Sequencing method

Abstract

The invention provides a hydrogel-based sequencing method, which is suitable for a Flowcell sequencing system and also supports an open sequencing system, and the cost can be effectively reduced while the sequencing efficiency and accuracy are ensured.

Description

Sequencing methods Technical Field

[0001] The present invention relates to the field of sequencing. Specifically, the present invention provides a hydrogel-based sequencing method that is applicable to flow cell sequencing systems and also supports open sequencing systems, and can effectively reduce costs while ensuring sequencing efficiency and accuracy. Background Art

[0002] With the advancement of sequencing technology and increasing public awareness, high-throughput gene sequencing has been widely used in various fields, including scientific research, precision medicine, social health, and public safety. Although sequencing costs have continued to decline in recent years, the price of over 1,000 RMB per WGS (whole genome sequencing, 100Gb of data) still hinders its wider application. Existing high-throughput sequencing (primarily referring to massively parallel sequencing (MPS)) technologies primarily utilize two approaches to achieve fluid exchange and reactions. The first is the flow cell approach, which uses syringe pumps and rotary valves to draw reagents from different locations and inject them into the flow channels of the chip, displacing the old reagents and replacing them with new ones. This completes the fluid exchange, allowing the replaced active ingredients to undergo biochemical reactions. The second is the immersion sequencing (DIP) approach, which pumps reagents into reagent reservoirs, transferring the sequencing chip between different reservoirs. Each time the chip is immersed in new reagents, diffusion exchanges the fluid and active ingredients. To effectively replace reagents, the flowcell solution requires a high replacement ratio (i.e., the ratio of the volume of new reagent pumped into the flow channel to the flow channel volume to completely replace the old reagent), generally requiring a ratio of 2-3 or more. Furthermore, the reagents are disposable, so while the reaction efficiency is high, the cost is also correspondingly high. Immersion sequencing solutions allow for the reuse of sequencing reagents, effectively reducing reagent costs. However, the immersion sequencing solution has the disadvantage that the chip is prone to drying out during the sequencing process. Using immersion sequencing solutions for some steps can increase costs or affect quality, and repeated use of reagents carries the risk of quality degradation.

[0003] Therefore, ensuring sequencing efficiency and accuracy while effectively reducing costs is a major challenge for high-throughput sequencing and a problem that needs to be solved urgently.

[0004] SUMMARY OF THE INVENTION

[0005] This application provides a hydrogel-based sequencing method that supports not only traditional flow cell-based sequencing systems but also open sequencing systems and large-scale sequencing chips. This method reduces reagent loss and avoids damage to target nucleic acid molecules on the slide surface caused by drying and crystallization. It also eliminates the need for complex fluidics and temperature control and effectively reduces reagent usage, enabling single-use sequencing and lowering costs.

[0006] In one aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting a labeled nucleotide with the target nucleic acid molecule to allow incorporation of the nucleotide; (2) detecting the label of the incorporated nucleotide; and (3) removing the label; wherein at least one of steps (1) and (3) is performed in a gel state; optionally, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule.

[0007] In certain embodiments, the nucleotide incorporation in step (1) forms a phosphodiester bond. In certain embodiments, step (3) allows the complementary strand of the target nucleic acid molecule to undergo an extension reaction.

[0008] In certain embodiments, the label is a fluorescent label.

[0009] In certain embodiments, the tag is an affinity tag, and step (2) includes step (2a), adding a luciferase carrying an affinity tag to perform an affinity reaction with the product of step (1); step (2b), adding a specific substrate to perform a catalytic reaction with the product of step (2a); and step (2c), detecting the product of step (2b). In certain embodiments, steps (2a) and (2b) are performed in a gel state.

[0010] In certain embodiments, when step (3) is performed in a gel state, the steps include: (3a) contacting a reaction composition comprising a removal reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having reversible thermo-induced gelling properties, and the reaction composition is in a liquid state; (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an excision reaction to allow removal of the label on the nucleotide; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0011] In another aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting a nucleotide carrying a blocking group with the target nucleic acid molecule to allow incorporation of the nucleotide; (2) detecting the incorporation of the nucleotide; and (3) removing the blocking group; wherein at least one of steps (1) and (2) is performed in a gel state; and optionally, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule. Preferably, the nucleotide incorporation in step (1) forms a phosphodiester bond. Preferably, step (3) allows the complementary strand of the target nucleic acid molecule to undergo an extension reaction.

[0012] In certain embodiments, step (2) includes step (2a), adding an affinity reagent carrying a fluorescent marker to undergo an affinity reaction with the product of step (1); and step (2b), detecting the product of step (2a). Preferably, step (2a) is carried out in a gel state. Preferably, the affinity reagent is an antibody against nucleotides A\T\C\G\U with a blocking group, and the antibody can specifically recognize the nucleotides and undergo an affinity reaction with them.

[0013] In certain embodiments, step (3) is performed in a gel state and comprises the following steps: (3a) contacting a reaction composition comprising a removal reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having reversible thermo-induced gelling properties, and the reaction composition is in a liquid state; (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an excision reaction to allow removal of blocking groups on the nucleotides; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0014] In another aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting a labeled nucleotide with the target nucleic acid molecule to allow incorporation of the nucleotide; (2) detecting the label of the incorporated nucleotide; (3) removing the incorporated nucleotide and the label carried by the nucleotide; (4) contacting an unlabeled nucleotide with the target nucleic acid molecule to allow incorporation of the unlabeled nucleotide; wherein at least one of steps (1), (3) and (4) is performed in a gel state; optionally, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule. In certain embodiments, the label is a fluorescent label.

[0015] In certain embodiments, the nucleotide incorporation described in step (1) only follows the principle of base complementary pairing and binds to the 3' end of the target nucleic acid complementary chain without forming a phosphodiester bond. In certain embodiments, the nucleotide incorporation described in step (4) undergoes a polymerization reaction to form a phosphodiester bond. In certain embodiments, the nucleotide incorporated in step (4) has a blocking group. In certain embodiments, step (3) also includes removing the blocking group remaining at the 3' end of the target nucleic acid complementary chain, which will allow the extension reaction of the target nucleic acid molecule complementary chain.

[0016] In certain embodiments, when step (3) is performed in a solution state, it includes: removing the incorporated labeled nucleotides using an elution reagent under appropriate conditions; preferably, it also includes removing the remaining blocking groups.

[0017] In certain embodiments, when step (3) is performed in a gel state, the method comprises the following steps: (3a) contacting a reaction composition comprising an elution reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermogelling property, and the reaction composition is in a liquid state; (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an elution reaction to allow removal of the incorporated nucleotides and the carried label; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition. In such embodiments, the method may further comprise, for example, removing the blocking group after the incorporation reaction of step (4), and then performing the nucleotide incorporation of step (1) in the next round of reaction.

[0018] In certain embodiments, when step (4) is performed in a gel state, the steps include: (4a) contacting a reaction composition comprising unlabeled nucleotides with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having reversible thermogel properties and is in a liquid state; preferably, the unlabeled nucleotides have blocking groups; (4b) providing a preset temperature to convert the reaction composition into a gel, and performing a polymerization reaction to allow the incorporation of nucleotides; (4c) changing the temperature to convert the reaction composition into a liquid state, and optionally removing the reaction composition.

[0019] In certain embodiments, in any of the above aspects, when step (1) is performed in a gel state, the method further comprises the following steps: (1a) contacting a reaction composition comprising nucleotides carrying a label or blocking group with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogel property and is in a liquid state; (1b) providing a preset temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of the nucleotides; and (1c) changing the temperature to convert the reaction composition into a liquid state. In certain embodiments, after converting the reaction composition into a liquid state in step (1c), the method further comprises: removing the reaction composition or directly proceeding to the detection reaction of step (2).

[0020] In certain embodiments, in any of the above aspects, the labeled nucleotide in step (1) further comprises an independent blocking group; or, the label comprised by the labeled nucleotide can be used as a blocking group.

[0021] In certain embodiments, in any of the above aspects, before, during, or after any step before step (3) (for example, after step (1) and before step (2)), the method further comprises: a step of contacting a nucleotide carrying a blocking group and not labeled with the target nucleic acid molecule; the step is optionally performed in a gel state. In certain embodiments, after step (1) and before step (2), the method comprises the following steps: (i) contacting a reaction composition comprising a nucleotide carrying a blocking group and not labeled with the target nucleic acid molecule, the reaction composition comprising a polymer having a reversible thermogelling property, and the reaction composition being in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow the incorporation of nucleotides; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0022] In certain embodiments, in any of the above aspects, prior to step (1), the method further comprises: a step of contacting a sequencing primer with the target nucleic acid molecule to allow hybridization; the step is optionally performed in a gel state. Preferably, prior to step (1), the method comprises the following steps: (i) contacting a reaction composition comprising a sequencing primer with the target nucleic acid molecule, the reaction composition comprising a polymer having a reversible thermogelling property, and the reaction composition being in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel so that the sequencing primer hybridizes to the target nucleic acid molecule; preferably, the preset temperature allows primer hybridization to occur; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0023] In certain embodiments, in any of the above aspects, the polymer having reversible thermo-induced gelling properties responds to temperature changes by changing from a liquid to a gel when the temperature rises. Preferably, the concentration of the polymer is about 0.5-30% (w / w), for example, about 10-30% (w / w), about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w). Preferably, the polymer has a gelling concentration of about 0.5%-25% (w / w). Preferably, the polymer has a gelling temperature of about 10-65°C. Preferably, the polymer is a block copolymer, a graft copolymer and a homopolymer. Preferably, the polymer is selected from Pluronic block polymers (e.g. Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly(ε-caprolactone) (mPEG-PCL), poly(N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA).

[0024] In certain embodiments, in any of the above aspects, (a) the temperature at which the gel state is converted into the liquid state is about 0-30°C (e.g., about 0-10°C, such as about 2-8°C, such as about 4°C; such as about 20-30°C, such as about 20-25°C, such as about 20°C or about 25°C); (b) the preset temperature at which the reaction composition comprising nucleotides (e.g., nucleotides carrying labels, unlabeled nucleotides, or nucleotides carrying blocking groups and not labeled) is converted into a gel is about 50-60°C, such as about 55-60°C, such as about 55°C; (c) the preset temperature at which the reaction composition comprising a removal reagent or an elution reagent is converted into a gel is about 50-60°C, such as about 55-60°C, such as about 55°C; and / or, (d) the preset temperature at which the reaction composition comprising a sequencing primer is converted into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, such as about 37°C.

[0025] In certain embodiments, in any of the above aspects, the target nucleic acid molecule is immobilized on a solid support, such as a chip.

[0026] In certain embodiments, in any of the above aspects, the method is open sequencing. Preferably, the target nucleic acid molecule is immobilized on the surface of an open sequencing slide. Preferably, the method comprises contacting or immersing the open sequencing slide with the immobilized target nucleic acid molecule with one or more sequencing reagents required for the sequencing reaction, wherein the one or more sequencing reagents are each placed in a separate reaction vessel.

[0027] In certain embodiments, in any of the above aspects, the method is flow cell sequencing. Preferably, the target nucleic acid molecule is immobilized on the surface of a flow cell. Preferably, the method comprises introducing one or more sequencing reagents required for the sequencing reaction into the flow cell to which the target nucleic acid molecule is immobilized.

[0028] On the other hand, the present invention provides a kit comprising: (a) a composition comprising nucleotides carrying a label and a polymer having reversible thermo-gelling properties; and / or, (b) a composition comprising a removal reagent and a polymer having reversible thermo-gelling properties. In certain embodiments, the kit further comprises: (c) a composition comprising nucleotides carrying a blocking group and not carrying a label and a polymer having reversible thermo-gelling properties. In certain embodiments, the kit further comprises: (d) a composition comprising a sequencing primer and a polymer having reversible thermo-gelling properties. In certain embodiments, the polymer having reversible thermo-gelling properties described in any one of (a)-(d) may be the same or different. In certain embodiments, the kit further comprises other reagents required for sequencing, such as a washing solution.

[0029] On the other hand, the present invention provides a kit comprising: (1) a composition comprising a nucleotide carrying a blocking group and a polymer having reversible thermogelling properties; and / or, (2) a composition comprising an affinity reagent carrying a fluorescent marker and a polymer having reversible thermogelling properties. In certain embodiments, the kit further comprises: (3) a composition comprising a removal reagent and a polymer having reversible thermogelling properties. In certain embodiments, the kit further comprises: (4) a composition comprising a sequencing primer and a polymer having reversible thermogelling properties. In certain embodiments, the polymer having reversible thermogelling properties described in any one of (1)-(4) may be the same or different. In certain embodiments, the kit further comprises other reagents required for sequencing, such as a washing solution.

[0030] On the other hand, the present invention provides a kit comprising: (i) a composition comprising labeled nucleotides and a polymer having reversible thermogel properties; (ii) a composition comprising an elution reagent and a polymer having reversible thermogel properties; and / or, (iii) a composition comprising unlabeled nucleotides and a polymer having reversible thermogel properties; preferably, the unlabeled nucleotides carry a blocking group. In certain embodiments, the kit further comprises: (iv) a composition comprising unlabeled nucleotides carrying a blocking group and a polymer having reversible thermogel properties. In certain embodiments, the kit further comprises: (v) a composition comprising a sequencing primer and a polymer having reversible thermogel properties. In certain embodiments, the polymer having reversible thermogel properties described in any one of (i)-(v) may be the same or different. In certain embodiments, the kit further comprises other reagents required for sequencing, such as a washing solution.

[0031] In certain embodiments, in any of the above aspects, the polymer having reversible thermo-induced gelling properties responds to temperature changes by changing from a liquid to a gel when the temperature rises. Preferably, the concentration of the polymer is about 0.5-30% (w / w), for example, about 10-30% (w / w), about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w). Preferably, the polymer has a gelling concentration of about 0.5%-25%. Preferably, the polymer has a gelling temperature of about 10-65°C. Preferably, the polymer is a block copolymer, a graft copolymer and a homopolymer. Preferably, the polymer is selected from Pluronic block polymers (e.g. Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly(ε-caprolactone) (mPEG-PCL), poly(N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA).

[0032] In certain embodiments, in any of the above aspects, the temperature at which the composition described in any one of (a)-(d), (1)-(4), (i)-(v) is converted from a gel state to a liquid state is about 0-30°C (e.g., about 0-10°C, for example, about 2-8°C, for example, about 4°C; for example, about 20-30°C, for example, about 20-25°C, for example, about 20°C or about 25°C); the preset temperature at which the reaction composition described in (a), (1) or (i) is converted into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C; the preset temperature at which the reaction composition described in (2) is converted into a gel is about 30-40°C, for example, about 30-37°C, about 32-37°C, about 35- 37°C, for example, about 35°C; the preset temperature for converting the reaction composition described in (b), (3) or (ii) into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C; the preset temperature for converting the reaction composition described in (iii) into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C; the preset temperature for converting the reaction composition described in (c) or (iv) into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C; and / or, the preset temperature for converting the reaction composition described in (d), (4) or (v) into a gel is about 35-45°C, for example, about 37-45°C, about 37-42°C, for example, about 37°C. The temperature ranges of the above embodiments do not constitute a specific limitation on the temperature setting of the present invention. The temperature setting is based on the actual temperature required by the reaction reagents. For example, if the suitable reaction temperature of the polymerase incorporated into the reaction is 37°C, then the gel temperature in step (a) is set to 37°C; if the temperature for primer hybridization is 60°C, then the gel temperature in the corresponding step is set to 60°C, etc.

[0033] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the states of reagents containing 20% ​​PF127 or 1% Methocel A15C at different temperatures.

[0035] FIG2 shows the cutting effect of the RR-gel reagent in Example 1.

[0036] FIG3 shows the Barcode sequencing of the RR-gel reagent in Example 1. FIG.

[0037] FIG4 shows Read1 sequencing of the Hot-gel reagent in Example 2.

[0038] FIG5 shows the Barcode sequencing of Hot-gel, Cold-gel and RR-gel reagents in Example 3.

[0039] FIG6 shows the Read1 sequencing results of Hot-gel, Cold-gel, and RR-gel reagents in Example 4.

[0040] FIG. 7 shows the primer hybridization reaction on the primer-gel in Example 5.

[0041] Detailed Description of the Invention

[0042] The present invention provides a hydrogel-based sequencing method and a kit for the sequencing method.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference.

[0044] Where a numerical range is provided, it is understood that each intermediate value (e.g., any integer value) between the upper and lower limits of the range is included within the scope of the invention. When the term "about" is used to describe a measurable value (e.g., concentration, temperature, etc.), it is intended to include a range of ±10%, ±5%, ±2%, or ±1% of the given value.

[0045] Sequencing methods

[0046] In a first aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:

[0047] (1) contacting the target nucleic acid molecule with a labeled nucleotide to allow incorporation of the nucleotide;

[0048] (2) detecting the label incorporated into the nucleotide;

[0049] (3) Remove the mark.

[0050] According to one embodiment of the present invention, step (1) is performed in a gel state.

[0051] According to another embodiment of the present invention, steps (1) and (3) are performed in a gel state.

[0052] In this article, the process of performing the reaction in a gel state refers to a gel-like reaction mixture. In certain embodiments, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule. In certain embodiments, the nucleotides described in step (1) are incorporated to form phosphodiester bonds. It will be understood by those skilled in the art that further extension of the complementary strand of the target nucleic acid molecule will be allowed after step (3).

[0053] In certain embodiments, the label is a fluorescent label.

[0054] In certain embodiments, the label is an affinity label, and step (2) includes step (2a), adding a luciferase carrying an affinity label to carry out an affinity reaction with the product of step (1); step (2b), adding a specific substrate to carry out a catalytic reaction with the product of step (2a); and step (2c), detecting the product of step (2b).

[0055] In certain embodiments, steps (2a) and (2b) are reacted in a gel state.

[0056] In certain embodiments, when step (2a) is performed in a gel state, the steps include: (i) contacting a reaction composition comprising an affinity-tagged luciferase with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermogelling property, and the reaction composition is in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel, and performing an affinity reaction; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0057] In certain embodiments, when step (2b) is performed in a gel state, the steps include: (i) contacting a reaction composition comprising a specific substrate with the product of the previous step, wherein the reaction composition further comprises a polymer having reversible thermo-induced gelling properties, and the reaction composition is in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel, and performing a catalytic reaction; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0058] In a second aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:

[0059] (1) contacting a nucleotide carrying a blocking group with the target nucleic acid molecule to allow incorporation of the nucleotide;

[0060] (2) detecting incorporated nucleotides;

[0061] (3) Removal of blocking groups;

[0062] Wherein, at least one of steps (1) and (2) is performed in a gel state.

[0063] According to one embodiment of the present invention, step (1) is performed in a gel state.

[0064] According to another embodiment of the present invention, at least one of the steps (2) is performed in a gel state.

[0065] According to another embodiment of the present invention, steps (1) and (2) are performed in a gel state.

[0066] According to another embodiment of the present invention, steps (1), (2) and (3) are performed in a gel state.

[0067] In this article, the process of performing the reaction in a gel state refers to a gel-like reaction mixture. In certain embodiments, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule. In certain embodiments, the nucleotides described in step (1) are incorporated to form phosphodiester bonds. In certain embodiments, step (3) allows the complementary strand of the target nucleic acid molecule to undergo an extension reaction.

[0068] In certain embodiments, step (2) includes step (2a), adding an affinity reagent carrying a fluorescent marker to perform an affinity reaction with the product of step (1); and step (2b), detecting the product of step (2a).

[0069] In certain embodiments, step (2a) is reacted in a gel state. In certain embodiments, when step (2a) is carried out in a gel state, the steps include: (i) contacting a reaction composition containing an affinity reagent carrying a fluorescent marker with the product of the previous step, wherein the reaction composition further contains a polymer having a reversible thermo-gelling property, and the reaction composition is in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel, and performing an affinity reaction; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0070] In certain embodiments, the affinity reagent is an antibody to the nucleotides A\T\C\G\U with a blocking group, and the antibody can specifically recognize the nucleotide and react with it. The method of using an affinity reagent to identify the incorporated base is specifically described in a disclosed patent (US10851410B2). In such embodiments, the fluorescent group is not directly labeled on the incorporated nucleotide, but is labeled on an affinity reagent (e.g., an antibody), which can specifically bind to the base, sugar, cleavable blocking group, or a combination of these components incorporated into the nucleotide, so that the type of nucleotide incorporated can be identified by the affinity reagent.

[0071] In a third aspect, the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:

[0072] (1) contacting the target nucleic acid molecule with a labeled nucleotide to allow incorporation of the nucleotide;

[0073] (2) detecting the label incorporated into the nucleotide;

[0074] (3) removal of incorporated nucleotides and the labels they carry;

[0075] (4) contacting the target nucleic acid molecule with an unlabeled nucleotide to allow incorporation of the unlabeled nucleotide.

[0076] According to one embodiment of the present invention, step (1) is performed in a gel state.

[0077] According to another embodiment of the present invention, steps (1) and (4) are performed in a gel state.

[0078] According to another embodiment of the present invention, steps (1), (3) and (4) are performed in a gel state.

[0079] Herein, the process of performing the reaction in a gel state refers to the reaction mixture being in a gel state. In certain embodiments, the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule.

[0080] In certain embodiments, the label is a fluorescent label.

[0081] In certain embodiments, the nucleotide incorporation described in step (1) is only bound to the 3' end of the complementary strand of the target nucleic acid according to the principle of base pairing, without forming a phosphodiester bond. Specific DNA polymerases and / or buffers can be used to prevent the formation of phosphodiester bonds, wherein the DNA polymerases are substantially inactive in catalyzing the formation of phosphodiester bonds, and the buffers are substantially free of divalent cations that promote the formation of phosphodiester bonds. Such DNA polymerases and / or buffers are well known to those skilled in the art.

[0082] In certain embodiments, the nucleotide incorporation in step (4) undergoes a polymerization reaction to form a phosphodiester bond. Those skilled in the art will appreciate that, since step (3) removes the originally incorporated nucleotides, the purpose of step (4) is to incorporate the nucleotides removed in step (3). Therefore, the nucleotides incorporated in step (4) are unlabeled nucleotides (unlabeled nucleotides) to complete the extension reaction of the complementary strand of the target nucleic acid molecule. In certain embodiments, the nucleotides incorporated in step (4) are nucleotides having a blocking group to ensure that only one nucleotide can be incorporated per extension reaction.

[0083] In certain embodiments, step (3) further comprises removing the blocking group remaining at the 3' end of the complementary strand of the target nucleic acid. The blocking group remaining refers to the modification carried by the 3' terminal nucleotide of the complementary strand of the target nucleic acid (i.e., the nucleotide adjacent to the removed nucleotide) after the nucleotide incorporated in step (3) and the label carried by it are removed, which can block the continued incorporation of other nucleotides. The adjacent nucleotides are incorporated by step (4) of the previous round. In certain embodiments, step (3) will allow the extension reaction of the complementary strand of the target nucleic acid molecule.

[0084] In certain embodiments, in any of the above aspects, the gel state described herein refers to contacting a reaction composition containing a polymer having a reversible thermogel property with the surface of a solid support on which a target nucleic acid molecule is fixed, providing a preset temperature to convert the reaction composition into a gel, and providing other conditions required for the reaction to carry out the corresponding sequencing biochemical reaction. After the reaction, the temperature is changed to convert the reaction composition into a liquid state, and washed, so that the next step of the reaction can be entered. The sequencing biochemical reaction described herein may include an incorporation reaction (including a base extension reaction or a polymerization reaction), a regeneration reaction to remove a fluorescent group and / or a blocking group, sequencing primer hybridization, and a reaction to detect the incorporation of nucleotides (e.g., an affinity reaction and a catalytic reaction in a bioluminescent sequencing method, an affinity reaction of a fluorescently labeled affinity reagent), etc. In certain embodiments, the contacting refers to dripping or spraying the liquid reaction composition onto the surface of the solid support.

[0085] In certain embodiments, in any of the above aspects, the sequencing reaction can be performed in partitions on the same solid phase chip. For example, a sequencing chip can be divided into several areas, the first area performs the incorporation reaction, the second area can perform signal detection, and the third area can perform the elution reaction, etc. Corresponding to the partition sequencing method, the solid support is preferably a semiconductor detection chip, the temperature of the solid support is preferably controlled in partitions, and the detection signal can be excited fluorescence or bioluminescence. In certain embodiments, the sequencing reaction can be carried out in partitions on the same solid phase chip, for example, a liquid reaction composition is dropped or sprayed onto different areas on the surface of the solid phase support, an incorporation reaction reagent is dropped or sprayed onto the first area to carry out an incorporation reaction, a scanning reagent is dropped or sprayed onto the second area to carry out signal detection, an elution or regeneration reagent (the regeneration reagent is an excision reagent) is dropped or sprayed onto the third area to carry out an elution or regeneration reaction (the regeneration reaction is an excision reaction), etc. Corresponding to the partition sequencing method, the solid support is preferably a semiconductor detection chip, and the temperature of the solid support is preferably controlled in partitions. The detection signal can be excited fluorescence or bioluminescence. When the detection signal is bioluminescence, the added or sprayed scanning reagent is replaced with a modified luciferase or enzyme-catalyzed substrate. The bioluminescence sequencing method and sequencing reagent are described in detail in patent US20220205036A1. In certain embodiments, the sequencing reaction can be performed in partitions on the same solid phase chip, by dropping or spraying the liquid reaction composition onto different areas of the solid phase support surface, and then using an air knife to remove residual reagents on the solid phase surface after the reaction.

[0086] Detection of nucleotide incorporation

[0087] It is easy to understand that in any of the above aspects, the incorporation of nucleotides is achieved by detecting the labeling carried directly or indirectly. In the case of using labeled nucleotides, the signal directly or indirectly generated by the labeling carries the identity information of the nucleotide, and the nucleotide at the position of the complementary pairing with the nucleotide on the sequenced nucleic acid molecule can be known by the signal. In addition, unlabeled nucleotides can also be used. In the case of using unlabeled nucleotides, affinity reagents (such as antibodies) carrying labels are used to specifically bind to bases, sugars, cleavable blocking groups or a combination of these components incorporated into the nucleotides to achieve indirect labeling of incorporated nucleotides. The term "label" refers to any molecule capable of detection that can directly or indirectly generate a detectable signal (including but not limited to optical signals, electrical signals, electromagnetic signals, radioactive signals, etc.).

[0088] In certain embodiments, the label is a fluorescent label. The mode of detecting the fluorescent label is well known in the art. For example, it can be achieved by a device that detects the wavelength of fluorescence. Such a device is well known in the art. For example, such a device can be a confocal scanning microscope, which scans the surface of a solid support with a laser so as to image the fluorophore directly bound to the sequenced nucleic acid molecule. In addition, each signal generated can be observed, for example, with a sensitive 2-D detector such as a charge coupled detector (CCD). Other techniques such as scanning near-field optical microscopy (SNOM) can also be used, or a semiconductor detection chip can be used to convert the optical signal into an electrical signal for transmission and detection using a photodiode.

[0089] In certain embodiments, the label is an affinity label. Affinity labels are used in sequencing technologies based on bioluminescent reactions, as described in, for example, US20220205036A1. The principle of bioluminescence detection includes that the nucleotide to be incorporated is not directly labeled with a fluorescent signal, but is labeled with an affinity substance such as biotin or digoxin. After the polymerization reaction, a pairing member of the above affinity substance with luciferase is added to bind the luciferase to the incorporated nucleotide, and then a substrate of the luciferase is added to generate a light signal to identify the identity of the incorporated nucleotide.

[0090] In certain embodiments, the affinity tag is any member of a molecular pair that can specifically bind to each other. Specific binding between the pair members enables the connection between the nucleotide and the luciferase. In certain embodiments, the label carried by the nucleotide is selected from biotin, digoxigenin, N3G or FITC, and the luciferase carries a pair member corresponding to the label. For example, if the label carried by the nucleotide is biotin, the luciferase can be a luciferase labeled with streptavidin; if the label carried by the nucleotide is digoxigenin, the luciferase can be a luciferase labeled with a digoxigenin antibody. The sources of the luciferase include but are not limited to organisms such as firefly, gaussia, and Renilla.

[0091] Sequencing reaction

[0092] It will be readily understood that, in any of the above aspects, the incorporation of nucleotides herein refers to incorporation into the complementary strand of the target nucleic acid molecule, with or without the formation of a phosphodiester bond. Nucleotides are incorporated sequentially along the 5' to 3' direction and follow the Watson-Crick base pairing rules. Therefore, the sequence of the target nucleic acid molecule can be inferred by detecting the type of incorporated nucleotides and following the Watson-Crick base pairing rules.

[0093] It is easy to understand that step (1) in one cycle will only allow one nucleotide to be incorporated into the complementary chain of the target polynucleotide, so the nucleotide to be incorporated has a modification that prevents extension, which prevents the polymerase from continuing to catalyze the incorporation of another nucleotide after incorporating the nucleotide containing the modification into the polynucleotide chain being synthesized.

[0094] In certain embodiments, the modification may be a separate blocking group (e.g., a 3' blocking group), which is typically located on the ribose ring of the nucleotide to block interaction with the 3' hydroxyl group. In such embodiments, the labeled nucleotide described in step (1) further comprises a separate blocking group.

[0095] In certain embodiments, the modification may also be a label that itself has a blocking effect. Without being bound by theory, such a label may have a size or structure sufficient to block the incorporation of other nucleotides into the polynucleotide chain. The blocking may be caused by steric hindrance, or may be caused by a combination of size, charge, and structure. In such embodiments, the label contained in the labeled nucleotide described in step (1) can serve as a blocking group.

[0096] In certain embodiments, the label carried by the labeled nucleotide may be chemically bonded to the nucleotide, or may be linked to the nucleotide in the form of a complex, without being bound by theory.

[0097] It is easy to understand that in any of the above aspects, further incorporation of nucleotides (i.e., continued extension of the target polynucleotide complementary chain) will be allowed after the completion of one cycle, so step (3) includes removing any modifications that prevent the target polynucleotide complementary chain from continuing to extend.

[0098] When the nucleotide incorporated in step (1) forms a phosphodiester bond, such as in the first or second aspects, step (3) comprises removing a modification on the incorporated nucleotide that prevents extension.

[0099] When the nucleotide incorporation described in step (1) does not form a phosphodiester bond, for example, in the third aspect, step (3) includes directly removing the incorporated nucleotide. If the incorporated nucleotide is removed in step (3), the step (4) will be further included to contact the unlabeled nucleotide with the target nucleic acid molecule to allow the incorporation of the unlabeled nucleotide and complete the extension reaction of the complementary chain of the target nucleic acid molecule (i.e., re-incorporate the nucleotide removed in step (3)). The unlabeled nucleotide described in step (4) should have a blocking group to ensure that only one nucleotide is incorporated in this step. In addition, in step (3), the blocking group carried by the nucleotide adjacent to the removed nucleotide (i.e., the unlabeled nucleotide with a blocking group incorporated by step (4) of the previous round of reaction) will also be removed to allow further incorporation in this round of step (4). Alternatively, after step (4), the blocking group of the nucleotide incorporated in step (4) is removed to allow the next round of sequencing reaction cycle to proceed.

[0100] In certain embodiments, when the incorporated nucleotide forms a phosphodiester bond and the incorporated nucleotide comprises an independent blocking group and a label, for example in the first aspect, step (3) further comprises removing the blocking group to allow extension. The removal of the blocking group and the removal of the label can be performed simultaneously or sequentially in any order. The removal of the blocking group and the removal of the label can use the same reagent or different reagents.

[0101] In certain embodiments, when the incorporated nucleotide forms a phosphodiester bond and the incorporated nucleotide comprises a label that can serve as a blocking group, such as in the first aspect, removal of the label in step (3) will also remove its blocking effect to allow extension.

[0102] In certain embodiments, when the incorporated nucleotide does not form a phosphodiester bond, such as in the third aspect, the removal of the incorporated nucleotide in step (3) also removes the label attached thereto, and also includes removing the remaining blocking group. It is understood that, under suitable conditions, an elution reagent is used to remove the nucleotide and the label it carries, as well as to remove the remaining blocking group.

[0103] In some embodiments, for example in the first aspect, the method of the present invention comprises the following steps: (1) contacting a nucleotide carrying a blocking group and a label with the target nucleic acid molecule to allow incorporation of the nucleotide and formation of a phosphodiester bond; (2) detecting the label of the incorporated nucleotide; (3) removing the blocking group and the label from the incorporated nucleotide to allow extension; wherein at least one of steps (1) and (3) is performed in a gel state.

[0104] In certain other embodiments, for example in the first aspect, the method of the present invention comprises the following steps: (1) contacting a nucleotide carrying a label with the target nucleic acid molecule to allow incorporation of the nucleotide and formation of a phosphodiester bond, wherein the label can serve as a blocking group; (2) detecting the label of the incorporated nucleotide; (3) removing the label from the incorporated nucleotide and removing its blocking effect to allow extension; wherein at least one of steps (1) and (3) is performed in a gel state.

[0105] In certain other embodiments, such as in the third aspect, the method of the present invention comprises the following steps: (1) contacting a labeled nucleotide with the target nucleic acid molecule to allow incorporation of the nucleotide but without forming a phosphodiester bond; (2) detecting the label of the incorporated nucleotide; (3) removing the labeled incorporated nucleotide and the remaining blocking group using an elution reagent under suitable conditions; (4) contacting an unlabeled nucleotide having a blocking group with the target nucleic acid molecule to allow incorporation of the unlabeled nucleotide and form a phosphodiester bond; wherein at least one of steps (1), (3) and (4) is performed in a gel state.

[0106] In certain embodiments, in the sequencing reaction of any of the above aspects, under the action of a polymerase, nucleotides (e.g., nucleotides carrying blocking groups and / or labels) are incorporated into the complementary strand of the target nucleic acid molecule, and this process is an incorporation reaction. The incorporation reaction may or may not form a phosphodiester bond. When the incorporation reaction forms a phosphodiester bond, it may also be referred to as a polymerization reaction. There are many different polymerases, and it is easy for a person of ordinary skill in the art to determine the most suitable polymerase. Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase, or vent polymerase. It is also possible to use a polymerase that is engineered to have a specific property. In the sequencing method provided herein, the incorporation reaction can be carried out under a gel state.

[0107] In certain embodiments, in any of the above-described sequencing reactions, when the incorporated labeled nucleotide forms a phosphodiester bond, the reaction in which the modification (e.g., a blocking group and / or label) incorporated into the nucleotide is removed (e.g., this may be removal of an independent blocking group and / or label, or removal of a label with a blocking effect) to allow the next round of sequencing is referred to as an excision reaction. In the sequencing methods provided herein, the excision reaction can be performed in a gel state.

[0108] In certain embodiments, in the sequencing reaction of any of the above aspects, when the incorporated labeled nucleotides do not form phosphodiester bonds, the reaction to remove the incorporated nucleotides is referred to as an elution reaction. The elution reaction may also include removing remaining blocking groups. In the sequencing method provided herein, the elution reaction may be performed in a gel state.

[0109] In certain embodiments, in the first aspect, steps (1) and (3) are both performed in a gel state. In certain embodiments, only step (1) is performed in a gel state. In certain embodiments, only step (3) is performed in a gel state. In certain embodiments, the affinity reaction in step (2) is also performed in a gel state.

[0110] In certain embodiments, in the third aspect, steps (1), (3) and (4) are all performed in a gel state. In certain embodiments, only step (1) is performed in a gel state. In certain embodiments, only step (3) is performed in a gel state. In certain embodiments, only steps (1) and (4) are performed in a gel state.

[0111] In certain embodiments, in the second aspect, steps (1) and (2) are both performed in a gel state. In certain embodiments, only step (1) is performed in a gel state. In certain embodiments, only step (2) is performed in a gel state. In certain embodiments, steps (1)-(3) are all performed in a gel state.

[0112] In certain embodiments, in the first or third aspect, when step (1) is performed in a gel state, the steps include:

[0113] (1a) contacting a reaction composition containing labeled nucleotides with the target nucleic acid molecule, wherein the reaction composition contains a polymer having a reversible thermogelling property and is in a liquid state;

[0114] (1b) providing a preset temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of nucleotides; preferably, the preset temperature allows the incorporation reaction to occur;

[0115] (1c) Changing the temperature to convert the reaction composition into a liquid state.

[0116] In some embodiments, the labeled nucleotide further comprises a separate blocking group.

[0117] In other embodiments, the labeled nucleotide comprises a label that can serve as a blocking group.

[0118] In some embodiments, after converting the reaction composition into a liquid state in step (1c), the process further comprises: removing the reaction composition or directly entering into the detection reaction of step (2).

[0119] In other embodiments, step (1c) is omitted, and the detection reaction of step (2) is performed after step (1b).

[0120] In certain embodiments, when the incorporation described in step (1) forms a phosphodiester bond, step (3) is an excision reaction and can be achieved by using a removal reagent. In this article, "removal reagent" refers to a reagent that can remove the modification on the incorporated nucleotide. The modification can include an independent label and a blocking group, or it can be a label that can be used as a blocking group. The selection of the removal reagent depends on the modification carried by the incorporated nucleotide used.

[0121] In certain embodiments, when the incorporation described in step (1) does not form a phosphodiester bond, step (3) is an elution reaction and can be achieved by using an elution reagent. In this article, "elution reagent" refers to an elution reagent that can remove the incorporated nucleotides (and any modifications carried therewith) under suitable conditions. The elution reagent can also include a reagent that can remove the remaining blocking groups.

[0122] In certain embodiments, in the second aspect, when step (1) is performed in a gel state, the steps include:

[0123] (1a) contacting a reaction composition comprising a nucleotide carrying a blocking group with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermo-gelling property and is in a liquid state;

[0124] (1b) providing a preset temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of nucleotides; preferably, the preset temperature allows the incorporation reaction to occur;

[0125] (1c) Changing the temperature to convert the reaction composition into a liquid state.

[0126] In certain embodiments, in any of the above aspects, when step (3) is performed in a gel state, the step comprises:

[0127] (3a) contacting a reaction composition containing a removal reagent or an elution reagent with the product of the previous step, wherein the reaction composition further contains a polymer having a reversible thermo-induced gelling property, and the reaction composition is in a liquid state;

[0128] (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an excision reaction or an elution reaction to allow removal of the label or blocking group on the nucleotide or removal of the incorporated nucleotide; preferably, the preset temperature allows the excision reaction or the elution reaction to occur;

[0129] (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0130] In some embodiments, in the first aspect, step (3) is an excision reaction, and when the labeled nucleotide in step (1) further comprises a separate blocking group, step (3) comprises removing the blocking group and the label from the incorporated nucleotide using a removal reagent. In such embodiments, step (3) when performed in a gel state comprises the following steps:

[0131] (3a) contacting a reaction composition containing a removal reagent with the product of the previous step, wherein the reaction composition further contains a polymer having a reversible thermo-induced gelling property, and the reaction composition is in a liquid state; the removal reagent includes a reagent capable of removing the label and the blocking group;

[0132] (3b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an excision reaction to allow removal of the blocking group and the label on the nucleotide; preferably, the predetermined temperature allows the excision reaction to occur;

[0133] (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0134] In other embodiments, in the first aspect, step (3) is an excision reaction. When the label contained in the labeled nucleotide in step (1) can serve as a blocking group, step (3) includes using a removal reagent to remove the label from the incorporated nucleotide and simultaneously remove its blocking effect. In such embodiments, step (3) when performed in a gel state includes the following steps:

[0135] (3a) contacting a reaction composition comprising a removal reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermo-induced gelling property, and the reaction composition is in a liquid state; the removal reagent comprises a reagent capable of removing the label;

[0136] (3b) providing a predetermined temperature to convert the reaction composition into a gel and performing an excision reaction to allow removal of the label on the nucleotide, wherein the removal of the label will simultaneously remove its blocking effect; preferably, the predetermined temperature allows the excision reaction to occur;

[0137] (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0138] In other embodiments, in the second aspect, step (3) is an excision reaction comprising removing the blocking group from the incorporated nucleotide using a removal reagent. In such embodiments, step (3) when performed in a gel state comprises the following steps:

[0139] (3a) contacting a reaction composition comprising a removal reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermo-induced gelling property, and the reaction composition is in a liquid state; the removal reagent comprises a reagent capable of removing a blocking group;

[0140] (3b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an excision reaction to allow removal of the blocking group on the nucleotide; preferably, the predetermined temperature allows the excision reaction to occur;

[0141] (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0142] In other embodiments, in the third aspect, step (3) is an elution reaction, which includes removing the incorporated nucleotides and the remaining blocking groups using an elution reagent. In such embodiments, step (3) when performed in a gel state includes the following steps:

[0143] (3a) contacting a reaction composition comprising an elution reagent with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermo-induced gelling property, and the reaction composition is in a liquid state; the elution reagent comprises a reagent capable of removing the incorporated nucleotide (and any modification carried by it) and the remaining blocking group, wherein the remaining blocking group can be a modification carried by the 3'-terminal nucleotide of the target nucleic acid complementary chain after the incorporated nucleotide is removed, which blocks the continued incorporation of other nucleotides;

[0144] (3b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an elution reaction to allow removal of incorporated nucleotides and removal of remaining blocking groups; preferably, the predetermined temperature allows the elution reaction to occur;

[0145] (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0146] In other embodiments, when step (3) is performed in a solution state, it includes: removing the incorporated labeled nucleotides and the remaining blocking groups using an elution reagent under appropriate conditions.

[0147] In certain embodiments, in the third aspect, when step (4) is performed in a gel state, the steps include:

[0148] (4a) contacting a reaction composition comprising unlabeled nucleotides with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogel property and is in a liquid state; preferably, the unlabeled nucleotides have a blocking group to ensure that only one nucleotide can be incorporated into the 3' end of the complementary chain of the target nucleic acid in each round of extension reaction;

[0149] (4b) providing a predetermined temperature to convert the reaction composition into a gel and performing a polymerization reaction to allow incorporation of nucleotides;

[0150] (4c) changing the temperature to convert the reaction composition into a liquid state, and optionally removing the reaction composition.

[0151] In certain embodiments, in the first or third aspect, before, during, or after any step prior to step (3) (e.g., after step (1) and before step (2)), the method further comprises: contacting a nucleotide carrying a blocking group and not labeled with the target nucleic acid molecule; said step is optionally performed in a gel state. In certain embodiments, the nucleotide incorporation in this step forms a phosphodiester bond.

[0152] In certain embodiments, in the first or third aspect, after step (1) and before step (2), the method comprises the following steps:

[0153] (i) contacting a reaction composition comprising a nucleotide carrying a blocking group and not having a label with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermo-gelling property and is in a liquid state;

[0154] (ii) providing a predetermined temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of nucleotides; preferably, the predetermined temperature allows the incorporation reaction to occur;

[0155] (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0156] In certain embodiments, in any of the above aspects, before step (1), the method further comprises: a step of contacting a sequencing primer with the target nucleic acid molecule to allow hybridization; the step is optionally performed in a gel state.

[0157] In certain embodiments, in any of the above aspects, before step (1), the method comprises the following steps:

[0158] (i) contacting a reaction composition comprising a sequencing primer with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogelling property and is in a liquid state;

[0159] (ii) providing a preset temperature to convert the reaction composition into a gel, so that the sequencing primer hybridizes to the target nucleic acid molecule; preferably, the preset temperature allows primer hybridization to occur;

[0160] (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

[0161] In certain embodiments, in any of the above aspects, the method is flow cell sequencing. The term "flow cell sequencing" refers to any sequencing method based on a flow cell on which the nucleic acid molecules to be tested are fixed. Typical flow cell sequencing includes, for example, Illumina's SBS sequencing technology. Taking Illumina's HiSeq 2500 platform as an example, the platform uses a flow cell to pump sequencing reaction reagents from a refrigeration device into the flow cell at a specific position at a specific time point, and flows through the sequencing chip under the control of a set of complex fluid control equipment, and is assisted by sophisticated temperature control facilities to ensure that the biochemical reaction is fully carried out in the shortest possible time.

[0162] In certain embodiments, in any of the above aspects, the target nucleic acid molecule is immobilized on the surface of a flow cell.

[0163] In certain embodiments, in any of the above aspects, the method comprises introducing one or more sequencing reagents required for the sequencing reaction into a flow cell immobilized with the target nucleic acid molecule to complete the corresponding sequencing biochemical reaction.

[0164] In certain embodiments, in any of the above aspects, the method is open sequencing.The term "open sequencing" refers to a sequencing method based on an immersion reaction scheme or a contact reaction scheme, see, for example, the method described in WO2019023951A1. Different from traditional flow channel sequencing, one or more sequencing reagents are introduced into a flow pool fixed with a target nucleic acid molecule. In open sequencing, the one or more sequencing reagents required for the sequencing reaction are each placed in a separate reaction vessel, and the solid support fixed with the nucleic acid molecule to be tested is brought into contact with the one or more sequencing reagents placed in a separate reaction vessel by moving, or immersed in the one or more sequencing reagents, to complete the corresponding sequencing biochemical reaction. In open sequencing, the solid support fixed with the target nucleic acid molecule can be referred to as an open sequencing slide.

[0165] In certain embodiments, in any of the above aspects, the target nucleic acid molecule is immobilized on the surface of an open sequencing slide.

[0166] In certain embodiments, in any of the above aspects, the method includes contacting or immersing the open sequencing slide having the target nucleic acid molecule fixed thereon with one or more sequencing reagents required for the sequencing reaction, respectively, wherein the one or more sequencing reagents are each placed in a separate reaction container.

[0167] In certain embodiments, in any of the aforementioned aspects, the methods provided herein are particularly suitable for use on open sequencing platforms. These methods offer the advantages of open sequencing platforms, such as reduced costs, simplified fluidics, and support for ultra-large chips, while also addressing some of the challenges of open sequencing platforms, such as chip drying, sample damage, and the inability to automate certain specialized processes (e.g., sample loading).

[0168] Thermogel polymer

[0169] As used herein, a polymer having reversible thermogel properties refers to a polymer that responds to temperature changes by changing from a liquid to a gel when the temperature is increased.

[0170] In certain embodiments, the concentration of the polymer is about 0.5-30% (w / w), for example, about 0.5-5% (w / w), 0.5-2% (w / w), about 5-10% (w / w), about 10-15% (w / w), about 15-20% (w / w), about 20-25% (w / w), about 25-30% (w / w).

[0171] In certain embodiments, the concentration of the polymer is about 10-30% (w / w), such as about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w). In certain embodiments, the polymer is selected from Pluronic block polymers.

[0172] In certain embodiments, the concentration of the polymer is about 0.5-10% (w / w), such as about 0.5-5% (w / w), about 0.5-2% (w / w), about 1-2% (w / w), such as about 1% (w / w). In certain embodiments, the polymer is selected from methylcellulose or hydroxypropyl methylcellulose.

[0173] In certain embodiments, the polymer is a block copolymer, a graft copolymer, and a homopolymer. In certain embodiments, the polymer is selected from Pluronic block polymers (e.g., Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g., Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly(ε-caprolactone) (mPEG-PCL), poly(N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA), and the like. Pluronic block polymers can be selected from, for example, Pluronic F38, P65, P68LF, P75, F77, P84, P85, F87, F88, F98, P103, P104, P105, F108, P123, F123, F127, 10R8, 17R8, 25R5, 25R8, such as Pluronic F127. Methylcellulose can be selected from, for example, Methocel A, Methocel A4C, Methocel A15C, Methocel A15 LV, Methocel A4M, such as Methocel A15C or Methocel A15 LV. Hydroxypropyl methylcellulose can be selected from, for example, Methocel E5LV, Methocel E3LV, Methocel E6LV, Methocel E15LV, Methocel E50LV, and Methocel K3LV.

[0174] In certain embodiments, the polymer has a gel concentration of about 0.5%-25% (w / w). In certain embodiments, the polymer has a gel concentration of about 0.5%-5% (w / w), about 5%-10% (w / w), about 10%-15% (w / w), about 15%-20% (w / w), or about 20%-25% (w / w).

[0175] In certain embodiments, the polymer has a gel temperature of about 10-65° C. In certain embodiments, the polymer has a gel temperature of about 10-15° C., about 15-20° C., about 20-25° C., about 25-30° C., about 30-35° C., about 35-40° C., about 40-45° C., about 45-50° C., about 50-55° C., about 55-60° C., or about 60-65° C.

[0176] In certain embodiments, the temperature at which the polymer having reversible thermogelling properties transforms from a gel state to a liquid state is about 0-30°C.

[0177] In certain embodiments, the temperature at which the polymer having reversible thermogelling properties transforms from a gel state to a liquid state is about 0-10° C., for example, about 2-8° C., such as about 4° C. In certain embodiments, the polymer is selected from Pluronic block polymers. In certain embodiments, the concentration of the polymer is about 10-30% (w / w), for example, about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w).

[0178] In certain embodiments, the temperature at which the polymer having reversible thermogelling properties transforms from a gel state to a liquid state is about 20-30° C., for example, about 20-25° C., for example, about 20° C. or about 25° C. In certain embodiments, the polymer is selected from methylcellulose or hydroxypropyl methylcellulose. In certain embodiments, the concentration of the polymer is about 0.5-10% (w / w), for example, about 0.5-5% (w / w), about 0.5-2% (w / w), about 1-2% (w / w), such as about 1% (w / w).

[0179] In certain embodiments, in any step of the method of the present application, the preset temperature refers to a temperature that allows the reaction composition containing a polymer with reversible thermogelatinization properties to transform from a liquid to a gel while allowing the desired reaction to occur. The desired reaction can be any one of the sequencing biochemical reactions, such as an incorporation reaction (including a base extension reaction or a polymerization reaction), a regeneration reaction to remove a fluorescent group and / or a blocking group, sequencing primer hybridization, and a reaction to detect the incorporation of nucleotides (for example, affinity reactions and catalytic reactions in bioluminescent sequencing methods, affinity reactions of fluorescently labeled affinity reagents), etc. Some exemplary preset temperatures are provided below, but those skilled in the art are fully capable of adjusting the preset temperature according to actual needs. For example, for an incorporation reaction, if the suitable reaction temperature of the polymerase is 37°C, the corresponding preset temperature can be set to 37°C. For example, for primer hybridization, if the suitable temperature is 60°C, the corresponding preset temperature can be set to 60°C.

[0180] For example, when the reaction composition is used for an incorporation reaction, the preset temperature should allow the incorporation reaction to occur. In certain embodiments, the preset temperature at which the reaction composition comprising nucleotides (e.g., labeled nucleotides, unlabeled nucleotides, or nucleotides carrying blocking groups and no labels) is converted into a gel is about 50-60° C., such as about 55-60° C., for example, about 55° C.

[0181] For example, when the reaction composition is used for an excision reaction, the preset temperature should allow for the removal of blocking groups and / or fluorescent groups incorporated into the nucleotides. In certain embodiments, the preset temperature at which the reaction composition comprising the removal reagent is converted into a gel is about 50-60° C., such as about 55-60° C., for example, about 55° C.

[0182] For example, when the reaction composition is used for an elution reaction, the preset temperature should allow for the removal of incorporated nucleotides and the removal of remaining blocking groups. In certain embodiments, the preset temperature at which the reaction composition comprising the elution reagent is converted into a gel is about 50-60° C., such as about 55-60° C., for example, about 55° C.

[0183] For example, when the reaction composition is used for primer hybridization (e.g., hybridization of a sequencing primer to an MDA strand produced by MDA amplification), the preset temperature should allow primer hybridization to occur. In certain embodiments, the preset temperature for converting the reaction composition comprising a sequencing primer into a gel is about 35-45° C., such as about 37-45° C., about 37-42° C., such as about 37° C.

[0184] For example, when the reaction composition is used for affinity reaction and catalytic reaction in a bioluminescent sequencing method, the preset temperature should allow the affinity reaction and catalytic reaction to occur, for example, about 30-40°C, for example, about 30-37°C, about 32-37°C, about 35-37°C, for example, about 35°C.

[0185] For example, when the reaction composition is used for an affinity reaction of a fluorescently labeled affinity reagent, the preset temperature should allow the affinity reaction to occur, for example, about 30-40°C, such as about 30-37°C, about 32-37°C, about 35-37°C, such as about 35°C.

[0186] Reaction composition

[0187] In any reaction step of the method of the present application, the reaction composition comprising the polymer having reversible thermogelling properties comprises any necessary ingredients that allow the corresponding reaction to occur.

[0188] In certain embodiments, when the reaction composition is used for an incorporation reaction, the necessary components may be an enzyme (e.g., a polymerase, such as a DNA polymerase), a primer, a buffer, or any combination thereof. In certain embodiments, the reaction composition for an incorporation reaction comprises a nucleotide to be incorporated and an enzyme (e.g., a polymerase, such as a DNA polymerase), wherein the nucleotide to be incorporated may be a nucleotide carrying a blocking group and a label, a nucleotide carrying a blocking group and no label, an unlabeled nucleotide, or any combination thereof.

[0189] In text, term " nucleotide " is expected to include natural nucleotide, non-natural nucleotide, ribonucleotide, deoxyribonucleotide, dideoxyribonucleotide and its analog.For example, this term generally refers to and comprises base part and is optionally attached to the nucleoside part (no matter ribose, deoxyribose or its analog) containing one or more phosphate moieties herein.This term can be used to refer to the monomeric unit present in polynucleotide, for example, to identify the subunit present in DNA or RNA chain.The term can also be used to refer to the monomeric molecule that is not necessarily present in polynucleotide, for example, can be incorporated into the molecule in polynucleotide in a template-dependent manner by polymerase.Exemplary nucleotide includes but is not limited to, monophosphate ribonucleotide (sometimes referred to as ribonucleoside monophosphate), diphosphate ribonucleotide (sometimes referred to as ribonucleoside diphosphate), triphosphate ribonucleotide (sometimes referred to as ribonucleoside triphosphate), monophosphate deoxynucleotide (sometimes referred to as deoxynucleoside monophosphate), diphosphate deoxynucleotide (sometimes referred to as deoxynucleoside diphosphate) and triphosphate deoxynucleotide (sometimes referred to as deoxynucleoside triphosphate). For clarity, when it is desired to distinguish RNA components from DNA components, the term "ribonucleotide" may be used to designate RNA nucleotides, such as ribouridine triphosphate, riboguanosine triphosphate, ribocytidine triphosphate, or riboadenosine triphosphate; and the term "deoxynucleotide" is used to designate RNA nucleotides, such as deoxythymidine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, or deoxyadenosine triphosphate.

[0190] In certain embodiments, the nucleotide to be incorporated is a dNTP or an analog thereof. In certain embodiments, the nucleotide to be incorporated is selected from deoxyadenosine triphosphate (dATP) or an analog thereof, deoxythymidine triphosphate (dTTP) or an analog thereof, deoxycytidine triphosphate (dCTP) or an analog thereof, deoxyguanosine triphosphate (dGTP) or an analog thereof. In certain embodiments, the analog refers to a synthetic nucleotide having a modified base moiety, a modified phosphodiester linkage, and / or a modified sugar moiety.

[0191] In certain embodiments, the nucleotide to be incorporated is a nucleotide carrying a label. In certain embodiments, the label is a fluorescent group.

[0192] In some embodiments, the nucleotides to be incorporated are nucleotides carrying separate blocking groups and labels.

[0193] Blocking group described in the application can stop the further incorporation of nucleotide at the complementary chain of described target nucleic acid molecule.Technical staff will understand how suitable blocking group is attached to the ribose ring of nucleotide to block the interaction with 3 ' hydroxyl.Blocking group can be directly attached to 3 ' position or can be attached to 2 ' position (blocking group has enough size or electric charge to block the interaction at 3 ' position).Or, blocking group can be attached to 3 ' and 2 ' position and can cut to expose 3 ' hydroxyl group.Suitable blocking group will be apparent to those skilled in the art.

[0194] In other embodiments, the nucleotide to be incorporated is a nucleotide that carries only a label that can serve as a blocking group. Without being bound by theory, such a label can have a size or structure that is sufficient to block the incorporation of other nucleotides into the polynucleotide chain. The blocking may be due to steric hindrance, or may be due to a combination of size, charge, and structure. Such labels will be apparent to those skilled in the art.

[0195] In certain embodiments, the blocking group and / or label can be linked to the nucleotide via a cleavable linker. In certain embodiments, the linker comprises one or more cleavable groups. In certain embodiments, the linker is selected from the group consisting of a disulfide linker, an acid-labile linker (e.g., a dialkoxybenzyl linker, a Sieber linker, an indole linker, a tert-butyl Sieber linker), an electrophilic cleavable linker, a nucleophilic cleavable linker, a photocleavable linker, or a combination thereof.

[0196] The types of disulfide linkers, acid-labile linkers (such as dialkoxybenzyl linkers, Sieber linkers, indole linkers, tert-butyl Sieber linkers), electrophilic cleavable linkers, nucleophilic cleavable linkers, and photocleavable linkers are obvious to those skilled in the art. For example, electrophilic cleavable linkers are typically cleaved by protons and include acid-sensitive cleavage. Suitable electrophilic cleavable linkers include modified benzyl systems such as trityl, p-alkoxybenzyl esters, and p-alkoxybenzyl amides. Other suitable electrophilic cleavable linkers include tert-butyloxycarbonyl (Boc) groups and acetal systems. In addition, it is also possible to consider using sulfur-philic metals such as nickel, silver, or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups to prepare suitable electrophilic cleavable linker molecules. Nucleophilic cleavage is a recognized method for preparing linker molecules. For example, groups that are unstable in water such as esters (i.e., can be simply cleaved at alkaline pH) and groups that are unstable to non-aqueous nucleophiles can be used. The photocleavable linker preferably activates the light required for cleavage without affecting other components of the modified nucleotide. For example, if a fluorescent group is used as a label, it is preferred that it absorbs light of a different wavelength than the light required to cleave the linker molecule.

[0197] In certain embodiments, the nucleotides to be incorporated are unlabeled nucleotides carrying blocking groups. The incorporation of such nucleotides can help compensate for incomplete incorporation reactions, ensuring synthesis efficiency and reducing the risk of incomplete reactions. Furthermore, because these nucleotides are unlabeled, signal interference caused by incomplete label excision or incomplete elution of the excised label is minimized, improving excision efficiency and reducing sequencing errors.

[0198] In certain embodiments, the nucleotides to be incorporated are unlabeled nucleotides. When the incorporated labeled nucleotides do not form phosphodiester bonds, the step of removing the label will simultaneously remove the incorporated nucleotides. In such embodiments, unlabeled nucleotides need to be re-added to allow the next round of sequencing reactions to proceed. In certain embodiments, the unlabeled nucleotides are nucleotides that do not carry a label but have a blocking group.

[0199] In certain embodiments, the nucleotides to be incorporated can form phosphodiester bonds.

[0200] In certain embodiments, the nucleotide to be incorporated is incapable of forming phosphodiester bonds.

[0201] Solid support

[0202] In certain embodiments, the target nucleic acid molecule is immobilized on a solid support.

[0203] In this article, the term "fixed" when referring to nucleic acid use means directly or indirectly attached to a solid support via a covalent bond or a non-covalent bond. In certain embodiments, the nucleic acid is covalently attached to a solid support. In certain embodiments, the nucleic acid is fixed to a solid support. In certain embodiments, the oligonucleotide to be used as a capture primer or amplification primer is fixed to a solid support so that the 3' end is available for enzymatic extension and at least a portion of the primer sequence is able to hybridize to a complementary nucleic acid sequence; the nucleic acid to be fixed is then hybridized to the oligonucleotide, in which case the fixed oligonucleotide or polynucleotide can be in a 3'-5' direction. In certain embodiments, the nucleic acid is fixed to a solid support and can include binding a nucleic acid binding protein to a solid support by amino modification, and capturing the nucleic acid molecule by the nucleic acid binding protein. Non-limiting examples of nucleic acid attachment to a solid support include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via a hydrogel or other porous polymer, etc.

[0204] In this article, the support can be made of various suitable materials. Such materials include, for example, inorganic substances, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to, cellulose, cellulose derivatives (e.g., nitrocellulose), acrylic resins, glass, silica gel, silicon dioxide, polystyrene, gelatin, polyvinyl pyrrolidone, copolymers of vinyl and acrylamide, cross-linked polystyrenes such as divinylbenzene (see, for example, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamide, latex, dextran, rubber, silicon, plastics, natural sponges, metal plastics, cross-linked dextran (e.g., Sephadex TM ), agarose gel (Sepharose TM ), and other supports known to those skilled in the art.

[0205] In certain embodiments, the support used to immobilize the nucleic acid molecules to be sequenced can be a solid support comprising an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.) that has been functionalized, for example, by applying an intermediate material containing reactive groups that allow for covalent attachment of biomolecules such as polynucleotides. In certain embodiments, the support is a glass or silicon wafer that has been surface-modified with a layer of avidin, amino, acrylamidosilane, or aldehyde chemical groups.

[0206] In this article, the solid support is not limited to its size, shape and configuration. In some embodiments, the solid support is a planar structure, such as a slide, chip, microchip and / or array. The surface of such a support can be in the form of a planar layer.

[0207] In certain embodiments, the support for immobilizing the nucleic acid molecules to be sequenced is a chip, such as a high-throughput sequencing chip.

[0208] Multiple copies of the target nucleic acid molecule

[0209] In order to enhance the detection signal of the label, those skilled in the art may select to form multiple copies of the target nucleic acid molecule to be measured, and its copy number is not limited. In such embodiments, multiple copies of the target nucleic acid molecule are connected to the solid support in the form of nucleic acid clusters. In certain embodiments, the nucleic acid clusters are prepared by a solid phase amplification method of bridge amplification. In certain embodiments, the nucleic acid clusters are prepared by rolling circle amplification, thereby forming DNA nanoballs (DNBs).

[0210] In certain embodiments, the target nucleic acid molecules are present on a nucleic acid array. In certain embodiments, each site on the array can include multiple copies of a single target nucleic acid molecule.

[0211] In certain embodiments, the target nucleic acid molecule may be a DNB formed from multiple copies of a single target nucleic acid molecule.

[0212] DNA nanoball (DNB) is a concatemer of target nucleic acid molecules comprising multiple copies. These nucleic acid copies are typically arranged one by one in a continuous linear chain of nucleotides, and this tandem repeat structure together with the single-stranded nature of DNA causes nanoball folding (folding) configuration. Generally speaking, the multiple copy target nucleic acid molecules in DNB each include a known linker sequence of sequence, so that they can be amplified or sequenced. The linker sequence of each target nucleic acid molecule is generally the same, but can also be different. DNB can be produced using, for example, rolling circle replication (RCA).

[0213] DNB can be loaded on the surface of a solid support as described herein. DNB can be attached to the surface of the solid support by any suitable method, and the non-limiting examples of such methods include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogel or other porous polymers, or a combination thereof. In certain embodiments, the solid support surface may have reactive functional groups that react with complementary functional groups on the polynucleotide molecules to form covalent bonds, such as by attaching cDNA to the same technique used on the microarray. DNB can also effectively attach to hydrophobic surfaces, such as clean glass surfaces with low concentrations of various reactive functional groups (such as -OH groups). In other embodiments, DNB can be adsorbed onto the surface. In this embodiment, the polynucleotide is fixed by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonds, van der Waals forces, etc.

[0214] Reagent test kit

[0215] In another aspect, the present invention provides a kit comprising:

[0216] (a) a composition comprising a labeled nucleotide and a polymer having reversible thermogelling properties, the composition optionally further comprising an enzyme (e.g., a polymerase, such as a DNA polymerase), a primer, and / or a buffer; and / or,

[0217] (b) A composition comprising a removal agent and a polymer having reversible thermogelling properties, the composition optionally comprising a buffer.

[0218] In some embodiments, the nucleotide carrying the label described in (a) further comprises an independent blocking group and is capable of forming a phosphodiester bond. In such embodiments, the removal reagent described in (b) comprises a reagent capable of removing the label and a reagent for removing the blocking group, and the two reagents can be the same or different.

[0219] In other embodiments, the label contained in the labeled nucleotide in (a) can serve as a blocking group and is capable of forming a phosphodiester bond. In such embodiments, the removal reagent in (b) comprises a reagent capable of removing the label, and it is understood by those skilled in the art that removal of the label will simultaneously remove its blocking effect.

[0220] In certain embodiments, the kit further comprises: (c) a composition comprising unlabeled nucleotides carrying blocking groups and a polymer having reversible thermogelling properties, the composition optionally further comprising an enzyme (e.g., a polymerase, e.g., a DNA polymerase), a primer, and / or a buffer. In certain embodiments, the unlabeled nucleotides carrying blocking groups in (c) are capable of forming phosphodiester bonds.

[0221] In certain embodiments, the kit further comprises: (d) a composition comprising a sequencing primer and a polymer having reversible thermogelling properties, wherein the composition optionally comprises a buffer.

[0222] In certain embodiments, the polymers having reversible thermogelling properties described in any of (a) to (d) may be the same or different.

[0223] In certain embodiments, the kit comprises: (a) and (b); (a) and (c); (b) and (c); (a) and (d); (b) and (d); (a), (b) and (c); (a), (b) and (d); or (a), (b), (c) and (d). In certain embodiments, the kit is used for sequencing, such as the sequencing method described herein. In certain embodiments, the kit further comprises other reagents for sequencing. In certain embodiments, when any of the foregoing kits is used for bioluminescent sequencing technology, the kit may further comprise: (e) a composition comprising an affinity-tagged luciferase and a polymer having reversible thermogelling properties, and / or (f) a composition comprising a specific substrate and a polymer having reversible thermogelling properties.

[0224] In certain embodiments, the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0-30° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0-10° C., such as about 2-8° C., for example, about 4° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20-30° C., such as about 20-25° C., for example, about 20° C. or about 25° C.

[0225] In certain embodiments, the preset temperature for converting the composition of (a) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (b) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (c) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (d) into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, for example, about 37°C. In certain embodiments, the preset temperature for converting the composition of (e) into a gel is about 30-40°C, such as about 30-37°C, about 32-37°C, about 35-37°C, for example, about 35°C. In certain embodiments, the preset temperature at which the composition of (f) is converted into a gel is about 30-40°C, such as about 30-37°C, about 32-37°C, about 35-37°C, such as about 35°C.

[0226] In certain embodiments, the kit described in any of the above embodiments may further comprise a washing solution.

[0227] In another aspect, the present invention also provides a kit comprising:

[0228] (1) a composition comprising a nucleotide carrying a blocking group and a polymer having reversible thermogelling properties, the composition optionally further comprising an enzyme (e.g., a polymerase, such as a DNA polymerase), a primer and / or a buffer; and / or,

[0229] (2) A composition comprising an affinity reagent carrying a fluorescent label (eg, an antibody recognizing a nucleotide) and a polymer having a reversible thermogelling property, wherein the composition optionally comprises a buffer.

[0230] In certain embodiments, (1) the nucleotide carrying a blocking group is capable of forming a phosphodiester bond.

[0231] In certain embodiments, (2) the affinity reagent is an antibody against the nucleotides A\T\C\G\U with a blocking group, and the antibody can specifically recognize the nucleotides and react with them with affinity.

[0232] In certain embodiments, the kit further comprises: (3) a composition comprising a removal reagent and a polymer having reversible thermogelling properties, wherein the composition optionally comprises a buffer. The removal reagent comprises a reagent capable of removing a blocking group.

[0233] In certain embodiments, the kit further comprises: (4) a composition comprising a sequencing primer and a polymer having reversible thermogelling properties, wherein the composition optionally comprises a buffer.

[0234] In certain embodiments, the kit comprises: (1) and (2); (1) and (3); (1) and (4); (2) and (3); (2) and (4); (1), (2) and (3); (1), (2) and (4); or (1), (2), (3) and (4).

[0235] In certain embodiments, the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0-30° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0-10° C., such as about 2-8° C., for example, about 4° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20-30° C., such as about 20-25° C., for example, about 20° C. or about 25° C.

[0236] In certain embodiments, the preset temperature for converting the composition of (1) into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (2) into a gel is about 30-40°C, for example, about 30-37°C, about 32-37°C, about 35-37°C, for example, about 35°C. In certain embodiments, the preset temperature for converting the composition of (3) into a gel is about 50-60°C, for example, about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (4) into a gel is about 35-45°C, for example, about 37-45°C, about 37-42°C, for example, about 37°C.

[0237] In another aspect, the present invention also provides a kit comprising:

[0238] (i) a composition comprising a labeled nucleotide and a polymer having reversible thermogelling properties, the composition optionally further comprising an enzyme (e.g., a polymerase, such as a DNA polymerase), a primer, and / or a buffer;

[0239] (ii) a composition comprising an eluting agent and a polymer having reversible thermogelling properties, said composition optionally comprising a buffer; and / or,

[0240] (iii) A composition comprising unlabeled nucleotides and a polymer having reversible thermogelling properties, optionally further comprising an enzyme (eg, a polymerase, such as a DNA polymerase), a primer, and / or a buffer.

[0241] In some embodiments, the nucleotide carrying the label described in (i) further comprises an independent blocking group and does not form a phosphodiester bond. In other embodiments, the label included in the nucleotide carrying the label described in (i) can be used as a blocking group and does not form a phosphodiester bond.

[0242] In certain embodiments, the unlabeled nucleotide in (iii) is capable of forming a phosphodiester bond. In certain embodiments, the unlabeled nucleotide in (iii) carries a blocking group.

[0243] In certain embodiments, the kit further comprises: (iv) a composition comprising unlabeled nucleotides carrying a blocking group and a polymer having reversible thermogelling properties, the composition optionally further comprising an enzyme (e.g., a polymerase, e.g., a DNA polymerase), a primer, and / or a buffer. In certain embodiments, (iv) the unlabeled nucleotides carrying a blocking group are capable of forming a phosphodiester bond.

[0244] In certain embodiments, the kit further comprises: (v) a composition comprising a sequencing primer and a polymer having reversible thermogelling properties, wherein the composition optionally comprises a buffer.

[0245] In certain embodiments, the polymers having reversible thermogelling properties described in any one of (i) to (v) may be the same or different.

[0246] In certain embodiments, the kit comprises: (i) and optionally (iv) and / or (v); (ii) and optionally (iv) and / or (v); (iii) and optionally (iv) and / or (v); (i) and (ii) and optionally (iv) and / or (v); (i) and (iii) and optionally (iv) and / or (v); (ii) and (iii) and optionally (iv) and / or (v); (i), (ii) and (ii) and optionally (iv) and / or (v).

[0247] In certain embodiments, the kit is used for sequencing, such as the sequencing method described herein. In certain embodiments, the kit further comprises other reagents for sequencing. In certain embodiments, when any of the foregoing kits is used for bioluminescent sequencing technology, the kit may further comprise: a composition comprising a luciferase carrying an affinity tag and a polymer having a reversible thermogelling property, and / or a composition comprising a specific substrate and a polymer having a reversible thermogelling property.

[0248] In certain embodiments, the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0-30° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0-10° C., such as about 2-8° C., for example, about 4° C. In certain embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20-30° C., such as about 20-25° C., for example, about 20° C. or about 25° C.

[0249] In certain embodiments, the preset temperature for converting the composition of (i) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (ii) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the reaction composition of (iii) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (iv) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In certain embodiments, the preset temperature for converting the composition of (v) into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, for example, about 37°C.

[0250] In certain embodiments, in any of the above aspects, the kit may further comprise a washing solution.

[0251] In certain embodiments, in any of the above aspects, the polymer with reversible thermo-induced gelling properties will respond to temperature changes by changing from a liquid to a gel when the temperature rises. In certain embodiments, the concentration of the polymer is about 0.5-30% (w / w), for example, about 10-30% (w / w), about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w). In certain embodiments, the polymer has a gelling concentration of about 0.5%-25%. In certain embodiments, the polymer has a gelling temperature of about 10-65°C. In certain embodiments, the polymer is a block copolymer, a graft copolymer, and a homopolymer. In certain embodiments, the polymer is selected from Pluronic block polymers (e.g., Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g., Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly(ε-caprolactone) (mPEG-PCL), poly(N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA).

[0252] The present invention also relates to the use of the kit described in any of the above aspects or any composition in the kit for sequencing. The present invention also relates to a sequencing method, which comprises using the kit described in any of the above aspects or any composition in the kit.

[0253] Advantageous Effects of the Invention

[0254] This application provides a hydrogel-based sequencing method that supports not only traditional flow cell-based sequencing systems but also open sequencing systems and large-scale sequencing chips. This method reduces reagent loss and avoids damage to target nucleic acid molecules on the slide surface caused by drying and crystallization. It also eliminates the need for complex fluidics and temperature control and effectively reduces reagent usage, enabling single-use sequencing and lowering costs. The method of this application has broad application prospects. Example

[0255] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0256] Preparation example:

[0257] I. Reagents:

[0258] II. Reagent preparation steps:

[0259] 1. Preparation of RR-gel reagent: Weigh 37.5 g of Pluronic F-127 and dissolve it in 150 mL of regeneration reagent (RR) to obtain RR-gel reagent (containing 20% ​​(w / w) PF127). Store at 4°C. Alternatively, weigh 1.515 g of Methocel A15C and dissolve it in 150 mL of regeneration reagent (RR) to obtain RR-gel reagent (containing 1% (w / w) Methocel A15C). Store at 4°C.

[0260] 2. Preparation of hot-gel reagent and cold-gel reagent: Weigh 37.5g Pluronic F-127, dissolve in 150mL sequencing reagent, and store at 4°C. Add 3mL dNTP mixture (fluorescently labeled) and 3mL sequencing enzyme to 144mL of the sequencing reagent prepared above (containing 20% ​​(w / w) PF127) to obtain hot-gel reagent, and store at 4°C. Add 6mL dNTP mixture II (without fluorescent label) and 3mL sequencing enzyme to 141mL of the sequencing reagent prepared above (containing 20% ​​(w / w) PF127) to obtain cold-gel reagent, and store at 4°C. Alternatively,

[0261] Weigh 1.515g of Methocel A15C and dissolve it in 150mL of sequencing reagent. Add 3mL of fluorescently labeled dNTP mix and 3mL of sequencing enzyme to 144mL of the above-prepared sequencing reagent (containing 1% (w / w) Methocel A15C) to obtain a hot-gel reagent and store at 4°C. Add 6mL of dNTP mix II (without fluorescent label) and 3mL of sequencing enzyme to 141mL of the above-prepared sequencing reagent (containing 1% (w / w) Methocel A15C) to obtain a cold-gel reagent and store at 4°C.

[0262] 3. Preparation of hot and cold reagents: Add 3 mL of dNTP mix (fluorescently labeled) and 3 mL of sequenase to 144 mL of sequencing reagent (excluding PF127 and Methocel A15C) to obtain the hot reagent and store at 4°C. Add 6 mL of dNTP mix II (non-fluorescently labeled) and 3 mL of sequenase to 141 mL of sequencing reagent (excluding PF127 and Methocel A15C) to obtain the cold reagent and store at 4°C.

[0263] 4. Results: As shown in Figure 1, a reagent containing 20% ​​(w / w) PF127 was in solution at 4°C and in a hydrogel at room temperature. When a reagent containing 20% ​​(w / w) PF127 was added dropwise to a slide and heated, it formed a hydrogel that did not flow when the slide was tilted. Similarly, when a reagent containing 1% (w / w) Methocel A15C was added dropwise to a slide and heated, it formed a hydrogel that exhibited a certain degree of elasticity.

[0264] III. Sequencing cycle steps:

[0265] 1. Prepare DNBs using the DNB preparation kit, load the DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, and store the loaded sequencing slide in Wash buffer 2.

[0266] 2. Rinse the sequencing slide with sequencing reagents, then add hot-gel at low temperature and place on a 55°C hot plate to form a hydrogel. After reacting for 1-3 minutes or 5-10 minutes, cool to 20°C, place horizontally at 4°C, or soak in Wash Buffer 2 at 4°C. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0267] 3. Add cold-gel dropwise at low temperature and place on a 55°C hot plate to form a hydrogel. After reacting for 1-3 minutes or 5-10 minutes, cool to 20°C or place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagent and rinse three times with Wash Buffer 2.

[0268] 4. Use the sequencer to take photos of the sequencing slides and basecall to complete cycle 1 sequencing.

[0269] 5. Rinse the sequencing slide with regeneration buffer, then add RR-gel at low temperature and place on a 55°C hot plate to form a hydrogel. After reacting for 1-3 minutes or 5-10 minutes, cool to 20°C, place horizontally at 4°C, or soak in Wash Buffer 2 at 4°C. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0270] 6. Repeat steps 2, 3, 4, and 5 to complete multiple sequencing cycles.

[0271] Example 1: RR-gel reagent Barcode sequencing

[0272] 1. Prepare DNBs using the DNB preparation kit, load DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, hybridize one strand of barcode sequencing primer, and store the loaded sequencing slide in Wash buffer 2.

[0273] 2. Rinse the sequencing slide with sequencing reagent, then add 3 mL of hot reagent under low temperature conditions, place it on a 55°C heating plate, react for 3 minutes, pour out the reagent, and rinse the sequencing slide three times with Wash buffer 2.

[0274] 3. Add 3 mL of cold reagent under low temperature conditions, place on a 55°C hot plate, react for 3 minutes, then discard the reagent and rinse the sequencing slide three times with Wash buffer 2.

[0275] 4. Set the sequencing type to SE1+barcode 10. Use the sequencer to take a photo of the sequencing slide and basecall the sequence to complete cycle 1 sequencing.

[0276] 5. Take another photo and basecall of the sequencing slide from step 4 to complete cycle 2 sequencing.

[0277] 6. Rinse the sequencing slide with regeneration buffer, then add RR-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 10 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0278] 7. Repeat steps 2, 3, 4, and 6 to complete cycle 3 to cycle 11. A total of 11 sequencing cycles are completed, and sequencing is completed.

[0279] 8. Analysis of Results: As shown in Figure 2, a 5-minute reaction on RR-gel (containing 20% ​​(w / w) PF127) followed by a further 5-minute reaction on RR-gel (containing 20% ​​(w / w) PF127) resulted in complete excision. A single 10-minute reaction on RR-gel (containing 20% ​​(w / w) PF127) showed better excision efficiency, comparable to the efficiency of two RR-gel reactions. As shown in Figure 3, SE1+barcode10 sequencing was performed using RR-gel, with a resolution rate of 79% for 441 FOVs and 81% for a single FOV.

[0280] Example 2: Hot-gel reagent Read 1 sequencing

[0281] 1. Prepare DNBs using the DNB preparation kit, load DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, hybridize one strand of sequencing primer 1, and store the loaded sequencing slide in Wash buffer 2.

[0282] 2. Rinse the sequencing slide with sequencing reagents, then add hot-gel (containing 1% (w / w) Methocel A15C) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 3 minutes, cool to 20°C and place horizontally or soak in Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0283] 3. Add 3 mL of cold reagent under low temperature conditions, place on a 55°C hot plate, react for 3 minutes, then discard the reagent and rinse the sequencing slide three times with Wash buffer 2.

[0284] 4. Set the sequencing type to SE7. Use the sequencer to take a photo of the sequencing slide and perform basecalling to complete cycle 1 sequencing.

[0285] 5. Rinse the sequencing slide with regeneration buffer, then add RR dropwise under low temperature conditions, place it on a 55°C hot plate, react for 5 minutes, discard the reagent, and rinse three times with Wash buffer 2.

[0286] 6. Repeat steps 2, 3, 4, and 5 to complete cycle 2 to cycle 8. Complete a total of 8 sequencing cycles (the 8th cycle is the calibration cycle) and end the sequencing.

[0287] 7. Result Analysis: As shown in Figure 4, SE7 sequencing can be completed using hot-gel, with relatively stable signal intensity and Unfilter Q30 maintained at around 80%.

[0288] Example 3: Barcode Sequencing Using Hot-gel, Cold-gel, and RR-gel Reagents

[0289] 1. Prepare DNBs using the DNB preparation kit, load DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, hybridize one strand of barcode sequencing primer, and store the loaded sequencing slide in Wash buffer 2.

[0290] 2. Rinse the sequencing slide with sequencing reagents, then add hot-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 8 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0291] 3. Add cold-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature and place on a 55°C hot plate to form a hydrogel. After reacting for 8 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagent and rinse three times with Wash Buffer 2.

[0292] 4. Set the sequencing type to SE1+barcode 10. Use the sequencer to take a photo of the sequencing slide and basecall the sequence to complete cycle 1 sequencing.

[0293] 5. Take another photo and basecall of the sequencing slide from step 4 to complete cycle 2 sequencing.

[0294] 6. Rinse the sequencing slide with regeneration buffer, then add RR-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 5 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0295] 7. Repeat steps 2, 3, 4, and 6 to complete cycle 3 to cycle 11. A total of 11 sequencing cycles are completed, and sequencing is completed.

[0296] 8. Result analysis: As shown in Figure 5, SE1+barcode10 sequencing can be completed using hot-gel, cold-gel, and RR-gel. The resolution rate of 36 FOVs is 72.25%, and the resolution rate of a single FOV is 75.53%.

[0297] Example 4: Hot-gel, Cold-gel and RR-gel reagents Read1 sequencing

[0298] 1. Prepare DNBs using the DNB preparation kit, load DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, hybridize one strand of sequencing primer, and store the loaded sequencing slide in Wash buffer 2.

[0299] 2. Rinse the sequencing slide with sequencing reagents, then add hot-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 8 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0300] 3. Add cold-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature and place on a 55°C hot plate to form a hydrogel. After reacting for 8 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagent and rinse three times with Wash Buffer 2.

[0301] 4. Set the sequencing type to SE6. Use the sequencer to take a photo of the sequencing slide and perform basecalling to complete cycle 1 sequencing.

[0302] 5. Rinse the sequencing slide with regeneration buffer, then add RR-gel (containing 20% ​​(w / w) PF127) dropwise at low temperature. Place on a 55°C hot plate to form a hydrogel. After reacting for 5 minutes, place horizontally at 4°C or soak in 4°C Wash Buffer 2. Once the hydrogel has converted to solution, discard the reagents and rinse three times with Wash Buffer 2.

[0303] 6. Repeat steps 2, 3, 4, and 5 to complete cycle 2 to cycle 7. Complete a total of 7 sequencing cycles (the 7th cycle is the calibration cycle) and end the sequencing.

[0304] 7. Result Analysis: As shown in Figure 6, SE6 sequencing can be completed using hot-gel, cold-gel, and RR-gel. The signal intensity is relatively stable, and the Unfilter Q30 remains at around 80%.

[0305] Example 5: Primer-gel hybridization reaction

[0306] 1. Weigh 5 g of Pluronic F-127 and dissolve it in 20 mL of sequencing primer 1 solution to obtain the Primer-gel reagent. Store at 4°C.

[0307] 2. Prepare DNBs using the DNB preparation kit, load DNBs using the DIPSEQ-T1 sequencing slide and DNB loading reagent, hybridize one strand of sequencing primer, and store the loaded sequencing slide in Wash buffer 2.

[0308] 3. Use the sequencer to take photos and basecall the sequencing slides.

[0309] 4. Rinse the sequencing slide with Wash Buffer 2, then add 3 mL of deionized formamide and incubate on a 37°C hot plate for 10 minutes to remove the first-strand sequencing primer. Discard the formamide and rinse three times with Wash Buffer 2. Add the Primer-gel reagent at low temperature and place on a 37°C hot plate to form a hydrogel. After incubation for 20 minutes, place the slide horizontally at 4°C or soak in Wash Buffer 2 at 4°C. Once the hydrogel has dissolved, discard the reagent and rinse three times with Wash Buffer 2.

[0310] 5. Use the sequencer again to take photos of the sequencing slides and basecall.

[0311] 6. Result Analysis: As shown in FIG7 , the primers in the hydrogel-forming reagent can hybridize to the DNB.

[0312] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings published, and that these variations are within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting the target nucleic acid molecule with a labeled nucleotide to allow incorporation of the nucleotide; (2) detecting the label incorporated into the nucleotide; (3) Removal of markings; wherein at least one of steps (1) and (3) is performed in a gel state; Optionally, the method comprises repeating the above steps sequentially to determine the sequence of the target nucleic acid molecule; Preferably, the nucleotide incorporation in step (1) forms a phosphodiester bond; Preferably, step (3) will allow the complementary strand of the target nucleic acid molecule to undergo an extension reaction.

2. The method according to claim 1, wherein The label is a fluorescent label.

3. The method according to claim 1, wherein The label is an affinity label, and the step (2) comprises: step (2a), adding luciferase carrying the affinity label to carry out an affinity reaction with the product of step (1); step (2b), adding a specific substrate to carry out a catalytic reaction with the product of step (2a); and step (2c), detecting the product of step (2b).

4. The method according to claim 3, wherein: The steps (2a) and (2b) are reacted in a gel state.

5. A method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting a nucleotide carrying a blocking group with the target nucleic acid molecule to allow incorporation of the nucleotide; (2) detecting incorporated nucleotides; (3) Removal of blocking groups; wherein at least one of steps (1) and (2) is performed in a gel state; Optionally, the method comprises repeating the above steps sequentially to determine the sequence of the target nucleic acid molecule; Preferably, the nucleotide incorporation in step (1) forms a phosphodiester bond; Preferably, step (3) will allow the complementary strand of the target nucleic acid molecule to undergo an extension reaction.

6. The method according to claim 5, wherein: The step (2) comprises: step (2a), adding an affinity reagent carrying a fluorescent marker to carry out an affinity reaction with the product of step (1); and step (2b), detecting the product of step (2a).

7. The method according to claim 6, wherein: The step (2a) is carried out in a gel state.

8. A method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting the target nucleic acid molecule with a labeled nucleotide to allow incorporation of the nucleotide; (2) detecting the label incorporated into the nucleotide; (3) removal of incorporated nucleotides and the labels they carry; (4) contacting an unlabeled nucleotide with the target nucleic acid molecule to allow incorporation of the unlabeled nucleotide; wherein at least one of steps (1), (3) and (4) is performed in a gel state; Optionally, the method comprises repeating the above steps sequentially to determine the sequence of the target nucleic acid molecule; Preferably, the nucleotide incorporation in step (1) does not form a phosphodiester bond; Preferably, the nucleotide incorporation in step (4) forms a phosphodiester bond; Preferably, the nucleotide incorporated in step (4) has a blocking group; Preferably, step (3) further comprises removing the blocking group remaining at the 3' end of the complementary strand of the target nucleic acid, thereby allowing an extension reaction of the complementary strand of the target nucleic acid molecule.

9. The method according to any one of claims 1 to 8, wherein: When step (1) is carried out in a gel state, the following steps are included: (1a) contacting a reaction composition comprising a nucleotide carrying a label or blocking group with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogel property and is in a liquid state; (1b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of nucleotides; (1c) Changing the temperature to convert the reaction composition into a liquid state.

10. The method according to claim 9, wherein after converting the reaction composition into a liquid state in step (1c), the method further comprises: Remove the reaction composition or directly proceed to the detection reaction in step (2).

11. The method according to any one of claims 1 to 7, wherein: When step (3) is carried out in a gel state, the following steps are included: (3a) contacting a reaction composition containing a removal agent with the product of the previous step, wherein the reaction composition further contains a polymer having a reversible thermo-gelling property, and the reaction composition is in a liquid state; (3b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an excision reaction to allow removal of the label or blocking group on the nucleotide; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

12. The method according to claim 8, wherein When step (3) is performed in a solution state, it includes: removing the incorporated labeled nucleotides using an elution reagent under appropriate conditions; preferably, it also includes removing the remaining blocking groups.

13. The method according to claim 8, wherein When step (3) is carried out in a gel state, the following steps are included: (3a) contacting a reaction composition containing an elution reagent with the product of the previous step, wherein the reaction composition further contains a polymer having a reversible thermogelling property, and the reaction composition is in a liquid state; (3b) providing a predetermined temperature to convert the reaction composition into a gel, and performing an elution reaction to allow removal of incorporated nucleotides and removal of remaining blocking groups; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

14. The method according to claim 8, wherein When step (4) is carried out in a gel state, the following steps are included: (4a) contacting a reaction composition comprising unlabeled nucleotides with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogel property and is in a liquid state; preferably, the unlabeled nucleotides have a blocking group; (4b) providing a predetermined temperature to convert the reaction composition into a gel and performing a polymerization reaction to allow incorporation of nucleotides; (4c) changing the temperature to convert the reaction composition into a liquid state, and optionally removing the reaction composition.

15. The method according to any one of claims 1 to 4 and 8, wherein: The labeled nucleotide in step (1) further comprises an independent blocking group; alternatively, the label contained in the labeled nucleotide can serve as a blocking group.

16. The method according to any one of claims 1 to 4 and 8, wherein: Before, during or after any step before step (3) (for example, after step (1) and before step (2)), the method further comprises: a step of contacting a nucleotide carrying a blocking group and not labeled with the target nucleic acid molecule; the step is optionally performed in a gel state.

17. The method according to claim 16, wherein After step (1) and before step (2), the method includes the following steps: (i) contacting a reaction composition comprising a nucleotide carrying a blocking group and not having a label with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermo-gelling property and is in a liquid state; (ii) providing a predetermined temperature to convert the reaction composition into a gel, and performing an incorporation reaction to allow incorporation of nucleotides; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

18. The method according to any one of claims 1 to 17, wherein: Prior to step (1), the method further comprises: a step of contacting a sequencing primer with the target nucleic acid molecule to allow hybridization; the step is optionally performed in a gel state; Preferably, before step (1), the method comprises the following steps: (i) contacting a reaction composition comprising a sequencing primer with the target nucleic acid molecule, wherein the reaction composition comprises a polymer having a reversible thermogelling property and is in a liquid state; (ii) providing a preset temperature to convert the reaction composition into a gel, so that the sequencing primer hybridizes to the target nucleic acid molecule; preferably, the preset temperature allows primer hybridization to occur; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.

19. The method according to any one of claims 1 to 18, wherein: The polymer having reversible thermogelling properties responds to temperature changes by changing from a liquid to a gel when the temperature is increased; Preferably, the concentration of the polymer is about 0.5-30% (w / w), such as about 10-30% (w / w), about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w); Preferably, the polymer has a gel-forming concentration of about 0.5%-25% (w / w); Preferably, the polymer has a gelling temperature of about 10-65°C; Preferably, the polymer is a block copolymer, a graft copolymer and a homopolymer; Preferably, the polymer is selected from Pluronic block polymers (e.g. Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxy polyethylene glycol-block-poly (ε-caprolactone) (mPEG-PCL), poly (N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA).

20. The method according to any one of claims 1 to 19, wherein: (a) the temperature at which the gel state is converted into the liquid state is about 0-30° C. (e.g., about 0-10° C., such as about 2-8° C., such as about 4° C.; such as about 20-30° C., such as about 20-25° C., such as about 20° C. or about 25° C.); (b) the predetermined temperature at which the reaction composition comprising nucleotides (e.g., labeled nucleotides, unlabeled nucleotides, or nucleotides carrying blocking groups and no label) is converted into a gel is about 50-60° C., such as about 55-60° C., for example, about 55° C.; (c) the predetermined temperature at which the reaction composition comprising the removal reagent or the elution reagent is converted into a gel is about 50-60° C., such as about 55-60° C., such as about 55° C.; and / or, (d) The preset temperature for converting the reaction composition containing sequencing primers into gel is about 35-45°C, such as about 37-45°C, about 37-42°C, such as about 37°C.

21. The method according to any one of claims 1 to 20, wherein: The target nucleic acid molecule is immobilized on a solid support, such as a chip.

22. The method according to any one of claims 1 to 21, wherein: The method is open sequencing; Preferably, the target nucleic acid molecule is fixed on the surface of an open sequencing slide; Preferably, the method comprises contacting or immersing the open sequencing slide having the target nucleic acid molecules fixed thereon with one or more sequencing reagents required for the sequencing reaction, respectively, wherein the one or more sequencing reagents are each placed in a separate reaction container.

23. The method according to any one of claims 1 to 21, wherein: The method is flow cell sequencing; Preferably, the target nucleic acid molecule is immobilized on the surface of a flow cell; Preferably, the method comprises introducing one or more sequencing reagents required for the sequencing reaction into a flow cell to which the target nucleic acid molecules are immobilized.

24. A kit comprising: (a) A composition comprising a labeled nucleotide and a polymer having a reversible thermogelling property; and / or, (b) a composition comprising a removal agent and a polymer having reversible thermogelling properties; Preferably, the kit further comprises: (c) a composition comprising a nucleotide carrying a blocking group and not labeled and a polymer having a reversible thermo-gelling property; Preferably, the kit further comprises: (d) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property; Preferably, the polymers having reversible thermogelling properties described in any one of (a) to (d) may be the same or different; Preferably, the kit further comprises other reagents required for sequencing, such as a washing solution.

25. A kit comprising: (1) a composition comprising a nucleotide carrying a blocking group and a polymer having reversible thermo-gelling properties; and / or, (2) A composition comprising an affinity reagent carrying a fluorescent label and a polymer having a reversible thermogelling property. Preferably, the kit further comprises: (3) A composition comprising a removal agent and a polymer having reversible thermogelling properties; Preferably, the kit further comprises: (4) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property; Preferably, the polymers having reversible thermogelling properties described in any one of (1) to (4) may be the same or different; Preferably, the kit further comprises other reagents required for sequencing, such as a washing solution.

26. A kit comprising: (i) a composition comprising a labeled nucleotide and a polymer having a reversible thermogelling property; (ii) a composition comprising an eluting agent and a polymer having reversible thermogelling properties; and / or, (iii) a composition comprising an unlabeled nucleotide and a polymer having a reversible thermogelling property; preferably, the unlabeled nucleotide carries a blocking group; Preferably, the kit further comprises: (iv) a composition comprising a nucleotide carrying a blocking group and not labeled and a polymer having a reversible thermogelling property; Preferably, the kit further comprises: (v) a composition comprising a sequencing primer and a polymer having reversible thermogelling properties; Preferably, the polymers having reversible thermogelling properties described in any one of (i) to (v) may be the same or different; Preferably, the kit further comprises other reagents required for sequencing, such as a washing solution.

27. The kit according to any one of claims 24 to 26, wherein The polymer having reversible thermogelling properties responds to temperature changes by changing from a liquid to a gel when the temperature is increased; Preferably, the concentration of the polymer is about 0.5-30% (w / w), such as about 10-30% (w / w), about 15-25% (w / w), about 18-22% (w / w), such as about 20% (w / w); Preferably, the polymer has a gel concentration of about 0.5% to 25%; Preferably, the polymer has a gelling temperature of about 10-65°C; Preferably, the polymer is a block copolymer, a graft copolymer and a homopolymer; Preferably, the polymer is selected from Pluronic block polymers (e.g. Pluronic F127), Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly(ε-caprolactone) (mPEG-PCL), poly(N-isopropylacrylamide-co-methacrylic acid) (pNIPAm-co-AA), poly(lactic acid-co-hydroxylactic acid)-polyethylene glycol-poly(lactic acid-co-hydroxylactic acid) (PLGA-PEG-PLGA).

28. The kit according to any one of claims 24 to 27, wherein: The temperature at which the composition described in any one of (a)-(d), (1)-(4), (i)-(v) is transformed from a gel state into a liquid state is about 0-30°C (e.g., about 0-10°C, for example, about 2-8°C, for example, about 4°C; for example, about 20-30°C, for example, about 20-25°C, for example, about 20°C or about 25°C); The predetermined temperature at which the reaction composition of (a), (1) or (i) is converted into a gel is about 50-60° C., for example, about 55-60° C., for example, about 55° C.; (2) The preset temperature for converting the reaction composition into a gel is about 30-40° C., for example, about 30-37° C., about 32-37° C., about 35-37° C., for example, about 35° C.; The predetermined temperature at which the reaction composition of (b), (3) or (ii) is converted into a gel is about 50-60° C., for example, about 55-60° C., for example, about 55° C.; (iii) the predetermined temperature at which the reaction composition is converted into a gel is about 50-60° C., for example, about 55-60° C., for example, about 55° C.; The predetermined temperature at which the reaction composition of (c) or (iv) is converted into a gel is about 50-60° C., such as about 55-60° C., such as about 55° C.; and / or, The preset temperature for converting the reaction composition described in (d), (4) or (v) into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, such as about 37°C.

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