0173]

[0174] Figure 1A It is illustrated how lithium cations (e.g., produced by the dissolution of lithium salts in water) and / or betaine advantageously increase the fluorescence "on" signal of each of the four tested nucleotides without increasing the fluorescence "off" signal. The "on" intensity is the median pixel count of all correctly called nucleotides from the imaging camera, and the "off" intensity is the median pixel count of the remaining three nucleotides (i.e., incorrect nucleotides) detected at the same feature from the imaging camera. It is noteworthy that the labeled dTTP nucleotides showed the greatest sensitivity to the different conditions, with the lowest "on" signal associated with the complete absence of lithium cations and betaine. The trends were similar for all four nucleotides. It is noteworthy that although both tested concentrations of LiCl gave essentially saturated "on" intensities, these results were further improved by the addition of betaine. This additive effect strongly suggests that lithium cations and betaine improve the binding system through different mechanisms.

[0175] Figure 1BThe use of lithium and betaine additives to increase confidence in correct base calls is demonstrated graphically. Here, purity values ​​are calculated by dividing the brightest nucleotide intensity value by the sum of the brightest nucleotide intensity and the second brightest nucleotide intensity values. To display on a scale of 0 to 1, an adjustment is made by subtracting 0.5 from the result and then multiplying by 2. As indicated, the inclusion of lithium ions in the imaging solution used to detect the ternary complex increases the purity value, particularly for the labeled dTTP nucleotide. In the absence of lithium, the addition of betaine advantageously increases the purity value of the dTTP ternary complex.

[0176] Separate experiments (data not shown) showed that increasing the LiCl concentration to 100 mM, 175 mM or 250 mM also provided high "on" signals, indicating a stabilizing effect on the ternary complex. However, 500 mM LiCl resulted in a significant decrease in the "on" signal and a decrease in purity compared to lower concentrations of LiCl, consistent with destabilization of the ternary complex.

[0177] These results indicate that lithium cations and betaine can provide ternary complex stabilization and can be included in aqueous assay fluids to allow for improved signal-to-noise ratios when detecting ternary complexes in the presence of uncomplexed labeled nucleotides.

[0178] Example II

[0179] Stabilization of the ternary complex over time

[0180] This example describes a time titration demonstrating improved detection of the ternary complex by washing with imaging buffer containing lithium and / or betaine for up to 5 minutes.

[0181] As described in Example 1, blocked 3'-ONH from 12 PCR reactions were 2 Primer template DNA for SBB TM The reaction was repeated except that the extension step was omitted so that the same template position was repeatedly examined. The ternary complex was allowed to form by washing in EXAM solution as described in Example 1. The IMG solution was then introduced into the flow cell and flowed at a flow rate of 1 μL / s for 20 seconds, 2 1 / 2 The washing time and the content of IMG solution used for 15 cycles of the test were shown in Table 2.

[0182] Table 2

[0183]

[0184] like Figure 2As shown, lithium (50 mM) and betaine (1 M) provided enhanced "on" signal intensity for the ternary complex, as shown in cycles 1-3 and 13-15. In the absence of lithium and betaine, the ternary complex dissociated and the ability to accurately distinguish the correct base was eliminated (as shown in cycles 4-6). Addition of 50 mM lithium (cycles 7-9) allowed sequencing, but further addition of 1 M betaine in combination with 50 mM lithium (cycles 1-3 and cycles 13-15) showed a synergistic effect, greatly improving the overall quality.

[0185] Figure 2 The results showed that betaine helps stabilize the ternary complex for a short duration, but 1 / 2 After 20 seconds, 2 1 / 2 At the time points of 15 minutes and 5 minutes, the overall intensity decays linearly, but Figure 3 As evidenced by the results shown, purity values ​​remained high and the "on" intensity was satisfactory for sequencing. The collected data showed that 50 mM LiCl and 1 M betaine provided ternary complex stabilization over time.

[0186] Sequencing reactions were performed with 0 mM, 5 mM and 50 mM LiCl and their quality was observed over the course of 20 cycles. Betaine was present at a concentration of 1 M in all conditions. FIG. 4A to FIG. 4C The results shown demonstrate that increasing the LiCl concentration up to 50 mM increases the interval between the "on" and "off" signals and produces more uniform intensities over 20 cycles. The "on" intensity of 50 mM LiCl is higher than that of 5 mM LiCl, indicating that the additional lithium helps to form and stabilize a larger population of ternary complexes. FIG. 5A to FIG. 5C It is shown that the purity is higher with the increase of LiCl concentration. The signal-to-noise ratio (SNR) is also improved with the increase of LiCl concentration to 50mM, as shown in FIG. 6A to FIG. 6C The results shown are shown. 7A to 7C As shown, when the LiCl concentration increased to 50 mM, the average error rate was also lower.

[0187] Example III

[0188] Stabilization of the ternary complex with an aqueous stabilizing solution containing lithium, calcium or betaine

[0189] This example illustrates the various combinations of lithium, calcium and betaine in SBB TM The effects of the steps on the stability of the ternary complex were examined.

[0190] As described in Example 1, blocked 3'-ONH from 12 PCR reactions were quantified in a flow cell. 2 Primer template DNA for SBB TM The reaction was repeated except that the extension step was omitted, thereby examining the same template position repeatedly with the following modifications. EXAM solution contained 20 mM Tricene pH 7.0, 3% sucrose, 50 mM KCl, 10 mM NH 4 (SO) 2 , 0.1% hydroxylamine, 0.1 mM EDTA, 0.1% Tween-80, 1 mM MgCl 2 、20U / ml Therminator TM DNA polymerase and Cy5-dNTP (400 nM each for Cy5-dATP, Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP). The IMG solution contained 20 mM Tricene pH 7.0, 3% sucrose, 50 mM KCl, 10 mM NH 4 (SO) 2 , 0.1% hydroxylamine, 0.1 mM EDTA, 0.1% Tween-80. Each solution also contained variable additives as shown in the table below.

[0191] The ternary complex was formed by flowing the EXAM solution into the flow cell containing the DNA beads. The flow cell was then flushed with 16 μl of IMG solution for 16 seconds. After the IMG flush, the first image of the DNA beads in the flow cell was acquired. After a 60 second delay, the second image of the beads was acquired from the flow cell. The intensity of the "on" and "off" signals in the two images was compared to determine the stability of the ternary complex.

[0192] Table 3 shows the variable content of 8 tests optionally containing lithium, calcium or betaine.

[0193] Table 3

[0194]

[0195] The percentage loss of signal intensity at each cycle (from the first image to the second image) is shown in Figure 8As shown. The percentage of signal loss of the ternary complex formed in the presence of four different homologous nucleotide types is represented by individual bars (open bars for dATP, dashed bars for dGTP, solid bars for dCTP and shaded bars for dTTP). The most dramatic signal loss (i.e., the lowest stability) was observed for the ternary complex incubated in the absence of betaine, calcium and lithium (see cycle 2). The addition of 1M betaine has a moderate effect on stabilizing the ternary complex formed with dATP, dGTP and dCTP, but does not seem to stabilize the ternary complex formed with dTTP (see cycle 4). When both 1M betaine and 50mM LiCl are present, the addition of 50mM LiCl alone (see cycle 3) improves stability, and a further increase in stability is observed for the ternary complex formed with dATP, dGTP and dCTP (see cycle 1). The combination of betaine and lithium also has a stabilizing effect on the ternary complex formed with dTTP, although not as obvious as the other three nucleotide types.

[0196] continue Figure 8 Compared with the results of lithium alone, the addition of 0.5 mM CaCl 2 (See cycle 5) leading to an even greater increase in the stability of the ternary complex formed with dATP, dGTP and dCTP. 1 M betaine and 0.5 mM CaCl 2 The presence of 50 mM LiCl and 0.5 mM CaCl also stabilized the ternary complex, with a stabilization effect slightly better than that observed for calcium alone. 2 The combination of (see cycle 7) has a stabilizing effect on the ternary complexes, especially those formed with dATP, dGTP and dCTP. All three additives 1 M betaine, 50 mM LiCl and 0.5 mM CaCl 2 The presence of (see cycle 8) also has a stabilizing effect on ternary complexes, especially those formed with dATP, dGTP and dCTP.

[0197] Fig. 9 Purity values ​​obtained from the second image (i.e., after 60 seconds of incubation in IMG solution) are shown. Cycle 2, run in the absence of lithium, betaine, or calcium, had the lowest purity score, indicating dissociation of the ternary complex. Addition of betaine alone resulted in low purity values ​​for the ternary complex formed with dTTP. Cycles performed in the presence of lithium (with or without betaine) or calcium (with or without betaine) produced complexes with high purity.

[0198] These results indicate that the presence of betaine, lithium or calcium during ternary complex formation and detection improves the test results, most likely by stabilizing the ternary complex. Independently, lithium and calcium appear to be the most effective. Adding betaine to lithium or calcium has a synergistic effect in improving the results. Compared to the complex formed with dTTP, the ternary complex formed with dATP, dGTP and dCTP is significantly more stable under most of the conditions tested. However, the ternary complex formed with dTTP appears to be stabilized by betaine or lithium alone or in combination with betaine.

[0199] Several other metals were evaluated for their potential to stabilize ternary complexes in EXAM and IMG solutions. When examining ternary complexes, potassium, doubling the KCl concentration to 100 mM or adding 50 mM sodium, could be used, but ternary complexes, especially those formed with Cy5-dTTP, were not as stable as those formed in the presence of similar concentrations of lithium or calcium. Strontium (10 mM SrCl 2 ) provided some stability to the ternary complexes formed with purine nucleotides (Cy5-dATP and Cy5-dGTP), but provided little or no stability to the ternary complexes formed with pyrimidine nucleotides under the conditions tested. Under the conditions tested, nickel (2.5 mM NiSO 4 ) and ytterbium (YbCl 3 ) did not provide very effective stabilization for the ternary complex.

[0200] Example IV

[0201] Stabilization of the ternary complex with polyethyleneimine

[0202] As described in Example 1, blocked 3'-ONH from 12 PCR reactions were 2 Primer template DNA for SBB TM The IMG solution contained 20 mM Tricine buffer (pH 8.42), 50 mM KCl, 0.1% Tween-80, 10 mM (NH 4 ) 2 SO 4 and 3% sucrose and the variable addition reagents as described below. EXAM solution contains the same fixed and variable reagents as IMG solution and also contains 20 U / ml Therminator TM DNA polymerase (New England Biolabs; Ipswich, MA), 1 mM MgCl 2and Cy5-dNTPs (400 nM for each of Cy5-dATP, Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP).

[0203] Table 4

[0204]

[0205] SBB was performed under conditions containing variable additives of IMG and EXAM as specified in Table 4. TM Reaction. Under all conditions, beads produce signals indicating stable ternary complex formation. However, it is observed that as PEI concentration increases, the ability to remove PEI and ternary complexes from beads by standard washing schemes decreases. High salt washing (4M NaCl) effectively removes most of PEI, and base discrimination can be performed after PEI is removed by high salt washing. However, compared with the background under SOP conditions (i.e., no PEI, standard washing), higher background is observed after PEI is removed by high salt washing. Heparin is also found to be an effective reagent for washing out PEI. After using EXAM and IMG solutions containing 0.001% 2k branched PEI, PEI can be removed using a washing solution containing at least 30 μg / ml heparin (testing washing solutions containing up to 100 μg / ml heparin and finding it to be effective).

[0206] Fig.10 The results of a stability test performed as follows are shown. The inspection and imaging steps were performed on beads with a primed template with T as the next template nucleotide. For one lane of the flow cell, the Exam and IMG buffers contained 0.001% branched PEI 25k (at Fig.10 A control lane was processed in parallel but lacked added PEI (in Fig.10 The imaging procedure was modified so that a standard image (in Fig.10 The beads were incubated in the dark for 1 min and a second image was acquired ( Fig.10 The beads were then incubated for an additional 1 minute ( Fig.10 2min in ). Fig.10 The results clearly show that the presence of PEI significantly improves the stability of the ternary complex at the three imaging time points.

[0207] Fig.11The results of stability testing performed as follows are shown. Inspection and imaging steps were performed on beads with various primed templates. The beads were distributed into 6 different lanes of the flow cell. The 6 flow cell lanes were processed in parallel, but the EXAM and IMG solutions used in each lane differed as follows: no PEI added (SOP), 0.01% branched PEI 800, 0.025% branched PEI 800, 0.05% branched PEI 800, 0.075% branched PEI 800, 0.1% branched PEI 800. The imaging step was modified so that a standard image was obtained, the beads were incubated in the dark for 1 minute, and a second image was obtained ( Fig.11 The beads were then incubated for an additional 1 min before acquiring the third image ( Fig.11 The data obtained from several different bead types were averaged so that for the bars labeled A, all beads producing a signal indicating dATP as the next correct nucleotide were averaged; for the bars labeled G, all beads producing a signal indicating dGTP as the next correct nucleotide were averaged; for the bars labeled C, all beads producing a signal indicating dCTP as the next correct nucleotide were averaged; and for the bars labeled Fig.11 The bars marked T in the , averaged over all beads that produced a signal indicative of dTTP as the next correct nucleotide. It is evident from the results that branched PEI 800 provides improved stability, especially when concentrations approach or exceed 0.075%.

[0208] Wash solutions containing 40 μg / ml to 100 μg / ml heparin effectively removed branched PEI 800 and ternary complexes from DNA beads (after delivery of EXAM and IMG with 0.1% branched PEI 800). Wash solutions containing 100 μM to 1 mM sodium hexametaphosphate effectively removed branched PEI 800 and ternary complexes from DNA beads (after delivery of EXAM and IMG with 0.075% branched PEI 800).

[0209] The result of this embodiment shows that polyethyleneimine can stabilize ternary complex.Compared with smaller PEI molecules, larger PEI molecules are effective at lower concentrations.However, larger PEI molecules are more difficult to remove from DNA beads than smaller PEI molecules.Available heparin and hexametaphosphate can be stripped from DNA beads smaller PEI molecules.

[0210] Throughout this application, various publications, patents and / or patent applications are referenced. The disclosures of these documents are hereby incorporated by reference into this application in their entirety.

[0211] A number of embodiments have been described. However, it should be understood that various modifications may be made. Therefore, other embodiments are within the scope of the following claims.

Claims

1. A method for detecting a primed template nucleic acid, the method comprising the following steps: (a) providing a mixture comprising a ternary complex, the ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next base of the primed template nucleic acid, wherein the mixture further comprises an excess of the same type of nucleotide present in the ternary complex; (b) Using Li + The excess nucleotides are replaced by a fluid, wherein Li + The concentration of is 5 mM to 250 mM; and (c) When the ternary complex is combined with the Li + When the fluid contacts the ternary complex, the ternary complex is detected.

2. The method of claim 1, further comprising (d) identifying the next correct base of the primed template nucleic acid based on the result of step (c).

3. The method of claim 2, further comprising (e) extending a primer of the primed template nucleic acid.

4. The method of claim 3, wherein the primer is extended by adding nucleotides comprising a reversible terminator portion.

5. The method of claim 1, wherein the primer of the primed template nucleic acid comprises a reversible terminator portion.

6. The method of claim 5, further comprising (e) modifying or removing the reversible terminator portion to deblock the primer.

7. The method of claim 6, further comprising (f) extending the deblocked primer.

8. The method of claim 7, wherein the deblocked primer is extended by adding nucleotides comprising a reversible terminator portion.

9. The method of any one of claims 1 to 8, wherein the ternary complex is immobilized on a solid support.

10. The method of claim 1, wherein the nucleotide homologue comprises an exogenous marker detected in step (c).

11. The method of claim 10, wherein the mixture comprises nucleotide homologs of at least two different base types suspected to be present in the template nucleic acid.

12. The method of claim 11, wherein the nucleotide homologues of the different base types respectively comprise different exogenous labels.

13. The method of claim 1, wherein the polymerase comprises an exogenous label that is detected in step (c).

14. The method of claim 1, wherein the mixture further comprises an excess of the same type of polymerase present in the ternary complex.

15. The method of claim 14, wherein step (c) comprises using the Li-containing + The fluid replaces the excess polymerase and the excess nucleotides.

16. The method of claim 1, wherein the excess nucleotides are soluble in the fluid.

17. The method of claim 1, wherein step (b) comprises delivering to the ternary complex a + and a fluid lacking said type of nucleotide present in said ternary complex, thereby replacing said excess nucleotide with said fluid.

18. The method of claim 1, wherein in step (c) the Li + Present at concentrations between 50 mM and 100 mM.

19. The method of claim 1, wherein the fluid further comprises betaine.

20. The method of claim 1, wherein the fluid further comprises polyethyleneimine.

21. A composition comprising a fluid, the fluid comprising a ternary complex and Li + wherein the ternary complex comprises a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid, and wherein the composition Li + The concentration ranges from 5 mM to 250 mM.

22. The composition of claim 21, wherein Li + Present at concentrations between 50 mM and 100 mM.

23. The composition of claim 21, wherein the fluid further comprises betaine.

24. The composition of claim 21, wherein the fluid further comprises polyethyleneimine.

25. The composition of claim 21, wherein the ternary complex is immobilized on a solid support.

26. The composition of claim 25, wherein the solid support comprises an array of features, and wherein each feature comprises an immobilized ternary complex comprising a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid at the feature.

27. The composition of any one of claims 21 to 26, wherein the nucleotides comprise an exogenous label.

28. The composition of claim 21, wherein the fluid further comprises nucleotide homologs of at least four different base types suspected to be present in the template nucleic acid.

29. The composition of claim 28, wherein the nucleotide homologues of the different base types respectively comprise different exogenous labels.

30. The composition of claim 21, wherein the polymerase comprises an exogenous label.

31. The composition of claim 21, wherein the primed template nucleic acid comprises a reversibly terminated primer.

32. The composition of claim 21, wherein the fluid is aqueous.

33. The composition of claim 21, wherein the fluid further comprises free nucleotides at a concentration lower than the concentration of the ternary complex in the fluid, the free nucleotides being of the same type present in the ternary complex.

34. The composition of claim 21, wherein the fluid further comprises free polymerase at a concentration lower than the concentration of the ternary complex in the fluid, the free polymerase being of the same type present in the ternary complex.

35. A method for detecting a primed template nucleic acid, the method comprising the following steps: (a) providing a ternary complex and Li + The fluid of claim 1, wherein the ternary complex comprises a primed template nucleic acid, a polymerase, and a nucleotide homolog of the next correct base of the primed template nucleic acid, and wherein the fluid contains Li + The concentration of is 5 mM to 250 mM; and (b) When the ternary complex contains Li + When the ternary complex is in the fluid, the ternary complex is detected.

36. The method of claim 35, further comprising (c) identifying the next correct base of the primed template nucleic acid based on the result of step (b).

37. The method of claim 36, further comprising (d) extending a primer of the primed template nucleic acid.

38. The method of claim 37, wherein the primer is extended by adding nucleotides comprising a reversible terminator portion.

39. The method of claim 35, wherein the primer of the primed template nucleic acid comprises a reversible terminator portion.

40. The method of claim 39, further comprising (d) modifying or removing the reversible terminator portion to deblock the primer.

41. The method of claim 40, further comprising (e) extending the deblocked primer by adding nucleotides comprising a reversible terminator portion.

42. A method as claimed in any one of claims 35 to 41, wherein the ternary complex is immobilised on a solid support.

43. The method of claim 35, wherein the nucleotide homologue comprises an exogenous marker detected in step (b).

44. The method of claim 43, wherein the fluid comprises nucleotide homologs of at least two different base types suspected to be present in the template nucleic acid.

45. The method of claim 44, wherein the nucleotide homologues of the different base types respectively comprise different exogenous labels.

46. ​​The method of claim 35, wherein the polymerase comprises an exogenous label that is detected in step (c).

47. The method of claim 35, wherein the fluid further comprises free nucleotides at a concentration lower than the concentration of the ternary complex in the fluid, the free nucleotides being of the same type present in the ternary complex.

48. The method of claim 35, wherein the fluid further comprises free polymerase at a concentration lower than the concentration of the ternary complex in the fluid, the free polymerase being of the same type present in the ternary complex.

49. The method of claim 35, wherein the fluid further comprises betaine.

50. The method of claim 35, wherein the fluid further comprises polyethyleneimine.

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