WO2024067674A1 - 测序方法 - Google Patents

测序方法 Download PDF

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Publication number
WO2024067674A1
WO2024067674A1 PCT/CN2023/121926 CN2023121926W WO2024067674A1 WO 2024067674 A1 WO2024067674 A1 WO 2024067674A1 CN 2023121926 W CN2023121926 W CN 2023121926W WO 2024067674 A1 WO2024067674 A1 WO 2024067674A1
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Prior art keywords
reaction
sequencing
gel
polymer
nucleotide
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PCT/CN2023/121926
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English (en)
French (fr)
Inventor
杨晋
宋重阳
邢承美
徐崇钧
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深圳华大智造科技股份有限公司
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Publication of WO2024067674A1 publication Critical patent/WO2024067674A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to the field of sequencing. Specifically, the present invention provides a hydrogel-based sequencing method, which is applicable to a flow cell sequencing system and also supports an open sequencing system, and can effectively reduce costs while ensuring sequencing efficiency and accuracy.
  • the second is the immersion sequencing (DIP) solution, which pumps reagents into the reagent tank, and the sequencing chip is transferred in different reagent tanks. Whenever it is immersed in a new reagent, the exchange of liquid and active ingredients is achieved through diffusion.
  • DIP immersion sequencing
  • the flowcell solution has a high replacement ratio (i.e., the ratio of the volume of new reagents pumped into the flow channel to the volume of the flow channel in order to completely replace the old reagents in the flow channel), which generally needs to reach 2-3 or more, and the reagents are disposable. Therefore, although the reaction efficiency is high, the cost is also relatively high.
  • the immersion sequencing solution can reuse sequencing reagents, so the reagent cost can be effectively reduced; however, the immersion sequencing solution has the problem that the chip is easy to dry during the sequencing process. Using the immersion sequencing solution in some steps will increase the cost or affect the quality, and there is a risk of quality being affected by repeated use of reagents.
  • the present application provides a hydrogel-based sequencing method, which not only supports the traditional sequencing system based on the flow cell, but also supports the open sequencing system and can support large-size sequencing chips.
  • the method can reduce reagent loss and avoid drying and crystallization damage to the target nucleic acid molecules on the surface of the slide; and does not require complex fluids and temperature control; it can also effectively reduce the amount of reagents used, thereby achieving single use and reducing costs.
  • 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 the incorporation of the nucleotide; (2) detecting the label of the incorporated nucleotide; (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.
  • step (1) forms a phosphodiester bond.
  • step (3) allows the complementary strand of the target nucleic acid molecule to undergo an extension reaction.
  • the label is a fluorescent label.
  • the label is an affinity label
  • the step (2) includes step (2a), adding a luciferase carrying an affinity label 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).
  • steps (2a) and (2b) are reacted in a gel state.
  • step (3) when step (3) is performed in a gel state, the steps include: (3a) contacting a reaction composition comprising a removal agent with the product of the previous step, wherein the reaction composition also comprises a polymer having a reversible thermo-gelling 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 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.
  • 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 the incorporation of the nucleotide; (2) detecting the incorporated nucleotide; (3) removing the blocking group; wherein at least one of steps (1) and (2) 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.
  • the nucleotide incorporation in step (1) forms a phosphodiester bond.
  • step (3) allows the complementary chain of the target nucleic acid molecule to undergo an extension reaction.
  • step (2) includes step (2a), adding an affinity reagent carrying a fluorescent marker to react with the product of step (1); and step (2b) detecting the product of step (2a).
  • step (2a) reacts in a gel state.
  • the affinity reagent is an antibody with a nucleotide A ⁇ T ⁇ C ⁇ G ⁇ U having a blocking group, and the antibody can specifically recognize the nucleotide and react with it.
  • step (3) is performed in a gel state and comprises the following steps: (3a) contacting a reaction composition comprising a removal agent with the product of the previous step, wherein the reaction composition further comprises a polymer having a reversible thermo-gelling 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 excision reaction to allow the removal of the blocking groups on the nucleotides; (3c) changing the temperature to convert the reaction composition into a gel; liquid state, and remove the reaction composition.
  • the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps: (1) contacting a nucleotide carrying a label with the target nucleic acid molecule to allow the 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 the 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.
  • the label is a fluorescent label.
  • the nucleotide incorporation described in step (1) only follows the principle of base complementary pairing and is bound to the 3' end of the target nucleic acid complementary chain without forming a phosphodiester bond.
  • the nucleotide incorporation described in step (4) undergoes a polymerization reaction to form a phosphodiester bond.
  • the nucleotide incorporated in step (4) has a blocking group.
  • step (3) also includes removing the blocking group left 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.
  • step (3) 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.
  • step (3) when step (3) is performed 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, the reaction composition further comprising a polymer having a reversible thermo-gelling 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 the removal of the incorporated nucleotides and the removal of the carried label; (3c) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.
  • the method may also include, 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.
  • 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 a reversible thermogel property and the reaction composition is in a liquid state; preferably, the unlabeled nucleotides have a blocking group; (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.
  • step (1) when step (1) is performed in a gel state, the steps include: (1a) contacting a reaction composition containing nucleotides carrying a label or blocking group with the target nucleic acid molecule, wherein the reaction composition contains a polymer having a reversible thermogel property and the reaction composition 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 the incorporation of nucleotides; (1c) changing the temperature to convert the reaction composition into a liquid state.
  • step (1c) After the reaction composition is converted into a liquid state, the method further comprises: removing the reaction composition or directly entering into the detection reaction of step (2).
  • 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.
  • the method 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 carrying a label with the target nucleic acid molecule; the step is optionally performed in a gel state.
  • the method comprises the following steps: (i) contacting a reaction composition comprising a nucleotide carrying a blocking group and not carrying a label with the target nucleic acid molecule, the reaction composition comprising 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 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.
  • 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.
  • 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 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.
  • the polymer having reversible thermo-induced gelling properties responds to temperature changes by changing from a liquid to a gel when the temperature rises.
  • 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).
  • the polymer has a gelling concentration of about 0.5%-25% (w/w).
  • the polymer has a gelling temperature of about 10-65°C.
  • the polymer is a block copolymer, a graft copolymer and a homopolymer.
  • 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( ⁇ -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).
  • Pluronic block polymers e.g. Pluronic F127
  • Tetronic block polymers hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly( ⁇ -caprolactone) (mPEG-PCL), poly(
  • the temperature at which (a) the gel state is converted into the liquid state is about 0 to 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 for converting the reaction composition containing nucleotides (e.g., nucleotides carrying labels, unlabeled nucleotides, or nucleotides carrying blocking groups and not labeled) into gel is about 50-60°C, such as about 55-60°C, such as about 55°C; (c) the preset temperature for converting the reaction composition containing removal reagents or elution reagents into 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
  • the target nucleic acid molecule is immobilized on a solid support, such as a chip.
  • the method is open sequencing.
  • the target nucleic acid molecule is fixed on the surface of an open sequencing slide.
  • the method comprises contacting or immersing the open sequencing slide with the target nucleic acid molecule fixed thereon with one or more sequencing reagents required for the sequencing reaction, respectively, and the one or more sequencing reagents are each placed in a separate reaction vessel.
  • the method is flow cell sequencing.
  • the target nucleic acid molecule is immobilized on the surface of a flow cell.
  • 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.
  • the present invention provides a kit comprising: (a) a composition comprising nucleotides carrying a label and a polymer having a reversible thermogelling property; and/or, (b) a composition comprising a removal reagent and a polymer having a reversible thermogelling property.
  • the kit further comprises: (c) a composition comprising nucleotides carrying a blocking group and not carrying a label and a polymer having a reversible thermogelling property.
  • the kit further comprises: (d) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property.
  • the polymer having a reversible thermogelling property described in any one of (a)-(d) may be the same or different.
  • the kit further comprises other reagents required for sequencing, such as a washing solution.
  • the present invention provides a kit comprising: (1) a composition comprising a nucleotide carrying a blocking group and a polymer having a reversible thermogelling property; and/or, (2) a composition comprising an affinity reagent carrying a fluorescent marker and a polymer having a reversible thermogelling property.
  • the kit further comprises: (3) a composition comprising a removal reagent and a polymer having a reversible thermogelling property.
  • the kit further comprises: (4) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property.
  • the polymer having a reversible thermogelling property described in any one of (1)-(4) may be the same or different.
  • the kit further comprises other reagents required for sequencing, such as a washing solution.
  • the present invention provides a kit comprising: (i) a nucleotide carrying a label and a (ii) a composition comprising an elution reagent and a polymer having a reversible thermogelling property; and/or, (iii) a composition comprising unlabeled nucleotides and a polymer having a reversible thermogelling property; preferably, the unlabeled nucleotides carry a blocking group.
  • the kit further comprises: (iv) a composition comprising nucleotides carrying a blocking group and not labeled and a polymer having a reversible thermogelling property.
  • the kit further comprises: (v) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property.
  • the polymer having a reversible thermogelling property described in any one of (i)-(v) may be the same or different.
  • the kit further comprises other reagents required for sequencing, such as a washing solution.
  • the polymer having reversible thermo-gelling properties responds to temperature changes by changing from a liquid to a gel when the temperature rises.
  • 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).
  • the polymer has a gelling concentration of about 0.5%-25%.
  • the polymer has a gelling temperature of about 10-65°C.
  • the polymer is a block copolymer, a graft copolymer and a homopolymer.
  • 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( ⁇ -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).
  • Pluronic block polymers e.g. Pluronic F127
  • Tetronic block polymers hydroxypropyl methylcellulose, methylcellulose (e.g. Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly( ⁇ -caprolactone) (mPEG-PCL), poly(
  • the temperature at which the composition described in any 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, such as 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
  • the temperature ranges of the above embodiments do not constitute specific limitations on the temperature setting of the present invention.
  • the temperature setting is based on the actual requirements of the reaction reagents. For example, if the suitable reaction temperature of the polymerase incorporated into the reaction is 37°C, then the gel temperature of step (a) Set to 37°C; the temperature of primer hybridization is 60°C, then the temperature of the gel in the corresponding step is set to 60°C, etc.
  • Figure 1 shows the state of the reagent containing 20% PF127 or 1% Methocel A15C at different temperatures.
  • FIG. 2 shows the cleavage effect of the RR-gel reagent in Example 1.
  • FIG. 3 shows the Barcode sequencing of the RR-gel reagent in Example 1.
  • FIG. 4 shows Read1 sequencing of the Hot-gel reagent in Example 2.
  • FIG. 5 shows the Barcode sequencing of Hot-gel, Cold-gel and RR-gel reagents in Example 3.
  • FIG6 shows the Read1 sequencing results of Hot-gel, Cold-gel and RR-gel reagents in Example 4.
  • FIG. 7 shows the Primer-gel hybridization reaction in Example 5.
  • the invention provides a hydrogel-based sequencing method and a kit for the sequencing method.
  • the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:
  • step (1) is performed in a gel state.
  • steps (1) and (3) are performed in a gel state.
  • the process of performing the reaction in a gel state means that the reaction mixture is in a gel state.
  • the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule.
  • the nucleotide incorporation in step (1) forms a phosphodiester bond. It will be appreciated by those skilled in the art that step (3) will allow for further extension of the complementary strand of the target nucleic acid molecule.
  • the label is a fluorescent label.
  • the label is an affinity label
  • 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).
  • steps (2a) and (2b) are reacted in a gel state.
  • step (2a) when step (2a) is performed in a gel state, the steps include: (i) contacting a reaction composition containing a luciferase carrying an affinity tag with the product of the previous step, wherein the reaction composition also contains 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.
  • step (2b) 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 also comprises 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 a catalytic reaction; (iii) changing the temperature to convert the reaction composition into a liquid state, and removing the reaction composition.
  • the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:
  • steps (1) and (2) is performed in a gel state.
  • step (1) is performed in a gel state.
  • At least one of the steps (2) is performed in a gel state.
  • steps (1) and (2) are performed in a gel state.
  • steps (1), (2) and (3) are performed in a gel state.
  • the process of carrying out in a gel state refers to that the reaction mixture is in a gel state.
  • the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule.
  • the nucleotides described in step (1) are incorporated to form phosphodiester bonds.
  • step (3) allows the complementary strand of the target nucleic acid molecule to undergo an extension reaction.
  • step (2) includes step (2a), adding an affinity reagent carrying a fluorescent marker, The product of step (1) is subjected to affinity reaction; and step (2b) is subjected to detection of the product of step (2a).
  • the step (2a) is reacted in a gel state.
  • step (2a) when step (2a) is carried out in a gel state, the following steps are included: (i) contacting a reaction composition containing an affinity reagent carrying a fluorescent marker with the product of the previous step, the reaction composition also containing a polymer with 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.
  • the affinity reagent is an antibody with a nucleotide A ⁇ T ⁇ C ⁇ G ⁇ U with a blocking group, and the antibody can specifically recognize the nucleotide and react with it in an affinity manner.
  • the method of using an affinity reagent to identify the incorporated base is specifically described in a disclosed patent (US10851410B2).
  • the fluorescent group is not directly labeled on the incorporated nucleotide, but is labeled on an affinity reagent (e.g., an antibody), and the affinity reagent can specifically bind to the base, sugar, cleavable blocking group, or a combination of these components incorporated into the nucleotide, so the type of nucleotide incorporated can be identified by the affinity reagent.
  • an affinity reagent e.g., an antibody
  • the present invention provides a method for sequencing a target nucleic acid molecule, comprising the following steps:
  • step (1) is performed in a gel state.
  • steps (1) and (4) are performed in a gel state.
  • steps (1), (3) and (4) are performed in a gel state.
  • the process of performing the process in a gel state refers to the reaction mixture being in a gel state.
  • the method comprises repeating the above steps in sequence to determine the sequence of the target nucleic acid molecule.
  • the label is a fluorescent label.
  • the nucleotide incorporation described in step (1) only follows the principle of base complementary pairing and is bound to the 3' end of the target nucleic acid complementary strand 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 free of the activity of catalyzing the formation of phosphodiester bonds, and the buffers are substantially free of divalent cations that promote the formation of phosphodiester bonds.
  • DNA polymerases and/or buffers are well known to those skilled in the art.
  • the nucleotide incorporation described in step (4) undergoes a polymerization reaction to form a phosphodiester bond.
  • step (3) removes the originally incorporated nucleotide
  • step (4) the purpose of step (4) is to incorporate the incorporated nucleotide.
  • the nucleotides removed in step (3) are therefore unlabeled nucleotides (nucleotides without labels) incorporated in step (4) to complete the extension reaction of the complementary strand of the target nucleic acid molecule.
  • the nucleotides incorporated in step (4) are nucleotides with blocking groups to ensure that only one nucleotide can be incorporated in each extension reaction.
  • step (3) further comprises removing the blocking group left over at the 3' end of the complementary strand of the target nucleic acid.
  • the blocking group left over refers to the modification carried by the 3' terminal nucleotide (i.e., the nucleotide adjacent to the removed nucleotide) of the complementary strand of the target nucleic acid 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.
  • step (3) will allow the extension reaction of the complementary strand of the target nucleic acid molecule.
  • 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, a sequencing primer hybridization, and a reaction to detect the incorporation of nucleotides (for example, an affinity reaction and a catalytic reaction in a bioluminescent sequencing method, an affinity reaction of a fluorescently labeled affinity reagent), etc.
  • the contact refers to dripping or spraying the liquid reaction composition onto the surface of the solid support.
  • the sequencing reaction can be performed in partitions on the same solid phase chip.
  • a sequencing chip is divided into several areas, the first area is for incorporation reaction, the second area can be for signal detection, and the third area can be for elution reaction, etc.
  • 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.
  • the sequencing reaction can be performed in partitions on the same solid phase chip, for example, a liquid reaction composition is dripped or sprayed onto different areas on the surface of the solid phase support, an incorporation reaction reagent is dripped or sprayed onto the first area to perform an incorporation reaction, a scanning reagent is dripped or sprayed onto the second area to perform signal detection, an elution or regeneration reagent (the regeneration reagent is an excision reagent) is dripped or sprayed onto the third area to perform an elution or regeneration reaction (the regeneration reaction is an excision reaction), etc.
  • 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.
  • the detection signal is bioluminescence
  • the dripped or sprayed scanning reagent is replaced with a modified luciferase or enzyme-catalyzed substrate.
  • the bioluminescence sequencing method and sequencing The reagents are described in detail in patent US20220205036A1.
  • the sequencing reaction can be performed in partitions on the same solid phase chip, and the liquid reaction composition is dripped or sprayed onto different areas of the surface of the solid phase support, and then the residual reagents after the reaction on the solid phase surface are removed by an air knife.
  • nucleotides incorporation of nucleotides is achieved by detecting the labels carried directly or indirectly.
  • the signal directly or indirectly generated by the label 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.
  • unlabeled nucleotides can also be used.
  • label refers to any molecule that can be detected, which can directly or indirectly generate a detectable signal (including but not limited to optical signals, electrical signals, electromagnetic signals, radioactive signals, etc.).
  • the label is a fluorescent label.
  • the mode of detecting the fluorescent label is well known in the art.
  • it can be realized by a device for detecting the wavelength of fluorescence.
  • a device for detecting the wavelength of fluorescence can be a device for detecting the wavelength of fluorescence.
  • a device for detecting the wavelength of fluorescence can be a device for detecting the wavelength of fluorescence.
  • a device for detecting the wavelength of fluorescence 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.
  • each signal generated can be observed, for example, with a sensitive 2-D detector, such as a charge coupled detector (CCD).
  • CCD charge coupled detector
  • Other techniques such as scanning near-field optical microscopy (SNOM) can also be used, for example, and semiconductor detection chips can be used to convert optical signals into electrical signals for transmission and detection using photodiodes.
  • the label is an affinity label.
  • Affinity labels are used in sequencing technologies based on bioluminescent reactions, see, for example, US20220205036A1.
  • the detection principle of bioluminescence includes that the fluorescent signal is not directly labeled on the incorporated nucleotide, but an affinity substance such as biotin or digoxin is labeled.
  • 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 luciferase is added to generate a light signal to identify the identity of the incorporated nucleotide.
  • the affinity tag is a member of any molecular pairing that can specifically bind to each other.
  • the specific binding between the pairing members realizes the connection between the nucleotide and the luciferase.
  • the label carried by the nucleotide is selected from biotin, digoxigenin, N3G or FITC, and the luciferase carries a pairing member corresponding to the label.
  • 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 luciferase source includes but is not limited to firefly, gaussia, Renilla and other organisms.
  • nucleotides described herein refers to incorporation into the complementary strand of the target nucleic acid molecule, which may or may not form a phosphodiester bond. Nucleotides are successively incorporated along the 5' to 3' direction and follow the Watson-Crick base complementary pairing rule. Therefore, the sequence of the target nucleic acid molecule can be inferred by the type of the detected incorporated nucleotides and according to the Watson-Crick base complementary pairing rule.
  • 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.
  • the modification may be a separate blocking group (e.g., a 3' blocking group) that is typically located on the ribose ring of the nucleotide to block interaction with the 3' hydroxyl group.
  • the labeled nucleotide described in step (1) also contains an independent blocking group.
  • the modification may also be a label that has a blocking effect itself.
  • 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.
  • the label contained in the nucleotide carrying the label described in step (1) can be used as a blocking group.
  • 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.
  • step (3) includes removing any modification that prevents the target polynucleotide complementary chain from continuing to extend.
  • step (3) comprises removing a modification on the incorporated nucleotide that prevents extension.
  • step (3) includes directly removing the incorporated nucleotide. If the incorporated nucleotide is removed in step (3), it will further include step (4) of contacting the target nucleic acid molecule with an unlabeled nucleotide 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-incorporating 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.
  • step (3) 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)
  • the blocking groups carried by the nucleotides will also be removed to allow further incorporation in this round of step (4).
  • step (4) the blocking groups of the nucleotides incorporated in step (4) are removed to allow the next round of sequencing reaction cycles to proceed.
  • step (3) further comprises removing the blocking group to allow extension.
  • the removal of the blocking group and the removal of the label may be performed simultaneously or sequentially in any order.
  • the removal of the blocking group and the removal of the label may use the same reagent or different reagents.
  • 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.
  • the removal of the incorporated nucleotide in step (3) will also remove the label attached thereto, and also include removing the remaining blocking groups. It is understood that under suitable conditions, the nucleotide and the label carried by it are removed using an eluting agent, as well as removing the remaining blocking groups.
  • 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.
  • 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 the incorporation of the nucleotide and the formation of a phosphodiester bond, wherein the label can be used 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.
  • the method of the present invention comprises the following steps: (1) contacting the target nucleic acid molecule with a labeled nucleotide to allow incorporation of the nucleotide but without forming a phosphodiester bond; (2) detecting the label of the incorporated nucleotide; (3) under suitable conditions, removing the labeled incorporated nucleotide using an elution reagent and removing the remaining blocking groups; (4) contacting the target nucleic acid molecule with an unlabeled nucleotide having a blocking group to allow incorporation of the unlabeled nucleotide and forming a phosphodiester bond; wherein at least one of steps (1), (3) and (4) is performed in a gel state.
  • nucleotides under the action of a polymerase, nucleotides (e.g., the incorporation reaction is carried out by a nucleotide (e.g., a nucleotide carrying a blocking group and/or a label) into the complementary strand of the target nucleic acid molecule.
  • the incorporation reaction may or may not form a phosphodiester bond.
  • the incorporation reaction forms a phosphodiester bond, it may also be referred to as a polymerization reaction.
  • polymerases 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. Polymerases that are engineered to have specific properties can also be used. In the sequencing method provided by the present application, the incorporation reaction can be carried out in a gel state.
  • the reaction of removing the modification (e.g., blocking group and/or label) on the incorporated nucleotide e.g., removing an independent blocking group and/or label, or removing a label with a blocking effect
  • the excision reaction can be performed in a gel state.
  • the reaction of removing the incorporated nucleotide is referred to as an elution reaction.
  • the elution reaction may also include removing the remaining blocking groups.
  • the elution reaction may be performed in a gel state.
  • both steps (1) and (3) are performed in a gel state.
  • only step (1) is performed in a gel state.
  • only step (3) is performed in a gel state.
  • the affinity reaction in step (2) is also performed in a gel state.
  • steps (1), (3) and (4) are all performed in a gel state.
  • only step (1) is performed in a gel state.
  • only step (3) is performed in a gel state.
  • only steps (1) and (4) are performed in a gel state.
  • 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.
  • step (1) when step (1) is performed in a gel state, it includes the following steps:
  • 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;
  • (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;
  • the labeled nucleotide further comprises an independent blocking group.
  • the label-bearing nucleotide comprises a label that can serve as a blocking group.
  • the process further comprises: removing the reaction composition or directly entering into the detection reaction of step (2).
  • step (1c) is omitted, and the detection reaction of step (2) is performed after step (1b).
  • step (3) is an excision reaction and can be achieved by using a removal reagent.
  • removal reagent refers to a reagent that can remove the modification on the incorporated nucleotide.
  • the modification can include independent labels and blocking groups, or it can also 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.
  • step (3) is an elution reaction and can be achieved by using an elution reagent.
  • elution reagent refers to an elution reagent that can remove the incorporated nucleotides (and any modifications carried therein) under suitable conditions.
  • the elution reagent can also include a reagent that can remove the remaining blocking groups.
  • step (1) when step (1) is performed in a gel state, the steps include:
  • reaction composition containing a nucleotide carrying a blocking group with the target nucleic acid molecule, wherein the reaction composition contains a polymer having a reversible thermo-gelling property and the reaction composition 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 nucleotides; preferably, the preset temperature allows the incorporation reaction to occur;
  • step (3) when step (3) is performed in a gel state, it includes the following steps:
  • 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-gelling 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 excision reaction or an elution reaction to allow removal of a label or blocking group on a nucleotide or removal of an incorporated nucleotide; preferably, the preset temperature allows the excision reaction or the elution reaction to occur;
  • step (3) is an excision reaction, and when the labeled nucleotide in step (1) further comprises an independent blocking group, step (3) comprises removing the blocking group and the label from the incorporated nucleotide using a removal reagent.
  • step (3) comprises the following steps when performed in a gel state:
  • 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-gelling property, and the reaction composition is in a liquid state;
  • the removal reagent includes a reagent capable of removing a label and a blocking group;
  • (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an excision reaction to allow the removal of blocking groups and labels on the nucleotides; preferably, the preset temperature allows the excision reaction to occur;
  • step (3) is an excision reaction, and when the label contained in the labeled nucleotide in step (1) can be used 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.
  • step (3) includes the following steps when performed in a gel state:
  • 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-gelling property, and the reaction composition is in a liquid state;
  • the removal reagent includes a reagent capable of removing the label;
  • (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, wherein the removal of the label will simultaneously remove its blocking effect; preferably, the preset temperature allows the excision reaction to occur;
  • step (3) is an excision reaction, comprising removing the blocking group from the incorporated nucleotide using a removal reagent.
  • step (3) comprises the following steps when performed in a gel state:
  • 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-gelling property, and the reaction composition is in a liquid state;
  • the removal reagent includes a reagent capable of removing a blocking group;
  • (3b) providing a preset temperature to convert the reaction composition into a gel, and performing an excision reaction to allow the removal of the blocking group on the nucleotide; preferably, the preset temperature allows the excision reaction to occur;
  • step (3) is an elution reaction, which includes removing the incorporated nucleotides and the remaining blocking groups using an elution reagent.
  • step (3) includes the following steps when performed in a gel state:
  • 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-gelling property, wherein the reaction composition is in liquid state;
  • the eluting 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;
  • (3b) providing a preset 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 preset temperature allows the elution reaction to occur;
  • step (3) 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.
  • step (4) when step (4) is performed in a gel state, it comprises the following steps:
  • 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;
  • the method before, during or after any step before step (3) (e.g., 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.
  • the nucleotide incorporation in this step forms a phosphodiester bond.
  • the method comprises the following steps:
  • 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 the reaction composition is in a liquid state;
  • the method before step (1), 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.
  • step (1) the method comprises the following steps:
  • 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 the reaction composition is in a liquid state;
  • the method is flow cell sequencing.
  • flow cell sequencing refers to any sequencing method based on a flow cell that fixes the nucleic acid molecules to be tested.
  • 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 can be fully carried out in the shortest possible time.
  • the target nucleic acid molecule is fixed to the surface of a flow cell.
  • the method includes 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.
  • the method is open sequencing.
  • 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.
  • one or more sequencing reagents are introduced into a flow pool fixed with a target nucleic acid molecule.
  • 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 moved to contact it with the one or more sequencing reagents placed in a separate reaction vessel, or it is immersed in the one or more sequencing reagents to complete the corresponding sequencing biochemical reaction.
  • the solid support fixed with the target nucleic acid molecule can be referred to as an open sequencing slide.
  • the target nucleic acid molecule is immobilized on the surface of an open sequencing slide.
  • the method comprises placing an open The sequencing slides are respectively contacted with or immersed in one or more sequencing reagents required for the sequencing reaction, and the one or more sequencing reagents are respectively placed in separate reaction containers.
  • the method provided by the present invention is particularly suitable for an open sequencing platform.
  • the method of the present invention has the advantages of an open scheme, such as reduced cost, simple fluids, and support for ultra-large chips, and solves some problems of the open scheme, such as chip drying, sample damage, and some special processes (such as sample loading) cannot be automated.
  • 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.
  • 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).
  • 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).
  • the polymer is selected from Pluronic block polymers.
  • 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).
  • the polymer is selected from methylcellulose or hydroxypropylmethylcellulose.
  • the polymer is a block copolymer, a graft copolymer, and a homopolymer.
  • 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( ⁇ -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), etc.
  • Pluronic block polymers e.g., Pluronic F127
  • Tetronic block polymers e.g., Tetronic block polymers, hydroxypropyl methylcellulose, methylcellulose (e.
  • Pluronic block polymers can be selected from Pluronic F38, P65, P68LF, P75, F77, P84, P85, F87, F88, F98, P103, P104, P105, F108, P123, F123, F127, 10R8, 17R8, 25R5, 25R8, for example Pluronic F127.
  • Methylcellulose can be selected from Methocel A, Methocel A4C, Methocel A15C, Methocel A15 LV, Methocel A4M, for example Methocel A15C or Methocel A15 LV.
  • Hydroxypropyl methylcellulose can be selected from Methocel E5LV, Methocel E3LV, Methocel E6LV, Methocel E15LV, Methocel E50LV and Methocel K3LV.
  • 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).
  • the polymer has a gelling temperature of about 10-65° C. In certain embodiments, the polymer has a gelling 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.
  • the temperature at which the polymer having reversible thermogelling property transforms from a gel state to a liquid state is about 0 to 30°C.
  • the temperature at which the polymer having reversible thermogelling properties is converted from a gel state to a liquid state is about 0 to 10° C., such as about 2 to 8° C., such as about 4° C.
  • the polymer is selected from Pluronic block polymers.
  • the concentration of the polymer is about 10 to 30% (w/w), such as about 15 to 25% (w/w), about 18 to 22% (w/w), such as about 20% (w/w).
  • the temperature at which the polymer having a reversible thermogelling property is converted from a gel state to a liquid state is about 20 to 30° C., for example, about 20 to 25° C., for example, about 20° C. or about 25° C.
  • the polymer is selected from methylcellulose or hydroxypropylmethylcellulose.
  • the concentration of the polymer is about 0.5 to 10% (w/w), for example, about 0.5 to 5% (w/w), about 0.5 to 2% (w/w), about 1 to 2% (w/w), such as about 1% (w/w).
  • the preset temperature refers to a temperature that allows the reaction composition containing a polymer having a reversible thermo-gelling property to be converted from a liquid state to a gel and allows 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, an affinity reaction and a catalytic reaction in a bioluminescent sequencing method, an affinity reaction of a fluorescently labeled affinity reagent), etc.
  • 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.
  • the preset temperature should allow the incorporation reaction to occur.
  • the preset temperature at which the reaction composition comprising nucleotides e.g., nucleotides carrying a label, unlabeled nucleotides, or nucleotides carrying a blocking group and not labeled
  • the preset temperature at which the reaction composition comprising nucleotides is converted into a gel is about 50-60° C., such as about 55-60° C., such as about 55° C.
  • the preset temperature should allow incorporation of nucleotides. Removal of blocking groups and/or fluorescent groups
  • 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, such as about 55°C.
  • the preset temperature should allow removal of incorporated nucleotides and removal of remaining blocking groups.
  • the preset temperature at which the reaction composition comprising an elution reagent is converted into a gel is about 50 to 60° C., such as about 55 to 60° C., such as about 55° C.
  • the preset temperature should allow primer hybridization to occur.
  • the preset temperature for converting the reaction composition containing sequencing primers into a gel is about 35-45° C., such as about 37-45° C., about 37-42° C., such as about 37° C.
  • 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.
  • the preset temperature should allow the affinity reaction to occur, such as about 30-40°C, such as about 30-37°C, about 32-37°C, about 35-37°C, such as about 35°C.
  • the reaction composition comprising the polymer having reversible thermogelling properties comprises any necessary ingredients that allow the corresponding reaction to occur.
  • the necessary components may be an enzyme (e.g., a polymerase, e.g., a DNA polymerase), a primer, a buffer, or any combination thereof.
  • the reaction composition for incorporation reaction comprises nucleotides to be incorporated and an enzyme (e.g., a polymerase, e.g., a DNA polymerase), and the nucleotides to be incorporated may be nucleotides carrying a blocking group and a label, nucleotides carrying a blocking group and no label, unlabeled nucleotides, or any combination thereof.
  • nucleotide is intended to include natural nucleotides, non-natural nucleotides, ribonucleotides, deoxyribonucleotides, dideoxyribonucleotides and their analogs.
  • the term generally refers to a nucleoside portion (regardless of ribose, deoxyribose or its analogs) comprising a base portion and optionally attached to a nucleoside portion containing one or more phosphate moieties herein.
  • the term can be used to refer to a monomer unit present in a polynucleotide, for example, to identify a subunit present in a DNA or RNA chain.
  • nucleotides include, but are not limited to, monophosphate ribonucleotides (sometimes referred to as ribonucleoside monophosphates), diphosphate ribonucleotides (sometimes referred to as ribonucleoside diphosphates), and oligonucleotides.
  • ribonucleotide may be used to designate RNA nucleotides, such as ribouridine triphosphate, riboguanosine triphosphate, ribocytidine triphosphate, or riboadenosine triphosphate
  • deoxynucleotide may be used to designate RNA nucleotides, such as deoxythymidine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, or deoxyadenosine triphosphate, when it is desired to distinguish RNA components from DNA components.
  • the nucleotide to be incorporated is a dNTP or an analog thereof.
  • 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.
  • the analog means a synthetic nucleotide with a modified base moiety, a modified phosphodiester connection and/or a modified sugar moiety.
  • the nucleotide to be incorporated is a nucleotide carrying a label.
  • the label is a fluorescent group.
  • the nucleotides to be incorporated are nucleotides carrying separate blocking groups and labels.
  • the blocking group described in the application can prevent the further incorporation of nucleotide in the complementary chain of the target nucleic acid molecule.
  • the technician will understand how to attach a suitable blocking group to the ribose ring of nucleotide to block the interaction with 3' hydroxyl.
  • the blocking group can be directly attached to the 3' position or can be attached to the 2' position (the blocking group has enough size or charge to block the interaction at the 3' position).
  • the blocking group can be attached to both 3' and 2' positions and can be cut to expose the 3' hydroxyl group. Suitable blocking groups will be apparent to those skilled in the art.
  • the nucleotide to be incorporated is a nucleotide that carries only a label that can be used as a blocking group.
  • 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 caused by steric hindrance, or may be caused by a combination of size, charge and structure.
  • the blocking group and/or label may be connected to the nucleotide via a cleavable linker.
  • the linker comprises one or more cleavable groups.
  • the linker is selected from: 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.
  • electrophilic cleavable linker The type of disulfide linker, acid-labile linker (such as dialkoxybenzyl linker, Sieber linker, indole linker, tert-butyl Sieber linker), electrophilic cleavable linker, nucleophilic cleavable linker, photocleavable linker It is obvious to those skilled in the art.
  • electrophilic cleavable linkers are usually 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.
  • Suitable electrophilic cleavable linkers include tert-butyloxycarbonyl (Boc) groups and acetal systems.
  • sulfur-philic metals such as nickel, silver or mercury in the cleavage of thioacetal 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 unstable in water such as esters (i.e., can be simply cleaved at alkaline pH) and groups unstable to non-aqueous nucleophiles can be used.
  • Photo-cleavable linkers preferably activate the light required for cleavage without affecting other components of the modified nucleotides. For example, if a fluorescent group is used as a label, it is preferred that it absorbs light of different wavelengths from the light required for cleavage of the linker molecule.
  • the nucleotide to be incorporated is a nucleotide carrying a blocking group and not labeled.
  • the incorporation of such nucleotides will help to supplement the incomplete incorporation reaction, ensure the synthesis efficiency, and reduce the risk of incomplete reaction; and because such nucleotides are not labeled, the signal interference caused by unclean excision of the label or unclean elution of the excised label is also reduced, the excision efficiency is improved, and the sequencing error rate is reduced.
  • the nucleotide to be incorporated is an unlabeled nucleotide.
  • the step of removing the label will simultaneously remove the incorporated nucleotide, and in such embodiments, unlabeled nucleotides need to be re-added to allow the next round of sequencing reactions to proceed.
  • the unlabeled nucleotide refers to a nucleotide that does not carry a label but has a blocking group.
  • the nucleotides to be incorporated can form phosphodiester bonds.
  • the nucleotide to be incorporated is incapable of forming phosphodiester bonds.
  • the target nucleic acid molecule is immobilized on a solid support.
  • fixing when used in reference to nucleic acids means directly or indirectly attached to a solid support via covalent or non-covalent bonds.
  • the nucleic acid is covalently attached to a solid support.
  • fixing the nucleic acid on a solid support can include fixing an oligonucleotide to be used as a capture primer or an amplification primer on 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.
  • fixing the nucleic acid on a solid support 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.
  • ways to attach nucleic acids to solid supports include nucleic acid hybridization, biotinylation, and the like. Binding to streptavidin, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, etc.
  • the support can be made of various suitable materials.
  • 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.
  • cellulose cellulose derivatives (e.g., nitrocellulose)
  • acrylic resins glass, silica gel, silicon dioxide, polystyrene
  • 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 covalent attachment of biomolecules such as polynucleotides.
  • the support is a glass or silicon slide with a layer of avidin, amino, acrylamide silane or aldehyde chemical groups modified on the surface.
  • the solid support is not limited to its size, shape and configuration.
  • the solid support is a planar structure, such as a slide, a chip, a microchip and/or an array.
  • the surface of such a support can be in the form of a planar layer.
  • the support for immobilizing the nucleic acid molecules to be sequenced is a chip, such as a high-throughput sequencing chip.
  • nucleic acid clusters are prepared by a solid phase amplification method of bridge amplification.
  • nucleic acid clusters are prepared by rolling circle amplification to form DNA nanoballs (DNBs).
  • the target nucleic acid molecule is present in a nucleic acid array.
  • each site on the array may include multiple copies of a single target nucleic acid molecule.
  • the target nucleic acid molecule may be a DNB formed by multiple copies of a single target nucleic acid molecule.
  • DNA nanoballs are concatemers containing multiple copies of a target nucleic acid molecule. These nucleic acid copies are typically Arranged one by one in a continuous linear chain of nucleotides, the tandem repeat structure together with the single-stranded nature of DNA causes the nanoball to fold (folding) configuration.
  • the multiple copies of the target nucleic acid molecules in the DNB each contain a known linker sequence, so that it can be amplified or sequenced.
  • the linker sequences of each target nucleic acid molecule are usually the same, but can also be different.
  • DNB can be produced using, for example, rolling circle replication (RCA).
  • DNB can be loaded on the surface of solid support as described herein.
  • DNB can be attached to the surface of solid support by any suitable method, and the non-limiting example of such method includes nucleic acid hybridization, biotin streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, physical restriction via hydrogel or other porous polymers, etc., or their combination.
  • the solid support surface may carry reactive functional groups, and the reactive functional groups react with complementary functional groups on polynucleotide molecules to form covalent bonds, for example, using the same method as the technology used to attach cDNA to microarray.
  • DNB can also be effectively attached to hydrophobic surfaces, for example, clean glass surfaces with various reactive functional groups (for example-OH groups) at low concentrations.
  • DNB can be adsorbed onto the surface.
  • polynucleotides are fixed by non-specific interactions with the surface, or by non-covalent interactions such as hydrogen bonds, van der Waals forces, etc.
  • the present invention provides a kit comprising:
  • composition comprising a nucleotide carrying a label and a polymer having a reversible thermogelling property, the composition optionally further comprising an enzyme (e.g. a polymerase, such as a DNA polymerase), a primer and/or a buffer; and/or,
  • an enzyme e.g. a polymerase, such as a DNA polymerase
  • composition comprising a removal agent and a polymer having reversible thermogelling properties, the composition optionally comprising a buffer.
  • the nucleotide carrying the label described in (a) also comprises an independent blocking group and is capable of forming a phosphodiester bond.
  • 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 may be the same or different.
  • the label contained in the nucleotide carrying the label in (a) can be used as a blocking group and can form a phosphodiester bond.
  • the removal reagent in (b) includes a reagent capable of removing the label, and those skilled in the art understand that the removal of the label will simultaneously remove its blocking effect.
  • the kit further comprises: (c) a composition comprising a nucleotide carrying a blocking group and not having a label and a polymer having a reversible thermogelling property, the composition optionally further comprising an enzyme (e.g., a polymerase, e.g., a DNA polymerase), a primer and/or a buffer.
  • an enzyme e.g., a polymerase, e.g., a DNA polymerase
  • the nucleotide carrying a blocking group and not having a label (c) is capable of forming a phosphodiester bond.
  • the kit further comprises: (d) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property, wherein the composition optionally comprises a buffer.
  • the polymers having reversible thermogelling properties described in any of (a) to (d) may be the same or different.
  • 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).
  • the kit is used for sequencing, such as the sequencing method described herein.
  • the kit further comprises other reagents for sequencing.
  • the kit may further comprise: (e) a composition comprising a luciferase carrying an affinity tag and a polymer having a reversible thermogelling property, and/or (f) a composition comprising a specific substrate and a polymer having a reversible thermogelling property.
  • the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0 to 30° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0 to 10° C., such as about 2 to 8° C., such as about 4° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20 to 30° C., such as about 20 to 25° C., such as about 20° C. or about 25° C.
  • the preset temperature at which the composition of (a) is converted into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In some embodiments, the preset temperature at which the composition of (b) is converted into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C. In some embodiments, the preset temperature at which the composition of (c) is converted into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C.
  • the preset temperature at which the composition of (d) is converted into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, for example, about 37°C. In some embodiments, the preset temperature at which the composition of (e) is converted 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.
  • the kit described in any of the above embodiments may further comprise a washing solution.
  • the present invention also provides a kit comprising:
  • composition comprising a nucleotide carrying a blocking group and a polymer having a reversible thermogelling property, the composition optionally further comprising an enzyme (e.g. a polymerase, such as a DNA polymerase), a primer and/or a buffer; and/or,
  • an enzyme e.g. a polymerase, such as a DNA polymerase
  • primer and/or a buffer e.g. a primer and/or a buffer
  • 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.
  • a fluorescent label eg, an antibody recognizing a nucleotide
  • a polymer having a reversible thermogelling property wherein the composition optionally comprises a buffer.
  • the nucleotide carrying a blocking group is capable of forming a phosphodiester bond.
  • the affinity reagent is an antibody against the nucleotides A ⁇ T ⁇ C ⁇ G ⁇ U with blocking groups, and the antibody can specifically recognize the nucleotides and react with them with affinity.
  • the kit further comprises: (3) a composition comprising a removal reagent and a polymer having a reversible thermogelling property, wherein the composition optionally comprises a buffer.
  • the removal reagent comprises a reagent capable of removing a blocking group.
  • the kit further comprises: (4) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property, wherein the composition optionally comprises a buffer.
  • 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).
  • the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0 to 30° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0 to 10° C., such as about 2 to 8° C., such as about 4° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20 to 30° C., such as about 20 to 25° C., such as about 20° C. or about 25° C.
  • the preset temperature for converting the composition of (1) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C.
  • the preset temperature for converting the composition of (2) 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.
  • the preset temperature for converting the composition of (3) into a gel is about 50-60°C, such as about 55-60°C, for example, about 55°C.
  • the preset temperature for converting the composition of (4) into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, for example, about 37°C.
  • the present invention also provides a kit comprising:
  • composition comprising a nucleotide carrying a label and a polymer having a reversible thermogelling property, the composition optionally further comprising an enzyme (e.g. a polymerase, such as a DNA polymerase), a primer and/or a buffer;
  • composition comprising an eluting agent and a polymer having reversible thermogelling properties, the composition optionally comprising a buffer; and/or,
  • composition comprising unlabeled nucleotides and a polymer having a reversible thermogelling property, optionally further comprising an enzyme (eg a polymerase, such as a DNA polymerase), a primer and/or a buffer.
  • an enzyme eg a polymerase, such as a DNA polymerase
  • the nucleotide carrying the label in (i) further comprises an independent blocking group and does not form a phosphodiester bond.
  • the label included in the nucleotide carrying the label in (i) can be used as a blocking group and does not form a phosphodiester bond.
  • the unlabeled nucleotide in (iii) is capable of forming a phosphodiester bond. In certain embodiments, the unlabeled nucleotide in (iii) carries a blocking group.
  • the kit further comprises: (iv) a composition comprising a nucleotide carrying a blocking group and not having a label and a polymer having a reversible thermogelling property, the composition optionally further comprising an enzyme (e.g., a polymerase, e.g., a DNA polymerase), a primer and/or a buffer.
  • an enzyme e.g., a polymerase, e.g., a DNA polymerase
  • the nucleotide carrying a blocking group and not having a label is capable of forming a phosphodiester bond.
  • the kit further comprises: (v) a composition comprising a sequencing primer and a polymer having a reversible thermogelling property, wherein the composition optionally comprises a buffer.
  • the polymers having reversible thermogelling properties described in any of (i) to (v) may be the same or different.
  • 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); (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).
  • the kit is used for sequencing, such as the sequencing method described herein.
  • the kit also includes other reagents for sequencing.
  • the kit may also include: a composition comprising a luciferase carrying an affinity tag and a polymer with a reversible thermogelling property, and/or a composition comprising a specific substrate and a polymer with a reversible thermogelling property.
  • the temperature at which any composition contained in the kit is converted from a gel state to a liquid state is about 0 to 30° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 0 to 10° C., such as about 2 to 8° C., such as about 4° C. In some embodiments, the temperature at which the composition is converted from a gel state to a liquid state is about 20 to 30° C., such as about 20 to 25° C., such as about 20° C. or about 25° C.
  • the preset temperature at which the composition of (i) is converted 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 at which the composition of (ii) is converted 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 at which the reaction composition of (iii) is converted 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 at which the reaction composition of (iv) is converted 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 at which the composition of (v) is converted into a gel is about 35-45°C, such as about 37-45°C, about 37-42°C, for example, about 37°C.
  • the kit may further comprise a washing solution.
  • 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.
  • 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).
  • the polymer has a gelling concentration of about 0.5%-25%.
  • the polymer has a gelling temperature of about 10-65°C.
  • the polymer is a block copolymer, a graft copolymer, and a homopolymer.
  • 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( ⁇ -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).
  • Pluronic block polymers e.g., Pluronic F127
  • Tetronic block polymers hydroxypropyl methylcellulose, methylcellulose (e.g., Methocel A15C, Methocel A15 LV), methoxypolyethylene glycol-block-poly( ⁇ -caprolactone) (mPEG-PC
  • 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.
  • the present application provides a hydrogel-based sequencing method, which not only supports traditional sequencing systems based on flow cells, but also supports open sequencing systems and large-size sequencing chips.
  • the method can reduce reagent loss and avoid damage to target nucleic acid molecules on the surface of the slide due to drying and crystallization; it does not require complex fluids and temperature control; it can also effectively reduce the amount of reagents used, thereby achieving single use and reducing costs.
  • the method of the present application has broad application prospects.
  • RR-gel reagent Preparation of RR-gel reagent: Weigh 37.5 g Pluronic F-127, dissolve in 150 mL regeneration reagent (RR) to obtain RR-gel reagent (containing 20% (w/w) PF127), and store at 4°C. Alternatively, weigh 1.515 g Methocel A15C, dissolve in 150 mL regeneration reagent (RR), and obtain RR-gel reagent (containing 1% (w/w) Methocel A15C), and store at 4°C.
  • Hot-gel reagent and cold-gel reagent Weigh 37.5 g of Pluronic F-127, dissolve it in 150 mL of sequencing reagent, and store it at 4°C. Add 3 mL of dNTP mixture (with fluorescent label) and 3 mL of sequencing enzyme to 144 mL of the sequencing reagent prepared above (containing 20% (w/w) PF127) to obtain hot-gel reagent, and store it at 4°C. Add 6 mL of dNTP mixture II (without fluorescent label) and 3 mL of sequencing enzyme to 141 mL of the sequencing reagent prepared above (containing 20% (w/w) PF127) to obtain cold-gel reagent, and store it at 4°C.
  • Pluronic F-127 dissolve it in 150 mL of sequencing reagent, and store it at 4°C.
  • Add 3 mL of dNTP mixture (with fluorescent label) and 3 mL of sequencing enzyme to 144 mL of the sequencing reagent prepared above (containing
  • the reagent containing 20% (w/w) PF127 is in solution at 4°C and in hydrogel at room temperature.
  • the reagent containing 20% (w/w) PF127 is added dropwise to the slide and forms a hydrogel after heating.
  • the hydrogel does not flow when the slide is tilted.
  • the reagent containing 1% (w/w) Methocel A15C is added dropwise to the slide and forms a hydrogel after heating.
  • the hydrogel has a certain elasticity.
  • Example 3 Barcode sequencing using Hot-gel, Cold-gel and RR-gel reagents
  • hot-gel, cold-gel and RR-gel can complete SE1+barcode10 sequencing, and the resolution rate of 36 fovs is 72.25%, and the resolution rate of a single fov is 75.53%.

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Abstract

本发明提供了基于水凝胶的测序方法，适用于Flowcell测序体系，也支持开放式测序体系，并且在保证测序效率和准确度的同时，可有效降低成本。

Description

测序方法 技术领域
本发明涉及测序领域。具体地，本发明提供了基于水凝胶的测序方法，适用于Flowcell测序体系，也支持开放式测序体系，并且在保证测序效率和准确度的同时，可有效降低成本。
背景技术
随着测序技术的发展和社会认知度的提高，高通量基因测序已广泛应用于科学研究、精准医学、社会卫生和公共安全等的各个领域。近年来，虽然测序成本不断下降，但每个WGS(全基因组测序，100Gb数据)超过1000元人民币的价格，依然阻碍了基因测序更广泛的应用。现有高通量测序(这里主要指大规模平行测序法，MPS)技术方案中，主要采用两种技术方案实现液体交换和反应。其一是Flowcell(流道式)方案，通过使用注射泵、旋转阀等方式，将试剂从不同的位置抽出来，注入芯片流道当中，推走旧的试剂，以新的试剂替换，完成液体交换，替换的有效成分可进行生化反应；其二是浸没测序(DIP)方案，通过将试剂泵入试剂槽中，测序芯片在不同的试剂槽当中转移，每当浸泡到新的试剂里面，通过扩散作用实现液体和活性成分的交换。Flowcell的方案，为了实现试剂的有效替换，替换比(即，为了完全替换流道中的旧试剂而泵入的新试剂的体积与流道容积的比值)较高，一般需达到2-3以上，且试剂为一次性使用，因此虽然反应效率较高，但成本相应的也比较高。浸没测序方案，可以重复利用测序试剂，因此试剂成本可以有效降低；但是浸没测序方案存在测序过程中，芯片容易干燥，部分步骤使用浸没测序方案，反而增加成本或者影响质量，且试剂重复使用，存在质量受影响的风险。
因此，保证测序效率和准确度的同时、有效降低成本，对高通量测序是一个很大的挑战，也是亟待解决的问题。
发明概述
本申请提供了基于水凝胶的测序方法，该方法不仅支持基于流动池的传统测序体系，也支持开放式测序体系，可支持大尺寸测序芯片。该方法可减少试剂损失，以及避免干燥、结晶损伤载片表面的靶核酸分子；并且无需复杂流体和温控；还可以有效降低试剂使用量，从而实现单次使用，并降低成本。
在一方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：(1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；(2)对掺入核苷酸的标记进行检测；(3)去除标记；其中，步骤(1)和(3)中的至少一个步骤在凝胶状态下进行；任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。
在某些实施方案中，步骤(1)中所述的核苷酸掺入形成磷酸二酯键。在某些实施方案中，步骤(3)将允许所述靶核酸分子互补链进行延伸反应。
在某些实施方案中，所述标记为荧光标记。
在某些实施方案中，所述标记为亲和标记，所述步骤(2)包括步骤(2a),加入携带亲和标记的荧光素酶，与步骤(1)的产物进行亲和反应；步骤(2b)，加入特异性底物，与步骤(2a)的产物进行催化反应；步骤(2c)，对步骤(2b)的产物进行检测。在某些实施方案中，所述步骤(2a)和(2b)在凝胶状态下反应。
在某些实施方案中，当步骤(3)在凝胶状态下进行时包括以下步骤：(3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上标记的去除；(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
另一方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：(1)将携带阻断基团的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；(2)对掺入核苷酸进行检测；(3)去除阻断基团；其中，步骤(1)和(2)中的至少一个步骤在凝胶状态下进行；任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。优选地，步骤(1)中所述的核苷酸掺入形成磷酸二酯键。优选地，步骤(3)将允许所述靶核酸分子互补链进行延伸反应。
在某些实施例中，所述步骤(2)包括步骤(2a),加入携带荧光标记的亲和试剂，与步骤(1)的产物进行亲和反应；步骤(2b)对步骤(2a)的产物进行检测。优选的，其中，所述步骤(2a)在凝胶状态下反应。优选的，其中，所述亲和试剂为带有阻断基团的核苷酸A\T\C\G\U的抗体，所述抗体可以特异性识别核苷酸，并与之发生亲和反应。
在某些实施方案中，步骤(3)在凝胶状态下进行并且包括以下步骤：(3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上阻断基团的去除；(3c)改变温度使所述反应组合物转化为 液态，并去除反应组合物。
另一方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：(1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；(2)对掺入核苷酸的标记进行检测；(3)去除掺入核苷酸及其携带的标记；(4)将未标记核苷酸与所述靶核酸分子接触，以允许所述未标记核苷酸的掺入；其中，步骤(1)、(3)和(4)中的至少一个步骤在凝胶状态下进行；任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。在某些实施方案中，所述标记为荧光标记。
在某些实施方案中，步骤(1)中所述的核苷酸掺入仅遵循碱基互补配对原则结合在靶核酸互补链的3’末端，不形成磷酸二酯键。在某些实施方案中，步骤(4)中所述的核苷酸掺入发生聚合反应，形成磷酸二酯键。在某些实施方案中，步骤(4)中所述掺入的核苷酸具有阻断基团。在某些实施方案中，步骤(3)还包括去除所述靶核酸互补链的3’末端遗留的阻断基团，将允许所述靶核酸分子互补链的延伸反应。
在某些实施方案中，当步骤(3)在溶液状态下进行时包括：利用洗脱试剂在合适条件下将掺入的带有标记的核苷酸去除；优选地还包括将遗留的阻断基团去除。
在某些实施方案中，当步骤(3)在凝胶状态下进行时包括以下步骤：(3a)将包含洗脱试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(3b)提供预设温度使所述反应组合物转化为凝胶，并进行洗脱反应以允许掺入核苷酸的去除以及携带的标记的去除；(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。在此类实施方案中，所述方法例如还可以包括在步骤(4)的掺入反应后去除阻断基团，然后进行下一轮反应中步骤(1)的核苷酸掺入。
在某些实施方案中，当步骤(4)在凝胶状态下进行时包括以下步骤：(4a)将包含未标记核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶(thermogel)性质的聚合物，并且所述反应组合物为液态；优选地，所述未标记的核苷酸具有阻断基团；(4b)提供预设温度使所述反应组合物转化为凝胶，并进行聚合反应以允许核苷酸的掺入；(4c)改变温度使所述反应组合物转化为液态，任选地去除反应组合物。
在某些实施方案中，上述任一方面中，当步骤(1)在凝胶状态下进行时包括以下步骤：(1a)将包含携带标记或阻断基团的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶(thermogel)性质的聚合物，并且所述反应组合物为液态；(1b)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；(1c)改变温度使所述反应组合物转化为液态。在某些实施方案中，其中步骤(1c)中 使所述反应组合物转化为液态后，进一步包括：去除反应组合物或直接进入步骤(2)的检测反应。
在某些实施方案中，上述任一方面中，步骤(1)中所述的携带标记的核苷酸还包含独立的阻断基团；或者，所述携带标记的核苷酸所包含的标记可用作阻断基团。
在某些实施方案中，上述任一方面中，在步骤(3)之前的任意步骤之前、之中或之后(例如，在步骤(1)之后且步骤(2)之前)，所述方法还包括：将携带阻断基团且不带标记的核苷酸与所述靶核酸分子接触的步骤；所述步骤任选地在凝胶状态下进行。在某些实施方案中，在步骤(1)之后且步骤(2)之前，所述方法包括以下步骤：(i)将包含携带阻断基团且不带标记的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；(ii)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；(iii)改变温度使所述反应组合物转化为液态，去除反应组合物。
在某些实施方案中，上述任一方面中，在步骤(1)之前，所述方法还包括：将测序引物与所述靶核酸分子接触以允许杂交的步骤；所述步骤任选地在凝胶状态下进行。优选地，在步骤(1)之前所述方法包括以下步骤：(i)将包含测序引物的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；(ii)提供预设温度使所述反应组合物转化为凝胶，以使得所述测序引物杂交至靶核酸分子；优选地，所述预设温度允许引物杂交的发生；(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在某些实施方案中，上述任一方面中，所述具有可逆热致成胶性质的聚合物在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应。优选地，所述聚合物的浓度为约0.5～30％(w/w)，例如约10～30％(w/w)，约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)。优选地，所述聚合物具有约0.5％-25％(w/w)的成胶浓度。优选地，所述聚合物具有约10-65℃的成胶温度。优选地，所述聚合物是嵌段共聚物、接枝共聚物和均聚物。优选地，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸)(pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)。
在某些实施方案中，上述任一方面中，(a)凝胶状态转化为液态的温度为约0～30℃ (例如约0～10℃，例如约2～8℃，例如约4℃；例如约20～30℃，例如约20～25℃，例如约20℃或约25℃)；(b)包含核苷酸(例如携带标记的核苷酸、未标记核苷酸、或携带阻断基团且不带标记的核苷酸)的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；(c)包含去除试剂或洗脱试剂的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；和/或，(d)包含测序引物的反应组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
在某些实施方案中，上述任一方面中，所述靶核酸分子固定于固相支持物，例如芯片。
在某些实施方案中，上述任一方面中，所述方法是开放式测序。优选地，所述靶核酸分子固定于开放式测序载片表面。优选地，所述方法包括将固定有靶核酸分子的开放式测序载片分别与测序反应所需的一种或多种测序试剂接触或浸泡于其中，所述一种或多种测序试剂各自置于分开的反应容器中。
在某些实施方案中，上述任一方面中，所述方法是流道式(Flowcell)测序。优选地，所述靶核酸分子固定于流动池(flow cell)表面。优选地，所述方法包括将测序反应所需的一种或多种测序试剂引入固定有靶核酸分子的流动池。
另一方面，本发明提供了一种试剂盒，其包括：(a)包含携带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；和/或，(b)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，所述试剂盒还包括：(c)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，所述试剂盒还包括：(d)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，(a)-(d)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同。在某些实施方案中，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
另一方面，本发明提供了一种试剂盒，其包含：(1)包含携带阻断基团的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；和/或，(2)包含携带荧光标记的亲和试剂以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，所述试剂盒还包括：(3)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，所述试剂盒还包括：(4)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，(1)-(4)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同。在某些实施方案中，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
另一方面，本发明提供了一种试剂盒，其包括：(i)包含携带标记的核苷酸以及具有 可逆热致成胶性质的聚合物的组合物；(ii)包含洗脱试剂以及具有可逆热致成胶性质的聚合物的组合物；和/或，(iii)包含未标记核苷酸以及具有可逆热致成胶性质的聚合物的组合物；优选地，所述未标记核苷酸带有阻断基团。在某些实施方案中，所述试剂盒还包括：(iv)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，所述试剂盒还包括：(v)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物。在某些实施方案中，(i)-(v)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同。在某些实施方案中，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
在某些实施方案中，上述任一方面中，所述具有可逆热致成胶性质的聚合物在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应。优选地，所述聚合物的浓度为约0.5～30％(w/w)，例如约10～30％(w/w)，约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)。优选地，所述聚合物具有约0.5％-25％的成胶浓度。优选地，所述聚合物具有约10-65℃的成胶温度。优选地，所述聚合物是嵌段共聚物、接枝共聚物和均聚物。优选地，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸)(pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)。
在某些实施方案中，上述任一方面中，(a)-(d)、(1)-(4)、(i)-(v)任一项中所述的组合物由凝胶状态转化为液态的温度为约0～30℃(例如约0～10℃，例如约2～8℃，例如约4℃；例如约20～30℃，例如约20～25℃，例如约20℃或约25℃)；(a)、(1)或(i)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；(2)所述的反应组合物转化为凝胶的预设温度为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃；(b)、(3)或(ii)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；(iii)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；(c)或(iv)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；和/或，(d)、(4)或(v)所述的反应组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。以上实施例的温度范围不构成对本发明的温度设置的具体限制，根据反应试剂实际需要的温度设定，例如，掺入反应的聚合酶适宜的反应温度为37℃，则步骤(a)的凝胶温度 设置为37℃；引物杂交的温度为60℃，则相应步骤中凝胶的温度设置为60℃等。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得明显。
附图说明
图1显示了含20％PF127或1％Methocel A15C的试剂在不同温度下的状态。
图2显示了实施例1中RR-gel试剂的切割效果。
图3显示了实施例1中RR-gel试剂的Barcode测序。
图4显示了实施例2中Hot-gel试剂的Read1测序。
图5显示了实施例3中Hot-gel、Cold-gel和RR-gel试剂的Barcode测序。
图6显示了实施例4中Hot-gel、Cold-gel和RR-gel试剂的Read1测序结果。
图7显示了实施例5中Primer-gel引物杂交反应。
发明详述
本发明提供了基于水凝胶的测序方法，以及用于该测序方法的试剂盒。
除非另有定义，否则本文中所用的所有技术和科学术语的含义与本发明所属领域的普通技术人员通常理解的含义相同。本文中提及的所有出版物均以引用方式并入本文。
在提供数值范围的情况下，应当理解，该范围的上限和下限之间的每个中间值(例如任何整数值)都包含在本发明的范围内。当术语“约”用于描述可测量的值(例如，浓度、温度等)时，意味着包含给定值的±10％、±5％、±2％或±1％的范围。
测序方法
第一方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：
(1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
(2)对掺入核苷酸的标记进行检测；
(3)去除标记。
根据本发明的一个实施例，其中步骤(1)在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(1)和(3)在凝胶状态下进行。
在本文中，所述在凝胶状态下进行是指反应混合物为凝胶状。在某些实施方案中，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。在某些实施方案中， 步骤(1)中所述的核苷酸掺入形成磷酸二酯键。本领域技术人员理解，步骤(3)之后将允许所述靶核酸分子互补链的进一步延伸。
在某些实施方案中，所述标记为荧光标记。
在某些实施方案中，所述标记为亲和标记，所述步骤(2)包括步骤(2a),加入携带亲和标记的荧光素酶，与步骤(1)的产物进行亲和反应；步骤(2b)，加入特异性底物，与步骤(2a)的产物进行催化反应；步骤(2c)，对步骤(2b)的产物进行检测。
在某些实施方案中，所述步骤(2a)和(2b)在凝胶状态下反应。
在某些实施方案中，当步骤(2a)在凝胶状态下进行时包括以下步骤：(i)将包含携带亲和标记的荧光素酶的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(ii)提供预设温度使所述反应组合物转化为凝胶，并进行亲和反应；(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在某些实施方案中，当步骤(2b)在凝胶状态下进行时包括以下步骤：(i)将包含特异性底物的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(ii)提供预设温度使所述反应组合物转化为凝胶，并进行催化反应；(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
第二方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：
(1)将携带阻断基团的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
(2)对掺入核苷酸进行检测；
(3)去除阻断基团；
其中，步骤(1)和(2)中的至少一个步骤在凝胶状态下进行。
根据本发明的一个实施例，其中步骤(1)在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(2)中的至少一个步骤在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(1)和(2)在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(1)、(2)和(3)在凝胶状态下进行。
在本文中，所述在凝胶状态下进行是指反应混合物为凝胶状。在某些实施方案中，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。在某些实施方案中，步骤(1)中所述的核苷酸掺入形成磷酸二酯键。在某些实施方案中，步骤(3)将允许所述靶核酸分子互补链进行延伸反应。
在某些实施例中，所述步骤(2)包括步骤(2a),加入携带荧光标记的亲和试剂， 与步骤(1)的产物进行亲和反应；步骤(2b)对步骤(2a)的产物进行检测。
在某些实施方案中，所述步骤(2a)在凝胶状态下反应。在某些实施方案中，当步骤(2a)在凝胶状态下进行时包括以下步骤：(i)将包含携带荧光标记的亲和试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；(ii)提供预设温度使所述反应组合物转化为凝胶，并进行亲和反应；(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在某些实施方案中，所述亲和试剂为带有阻断基团的核苷酸A\T\C\G\U的抗体，所述抗体可以特异性识别核苷酸，并与之发生亲和反应。所述利用亲和试剂识别掺入碱基的方法在公开的专利(US10851410B2)中有具体描述。在此类实施方案中，荧光基团不是直接标记在掺入核苷酸上，而是标记在亲和试剂(例如抗体)上，亲和试剂可以特异性结合掺入核苷酸中的碱基、糖、可裂解的阻断基团或这些组分的组合，因此可以通过亲和试剂来鉴定被掺入的核苷酸类型。
第三方面，本发明提供了一种对靶核酸分子测序的方法，其包括以下步骤：
(1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
(2)对掺入核苷酸的标记进行检测；
(3)去除掺入核苷酸及其携带的标记；
(4)将未标记核苷酸与所述靶核酸分子接触，以允许所述未标记核苷酸的掺入。
根据本发明的一个实施例，其中步骤(1)在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(1)和(4)在凝胶状态下进行。
根据本发明的另一个实施例，其中步骤(1)、(3)和(4)在凝胶状态下进行。
在本文中，所述在凝胶状态下进行是指反应混合物为凝胶状态。在某些实施方案中，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列。
在某些实施方案中，所述标记为荧光标记。
在某些实施方案中，步骤(1)中所述的核苷酸掺入仅遵循碱基互补配对原则结合在靶核酸互补链的3’末端，不形成磷酸二酯键。可以使用特定的DNA聚合酶和/或缓冲液以阻止磷酸二酯键的形成，所述DNA聚合酶基本上不具备催化形成磷酸二酯键的活性，所述缓冲液基本上不含促进磷酸二酯键形成的二价阳离子。此类DNA聚合酶和/或缓冲液是本领域技术人员熟知的。
在某些实施方案中，步骤(4)中所述的核苷酸掺入发生聚合反应，形成磷酸二酯键。本领域技术人员理解，由于步骤(3)去除了原本掺入的核苷酸，步骤(4)的目的在于掺入被 步骤(3)去除的核苷酸，因此步骤(4)掺入的为未标记核苷酸(不带标记的核苷酸)，完成所述靶核酸分子互补链的延伸反应。在某些实施方案中，步骤(4)掺入的为具有阻断基团的核苷酸，以保证每次延伸反应只能掺入一个核苷酸。
在某些实施方案中，步骤(3)还包括去除所述靶核酸互补链的3’末端上遗留的阻断基团。所述遗留的阻断基团是指，在步骤(3)中掺入核苷酸及其携带的标记被去除后，所述靶核酸互补链的3’末端核苷酸(也即与被去除的核苷酸相邻的核苷酸)携带的可以阻断其它核苷酸继续掺入的修饰。所述相邻的核苷酸由上一轮的步骤(4)掺入。在某些实施方案中，步骤(3)将允许所述靶核酸分子互补链的延伸反应。
在某些实施方案中，在上述任一方面中，本文中所述的在凝胶状态下进行是指，将含有具有可逆热致成胶(thermogel)性质的聚合物的反应组合物与固定有靶核酸分子的固相支持物的表面接触，提供预设温度使所述反应组合物转化为凝胶，并给予反应所需的其他条件以进行相应的测序生化反应。反应之后，改变温度使所述反应组合物转化为液态，并洗涤，从而可进入下一步的反应。本文中所述的测序生化反应可以包括掺入反应(包括碱基延伸反应或聚合反应)、去除荧光基团和/或阻断基团的再生反应、测序引物杂交、以及检测掺入核苷酸的反应(例如，生物自发光的测序方法中的亲和反应与催化反应，荧光标记的亲和试剂的亲和反应)等。在某些实施方案中，所述接触是指将该液态反应组合物滴加或喷涂至所述固相支持物表面。
在某些实施方案中，在上述任一方面中，所述测序反应在同一张固相芯片上可以分区进行，例如将一张测序芯片划分几个区域，第一个区域进行掺入反应，第二个区域可以进行信号检测，同时第三个区域可以进行洗脱反应等，与分区测序方法对应的，所述固体支持物优选半导体检测芯片，所述固体支持物的温度优选分区控制，所述检测信号可以为被激发的荧光或者生物自发光。在某些实施方案中，所述测序反应在同一张固相芯片上可以分区进行，例如将液态反应组合物滴加或喷涂至所述固相支持物表面不同的区域，第一个区域滴加或喷涂掺入反应试剂，进行掺入反应，第二个区域滴加或喷涂扫描试剂，进行信号检测，第三个区域滴加或喷涂洗脱或再生试剂(所述再生试剂为切除试剂)，可以进行洗脱或再生反应(所述再生反应为切除反应)等，与分区测序方法对应的，所述固体支持物优选半导体检测芯片，所述固体支持物的温度优选分区控制，所述检测信号可以为被激发的荧光或者生物自发光，当所述检测信号为生物自发光时，滴加或喷涂的扫描试剂替换为修饰的荧光素酶或酶催化底物，生物自发光的测序方法和测序 试剂在专利US20220205036A1中有详细描述。在某些实施方案中，所述测序反应在同一张固相芯片上可以分区进行，将所述液态反应组合物滴加或喷涂至所述固相支持物表面不同的区域，再利用风刀去除固相表面反应之后的残余试剂。
核苷酸掺入的检测
易于理解，在上述任一方面中，核苷酸的掺入通过对其直接或间接携带的标记的检测来实现。在使用经标记的核苷酸的情况下，由标记直接或间接产生的信号携带核苷酸的身份信息，并且通过该信号可获知被测序的核酸分子上与该核苷酸互补配对的位置的核苷酸。此外也可以使用未经标记的核苷酸，在使用未标记的核苷酸的情况下，使用携带标记的亲和试剂(例如抗体)来特异性结合掺入核苷酸中的碱基、糖、可裂解的阻断基团或这些组分的组合，以实现对掺入核苷酸的间接标记。术语“标记”是指能够检测的任何分子，其能够直接或间接产生可被检测的信号(包括但不限于光信号、电信号、电磁信号、放射信号等)。
在某些实施方案中，所述标记是荧光标记。检测荧光标记的方式是本领域熟知的。例如，可以通过检测荧光的波长的装置来实现。这样的装置是本领域熟知的。例如，这样的装置可以是共焦扫描显微镜，其用激光扫描固体支持物的表面，以便使直接结合被测序的核酸分子上的荧光团成像。另外，可以例如用灵敏的2-D探测器，如电荷偶连的探测器(CCD)观察所产生的每一种信号。还可以例如使用诸如扫描近场光学显微方法(SNOM)的其他技术，还可以使用半导体检测芯片，利用光电二极管将光信号转换为电信号传输检测。
在某些实施方案中，所述标记是亲和标记。亲和标记被用于基于生物自发光反应的测序技术，可参见例如US20220205036A1。生物自发光的检测原理包括，待掺入核苷酸上不直接标记荧光信号，而是标记生物素或地高辛等亲和物质，聚合反应之后，加入带有荧光素酶的上述亲和物质的配对成员，从而将荧光素酶结合至被掺入的核苷酸上，然后加入荧光素酶的底物产生光信号，以识别被掺入核苷酸的身份。
在某些实施方案中，所述亲和标记是任何能够彼此特异性结合的分子配对的成员。配对成员之间的特异性结合实现核苷酸与荧光素酶的连接。在某些实施方案中，所述核苷酸带有的标记选自生物素、地高辛、N3G或FITC，荧光素酶带有与所述标记物对应的配对成员。例如，所述核苷酸带有的标记是生物素，则荧光素酶可以是经链霉亲和素标记的荧光素酶；所述核苷酸带有的标记是地高辛，则荧光素酶可以是经地高辛抗体标记的荧光素酶。所述荧光素酶来源包括但不限于firefly，gaussia，Renilla等生物。
测序反应
易于理解，在上述任一方面中，本文中所述的核苷酸的掺入是指掺入至靶核酸分子的互补链中，可以形成或者不形成磷酸二酯键。核苷酸沿5’到3’的方向相继掺入，并且遵循Watson-Crick碱基互补配对规则，因此，可通过检测到的被掺入的所述核苷酸的类型，并依照Watson-Crick碱基互补配对规则推断靶核酸分子的序列。
易于理解，一次循环中的步骤(1)将仅允许一个核苷酸掺入所述靶多核苷酸的互补链中，因此待掺入的核苷酸具备阻止延伸的修饰，该修饰阻止聚合酶在将含有该修饰的核苷酸掺入到正在合成的多核苷酸链上后继续催化另一核苷酸的掺入。
在某些实施方案中，所述修饰可以是单独的阻断基团(例如3’阻断基团)，该阻断基团典型地位于核苷酸的核糖环以阻断与3’羟基的相互作用。在此类实施方案中，步骤(1)中所述的携带标记的核苷酸还含有独立的阻断基团。
在某些实施方案中，所述修饰也可以是自身具备阻断作用的标记，不受理论约束，这样的标记可以具有足以发挥阻断其他核苷酸掺入到多核苷酸链上的大小或结构。所述阻断可能是由于空间位阻造成的，或者可能是由于大小、电荷和结构的组合造成的。在此类实施方案中，步骤(1)中所述的携带标记的核苷酸所包含的标记可用作阻断基团。
在某些实施方案中，所述携带标记的核苷酸所携带的标记可以以化学键连接在核苷酸上，也可以与核苷酸以复合物的形式连接，不受理论约束。
易于理解，在上述任一方面中，一次循环结束后将允许核苷酸的进一步掺入(即，靶多核苷酸互补链的继续延伸)，因此步骤(3)包括去除阻止靶多核苷酸互补链继续延伸的任何修饰。
当步骤(1)中所述的核苷酸掺入形成磷酸二酯键时，例如在第一或第二方面中，步骤(3)包括去除掺入核苷酸上阻止延伸的修饰。
当步骤(1)中所述的核苷酸掺入不形成磷酸二酯键时，例如在第三方面中，步骤(3)包括直接去除掺入的核苷酸，若步骤(3)中去除掺入的核苷酸，则将进一步包括步骤(4)将未标记核苷酸与所述靶核酸分子接触，以允许所述未标记核苷酸的掺入，完成所述靶核酸分子互补链的延伸反应(即，重新掺入步骤(3)去除的核苷酸)。步骤(4)中所述的未标记核苷酸应当具有阻断基团，以保证该步骤只掺入一个核苷酸。此外，在步骤(3)中，与被去除的核苷酸相邻的核苷酸(即，由上一轮反应的步骤(4)掺入的未标记但具有阻断基团的 核苷酸)所携带的阻断基团也将被去除，以允许本轮步骤(4)的进一步掺入。或者，在步骤(4)之后，去除步骤(4)中掺入的核苷酸的阻断基团，以允许下一轮测序反应循环的进行。
在某些实施方案中，当掺入核苷酸形成磷酸二酯键且掺入核苷酸包含独立的阻断基团和标记时，例如在第一方面中，步骤(3)还包括去除阻断基团以允许延伸。所述去除阻断基团和去除标记可以同时进行，也可以以任意顺序相继进行。所述去除阻断基团和去除标记可以使用相同的试剂，也可以使用不同的试剂。
在某些实施方案中，当掺入核苷酸形成磷酸二酯键且掺入核苷酸包含可用作阻断基团的标记时，例如在第一方面中，步骤(3)中去除标记的同时也将去除其阻断作用以允许延伸。
在某些实施方案中，当掺入核苷酸没有形成磷酸二酯键的情况下，例如在第三方面中，步骤(3)中去除掺入核苷酸的同时也将去除与其连接的标记，同时还包括去除遗留的阻断基团。可以理解的，在适合的条件下，利用洗脱试剂去除核苷酸及其携带标记，以及去除遗留的阻断基团。
在一些实施方案中，例如在第一方面中，本发明的方法包括以下步骤：(1)将携带阻断基团和标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入并形成磷酸二酯键；(2)对掺入核苷酸的标记进行检测；(3)从掺入核苷酸中去除阻断基团以及标记，以允许延伸；其中，步骤(1)和(3)中的至少一个步骤在凝胶状态下进行。
在另一些某些实施方案中，例如在第一方面中，本发明的方法包括以下步骤：(1)将携带标记的核苷酸与所述靶核酸分子接触，允许核苷酸的掺入并形成磷酸二酯键，所述标记可用作阻断基团；(2)对掺入核苷酸的标记进行检测；(3)从掺入核苷酸中去除标记并去除其阻断作用，以允许延伸；其中，步骤(1)和(3)中的至少一个步骤在凝胶状态下进行。
在另一些某些实施方案中，例如在第三方面中，本发明的方法包括以下步骤：(1)将携带标记的核苷酸与所述靶核酸分子接触，允许核苷酸的掺入，但不形成磷酸二酯键；(2)对掺入核苷酸的标记进行检测；(3)在适合的条件下，利用洗脱试剂去除带有标记的掺入的核苷酸，并且去除遗留的阻断基团；(4)将未标记但具有阻断基团的核苷酸与所述靶核酸分子接触，允许所述未标记核苷酸的掺入，且形成磷酸二酯键；其中，步骤(1)、(3)和(4)中的至少一个步骤在凝胶状态下进行。
在某些实施方案中，在上述任一方面的测序反应中，在聚合酶作用下，核苷酸(例 如携带阻断基团和/或标记的核苷酸)被掺入靶核酸分子的互补链中，该过程即为掺入反应。掺入反应可以形成或不形成磷酸二酯键。当掺入反应形成磷酸二酯键时也可被称为聚合反应。存在很多不同的聚合酶，并且对本领域普通技术人员来说容易确定最适合的聚合酶。优选的酶包括DNA聚合酶I、Klenow片段、DNA聚合酶III、T4或T7 DNA聚合酶、Taq聚合酶或vent聚合酶。还可以使用通过工程方法改造成具有特定性质的聚合酶。本申请所提供的测序方法中，掺入反应可以在凝胶状态下进行。
在某些实施方案中，在上述任一方面的测序反应中，当掺入的携带标记的核苷酸形成磷酸二酯键时，将已掺入核苷酸上的修饰(例如，阻断基团和/或标记)切除(例如，可以是切除独立的阻断基团和/或标记，也可以是切除具备阻断作用的标记)以允许下一轮测序的反应被称为切除反应。本申请所提供的测序方法中，切除反应可以在凝胶状态下进行。
在某些实施方案中，在上述任一方面的测序反应中，当掺入的携带标记的核苷酸不形成磷酸二酯键时，将已掺入核苷酸去除的反应被称为洗脱反应。洗脱反应还可以包含去除遗留的阻断基团。本申请所提供的测序方法中，洗脱反应可以在凝胶状态下进行。
在某些实施方案中，在第一方面中，步骤(1)和(3)均在凝胶状态下进行。在某些实施方案中，仅步骤(1)在凝胶状态下进行。在某些实施方案中，仅步骤(3)在凝胶状态下进行。在某些实施方案中，步骤(2)中的亲和反应也在凝胶状态下进行。
在某些实施方案中，在第三方面中，步骤(1)、(3)和(4)均在凝胶状态下进行。在某些实施方案中，仅步骤(1)在凝胶状态下进行。在某些实施方案中，仅步骤(3)在凝胶状态下进行。在某些实施方案中，仅步骤(1)和(4)在凝胶状态下进行。
在某些实施方案中，在第二方面中，步骤(1)和(2)均在凝胶状态下进行。在某些实施方案中，仅步骤(1)在凝胶状态下进行。在某些实施方案中，仅步骤(2)在凝胶状态下进行。在某些实施方案中，步骤(1)-(3)均在凝胶状态下进行。
在某些实施方案中，在第一或第三方面中，当步骤(1)在凝胶状态下进行时包括以下步骤：
(1a)将包含携带标记的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
(1b)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；优选地，所述预设温度允许掺入反应的发生；
(1c)改变温度使所述反应组合物转化为液态。
在一些实施方案中，所述携带标记的核苷酸还包含独立的阻断基团。
在另一些实施方案中，所述携带标记的核苷酸所包含的标记可用作阻断基团。
在一些实施方案中，步骤(1c)中使所述反应组合物转化为液态后，进一步包括：去除反应组合物或直接进入步骤(2)的检测反应。
在另一些实施方案中，所述步骤(1c)省略，步骤(1b)之后进行步骤(2)的检测反应。
在某些实施方案中，当步骤(1)所述的掺入形成磷酸二酯键时，步骤(3)为切除反应，并且可以通过使用去除试剂来实现。在本文中，“去除试剂”是指能够去除掺入核苷酸上的修饰的试剂。所述修饰可以包括独立的标记和阻断基团，或者也可以是可用作阻断基团的标记。去除试剂的选择取决于所使用的掺入核苷酸所带有的修饰。
在某些实施方案中，当步骤(1)所述的掺入不形成磷酸二酯键时，步骤(3)为洗脱反应，并且可以通过使用洗脱试剂来实现。在本文中，“洗脱试剂”是指在合适的条件下，可以去除掺入的核苷酸(以及其携带的任何修饰)的洗脱试剂。所述洗脱试剂还可以包含能够去除遗留的阻断基团的试剂。
在某些实施方案中，在第二方面中，当步骤(1)在凝胶状态下进行时包括以下步骤：
(1a)将包含携带阻断基团的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
(1b)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；优选地，所述预设温度允许掺入反应的发生；
(1c)改变温度使所述反应组合物转化为液态。
在某些实施方案中，在上述任一方面中，当步骤(3)在凝胶状态下进行时包括以下步骤：
(3a)将包含去除试剂或洗脱试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；
(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应或洗脱反应以允许核苷酸上标记或阻断基团的去除或者掺入核苷酸的去除；优选地，所述预设温度允许切除反应或洗脱反应的发生；
(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在一些实施方案中，在第一方面中，步骤(3)为切除反应，当步骤(1)中的携带标记的核苷酸还包含独立的阻断基团时，步骤(3)包括使用去除试剂从掺入核苷酸中去除阻断基团以及标记。在此类实施方案中，步骤(3)在凝胶状态下进行时包括以下步骤：
(3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；所述去除试剂包括能够去除标记和阻断基团的试剂；
(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上阻断基团以及标记的去除；优选地，所述预设温度允许切除反应的发生；
(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在另一些实施方案中，在第一方面中，步骤(3)为切除反应，当步骤(1)中的携带标记的核苷酸所包含的标记可用作阻断基团时，步骤(3)包括使用去除试剂从掺入核苷酸中去除标记并同时去除其阻断作用。在此类实施方案中，步骤(3)在凝胶状态下进行时包括以下步骤：
(3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；所述去除试剂包括能够去除标记的试剂；
(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上标记的去除，所述标记的去除将同时去除其阻断作用；优选地，所述预设温度允许切除反应的发生；
(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在另一些实施方案中，在第二方面中，步骤(3)为切除反应，包括使用去除试剂从掺入核苷酸中去除阻断基团。在此类实施方案中，步骤(3)在凝胶状态下进行时包括以下步骤：
(3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；所述去除试剂包括能够去除阻断基团的试剂；
(3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上阻断基团的去除；优选地，所述预设温度允许切除反应的发生；
(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在另一些实施方案中，在第三方面中，步骤(3)为洗脱反应，其包括使用洗脱试剂去除掺入核苷酸以及遗留的阻断基团。在此类实施方案中，步骤(3)在凝胶状态下进行时包括以下步骤：
(3a)将包含洗脱试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含 具有可逆热致成胶性质的聚合物，所述反应组合物为液态；所述洗脱试剂包括能够去除掺入的核苷酸(以及其携带的任何修饰)以及遗留的阻断基团的试剂，其中所述遗留的阻断基团可以是在掺入核苷酸被去除后靶核酸互补链的3’末端核苷酸携带的阻断其它核苷酸继续掺入的修饰；
(3b)提供预设温度使所述反应组合物转化为凝胶，并进行洗脱反应以允许掺入核苷酸的去除以及遗留的阻断基团的去除；优选地，所述预设温度允许洗脱反应的发生；
(3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在另一些实施方案中，当步骤(3)在溶液状态下进行时包括：利用洗脱试剂在合适条件下将掺入的带有标记的核苷酸以及遗留的阻断基团去除。
在某些实施方案中，在第三方面中，当步骤(4)在凝胶状态下进行时包括以下步骤：
(4a)将包含未标记核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶(thermogel)性质的聚合物，并且所述反应组合物为液态；优选地，所述未标记的核苷酸具有阻断基团，以确保靶核酸互补链的3‘末端在每轮延伸反应中只能掺入一个核苷酸；
(4b)提供预设温度使所述反应组合物转化为凝胶，并进行聚合反应以允许核苷酸的掺入；
(4c)改变温度使所述反应组合物转化为液态，任选地去除反应组合物。
在某些实施方案中，在第一或第三方面中，在步骤(3)之前的任意步骤之前、之中或之后(例如，在步骤(1)之后且步骤(2)之前)，所述方法还包括：将携带阻断基团且不带标记的核苷酸与所述靶核酸分子接触的步骤；所述步骤任选地在凝胶状态下进行。在某些实施方案中，该步骤所述的核苷酸掺入形成磷酸二酯键。
在某些实施方案中，在第一或第三方面中，在步骤(1)之后且步骤(2)之前，所述方法包括以下步骤：
(i)将包含携带阻断基团且不带标记的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
(ii)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；优选地，所述预设温度允许掺入反应的发生；
(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在某些实施方案中，在上述任一方面中，在步骤(1)之前，所述方法还包括：将测序引物与所述靶核酸分子接触以允许杂交的步骤；所述步骤任选地在凝胶状态下进行。
在某些实施方案中，在上述任一方面中，在步骤(1)之前所述方法包括以下步骤：
(i)将包含测序引物的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
(ii)提供预设温度使所述反应组合物转化为凝胶，以使得所述测序引物杂交至靶核酸分子；优选地，所述预设温度允许引物杂交的发生；
(iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
在某些实施方案中，在上述任一方面中，所述方法是流道式(Flowcell)测序。术语“流道式测序”是指任何基于固定有待测核酸分子的流动池(flow cell)而进行的测序方法。典型的流道式测序包括例如Illumina的SBS测序技术，以illumina的HiSeq 2500平台为例，该平台使用流动池，将测序反应试剂在特定时间点的特定位置从制冷设备泵入流动池，在一套复杂的流体控制设备的控制下流经测序芯片，并且辅助以精细的温控设施，确保在尽可能短的时间内让生化反应得以充分进行。
在某些实施方案中，在上述任一方面中，所述靶核酸分子固定于流动池(flow cell)表面。
在某些实施方案中，在上述任一方面中，所述方法包括将测序反应所需的一种或多种测序试剂引入固定有靶核酸分子的流动池，以完成相应的测序生化反应。
在某些实施方案中，在上述任一方面中，所述方法是开放式测序。术语“开放式测序”是指基于浸泡式反应方案或接触式反应方案的测序方法，参见例如WO2019023951A1中描述的方法。有别于传统的流道式测序中将一种或多种测序试剂引入固定有靶核酸分子的流动池，在开放式测序中，测序反应所需的一种或多种测序试剂各自置于分开的反应容器中，通过移动固定有待测核酸分子的固相支持物使其与置于分开的反应容器中的所述一种或多种测序试剂接触，或者将其浸泡于所述一种或多种测序试剂中，以完成相应的测序生化反应。在开放式测序中，固定有靶核酸分子的固相支持物可称为开放式测序载片。
在某些实施方案中，在上述任一方面中，所述靶核酸分子固定于开放式测序载片表面。
在某些实施方案中，在上述任一方面中，所述方法包括将固定有靶核酸分子的开放 式测序载片分别与测序反应所需的一种或多种测序试剂接触或浸泡于其中，所述一种或多种测序试剂各自置于分开的反应容器中。
在某些实施方案中，在上述任一方面中，本发明提供的方法特别适用于开放式测序平台。本发明的方法既具备开放式方案的优点，如成本降低、流体简单、支持超大尺寸芯片，又解决了开放式方案存在的一些问题，例如芯片干燥、样本受损、部分特殊流程(如样本加载)不能自动化等。
Thermogel聚合物
本文中所述的具有可逆热致成胶(thermogel)性质的聚合物是指在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应的聚合物。
在某些实施方案中，所述聚合物的浓度为约0.5～30％(w/w)，例如约0.5～5％(w/w)，0.5～2％(w/w)，约5～10％(w/w)，约10～15％(w/w)，约15～20％(w/w)，约20～25％(w/w)，约25～30％(w/w)。
在某些实施方案中，所述聚合物的浓度为约10～30％(w/w)，例如约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)。在某些实施方案中，所述聚合物选自Pluronic嵌段聚合物。
在某些实施方案中，所述聚合物的浓度为约0.5～10％(w/w)，例如约0.5～5％(w/w)，约0.5～2％(w/w)，约1～2％(w/w)，如约1％(w/w)。在某些实施方案中，所述聚合物选自甲基纤维素或羟丙基甲基纤维素。
在某些实施方案中，所述聚合物是嵌段共聚物、接枝共聚物和均聚物。在某些实施方案中，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸)(pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)等。Pluronic嵌段聚合物例如可以选自Pluronic F38、P65、P68LF、P75、F77、P84、P85、F87、F88、F98、P103、P104、P105、F108、P123、F123、F127、10R8、17R8、25R5、25R8，例如为Pluronic F127。甲基纤维素例如可以选自Methocel A、Methocel A4C、Methocel A15C、Methocel A15 LV、Methocel A4M，例如为Methocel A15C或Methocel A15 LV。羟丙基甲基纤维素例如可以选自Methocel E5LV、Methocel E3LV、Methocel E6LV、Methocel E15LV、Methocel E50LV以及Methocel K3LV。
在某些实施方案中，所述聚合物具有约0.5％-25％(w/w)的成胶浓度(Gel concentration)。在某些实施方案中，所述聚合物具有约0.5％-5％(w/w)、约5％-10％(w/w)、约10％-15％(w/w)、约15％-20％(w/w)或约20％-25％(w/w)的成胶浓度。
在某些实施方案中，所述聚合物具有约10-65℃的成胶温度。在某些实施方案中，所述聚合物具有约10-15℃、约15-20℃、约20-25℃、约25-30℃、约30-35℃、约35-40℃、约40-45℃、约45-50℃、约50-55℃、约55-60℃或约60-65℃的成胶温度。
在某些实施方案中，所述具有可逆热致成胶性质的聚合物由凝胶状态转化为液态的温度为约0～30℃。
在某些实施方案中，所述具有可逆热致成胶性质的聚合物由凝胶状态转化为液态的温度为约0～10℃，例如约2～8℃，例如约4℃。在某些实施方案中，所述聚合物选自Pluronic嵌段聚合物。在某些实施方案中，所述聚合物的浓度为约10～30％(w/w)，例如约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)。
在某些实施方案中，所述具有可逆热致成胶性质的聚合物由凝胶状态转化为液态的温度为约20～30℃，例如约20～25℃，例如约20℃或约25℃。在某些实施方案中，所述聚合物选自甲基纤维素或羟丙基甲基纤维素。在某些实施方案中，所述聚合物的浓度为约0.5～10％(w/w)，例如约0.5～5％(w/w)，约0.5～2％(w/w)，约1～2％(w/w)，如约1％(w/w)。
在某些实施方案中，本申请方法的任一步骤中，所述预设温度是指，允许包含具有可逆热致成胶性质的聚合物的反应组合物由液态转化为凝胶、同时允许所需反应发生的温度。所需反应可以是测序生化反应中的任意一种，例如掺入反应(包括碱基延伸反应或聚合反应)、去除荧光基团和/或阻断基团的再生反应、测序引物杂交、以及检测掺入核苷酸的反应(例如，生物自发光的测序方法中的亲和反应与催化反应，荧光标记的亲和试剂的亲和反应)等。以下提供了一些示例性预设温度，然而本领域技术人员完全有能力根据实际需要对预设温度进行调整，例如对于掺入反应而言，如果聚合酶适宜的反应温度为37℃，相应预设温度可设置为37℃，例如对于引物杂交而言，如果适宜温度为60℃，相应预设温度可设置为60℃。
例如，当所述反应组合物用于掺入反应时，所述预设温度应当允许掺入反应的发生。在某些实施方案中，包含核苷酸(例如携带标记的核苷酸、未标记核苷酸、或携带阻断基团且不带标记的核苷酸)的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。
例如，当所述反应组合物用于切除反应时，所述预设温度应当允许掺入核苷酸中的 阻断基团和/或荧光基团的去除。在某些实施方案中，包含去除试剂的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。
例如，当所述反应组合物用于洗脱反应时，所述预设温度应当允许掺入核苷酸的去除以及遗留的阻断基团的去除。在某些实施方案中，包含洗脱试剂的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。
例如，当所述反应组合物用于引物杂交(例如测序引物杂交至MDA扩增产生的MDA链)时，所述预设温度应当允许引物杂交的发生。在某些实施方案中，包含测序引物的反应组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
例如，当所述反应组合物用于生物自发光的测序方法中的亲和反应与催化反应时，所述预设温度应当允许所述亲和反应与催化反应的发生，例如为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃。
例如，当所述反应组合物用于荧光标记的亲和试剂的亲和反应时，所述预设温度应当允许所述亲和反应的发生，例如为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃。
反应组合物
在本申请方法的任一反应步骤中，包含具有可逆热致成胶性质的聚合物的反应组合物包含允许相应反应发生的任何必要成分。
在某些实施方案中，当所述反应组合物用于掺入反应时，所述必要成分可以是酶(例如聚合酶，例如DNA聚合酶)、引物、缓冲液或其任意组合。在某些实施方案中，进行掺入反应的反应组合物包括待掺入的核苷酸以及酶(例如聚合酶，例如DNA聚合酶)，所述待掺入的核苷酸可以是携带阻断基团和标记的核苷酸，携带阻断基团且不带标记的核苷酸，未标记的核苷酸，或其任意组合。
在文本中，术语“核苷酸”预期包括天然核苷酸、非天然核苷酸、核糖核苷酸、脱氧核糖核苷酸、双脱氧核糖核苷酸及其类似物。例如，该术语在本文通常指包含碱基部分并且任选地附接到含有一个或更多个磷酸部分的核苷部分(无论核糖、脱氧核糖或其类似物)。该术语可被用于指存在于多核苷酸中的单体单元，例如，以鉴定存在于DNA或RNA链中的亚基。术语还可被用于指不必需存在于多核苷酸中的单体分子，例如，能够通过聚合酶以模板依赖的方式并入多核苷酸中的分子。示例性的核苷酸包括但不限于，单磷酸核糖核苷酸(有时称为核糖核苷单磷酸)、二磷酸核糖核苷酸(有时称为核糖核 苷二磷酸)、三磷酸核糖核苷酸(有时称为核糖核苷三磷酸)、单磷酸脱氧核苷酸(有时称为脱氧核苷单磷酸)、二磷酸脱氧核苷酸(有时称为脱氧核苷二磷酸)和三磷酸脱氧核苷酸(有时称为脱氧核苷三磷酸)。为了清楚，当期望将RNA成分与DNA成分区分时，术语“核糖核苷酸”可被用于指定RNA核苷酸，诸如核糖尿苷三磷酸、核糖鸟苷三磷酸、核糖胞苷三磷酸或核糖腺苷三磷酸；且术语“脱氧核苷酸”被用于指定RNA核苷酸，诸如脱氧胸苷三磷酸、脱氧鸟苷三磷酸、脱氧胞苷三磷酸或脱氧腺苷三磷酸。
在某些实施方案中，待掺入的核苷酸为dNTP或其类似物。在某些实施方案中，待掺入的核苷酸选自三磷酸脱氧腺苷(dATP)或其类似物、三磷酸脱氧胸苷(dTTP)或其类似物、三磷酸脱氧胞苷(dCTP)或其类似物、三磷酸脱氧鸟苷(dGTP)或其类似物。在某些实施方案中，所述类似物意为合成的具有修饰的碱基部分、修饰的磷酸二酯连接和/或修饰的糖部分的核苷酸。
在某些实施方案中，待掺入的核苷酸是携带标记的核苷酸。在某些实施方案中，所述标记为荧光基团。
在一些实施方案中，待掺入的核苷酸是携带独立的阻断基团和标记的核苷酸。
本申请所述阻断基团可阻止核苷酸在所述靶核酸分子的互补链的进一步掺入。技术人员将理解如何将合适的阻断基团附接至核苷酸的核糖环以阻断与3’羟基的相互作用。阻断基团可以直接附接在3’位置处或可以附接在2’位置处(阻断基团具有足够的大小或电荷以阻断在3’位置处的相互作用)。或者，阻断基团可以附接在3’和2’位置两者处并且可以切割以暴露3’羟基基团。合适的阻断基团对于本领域技术人员将是显而易见的。
在另一些实施方案中，待掺入的核苷酸是仅携带标记的核苷酸，所述标记可用作阻断基团。不受理论约束，这样的标记可以具有足以发挥阻断其他核苷酸掺入到多核苷酸链上的大小或结构。所述阻断可能是由于空间位阻造成的，或者可能是由于大小、电荷和结构的组合造成的。此类标记对于本领域技术人员将是显而易见的。
在某些实施方案中，所述阻断基团和/或标记均可通过可裂解的接头与所述核苷酸连接。在某些实施方案中，所述接头包含一个或多个可裂解的基团。在某些实施方案中，所述接头选自：二硫化物接头，酸不稳定的接头(如二烷氧基苄基接头、Sieber接头、吲哚接头、叔丁基Sieber接头)，亲电可裂解的接头，亲核可裂解的接头，可光裂解的接头或其组合。
所述二硫化物接头，酸不稳定的接头(如二烷氧基苄基接头、Sieber接头、吲哚接头、叔丁基Sieber接头)，亲电可裂解的接头，亲核可裂解的接头，可光裂解的接头的类型对 本领域技术人员而言是显而易见的。例如，亲电可切割的接头通常被质子切割，并且包括对酸敏感的切割。合适的亲电可切割的接头包括改性的苄基系统，如三苯甲基、对烷氧基苄基酯和对烷氧基苄基酰胺。其他合适的亲电子可切割的接头包括叔丁氧羰基(Boc)基团和乙缩醛系统。此外，还可以考虑在硫缩醛或其他含硫保护基团的切割中使用亲硫金属如镍、银或汞，以制备合适的亲电可切割的接头分子。亲核切割是制备接头分子的公认方法。例如，可以使用在水中不稳定的基团如酯(即，可以在碱性pH下简单地切割)和对非水性亲核试剂不稳定的基团。可光切割的接头优选激活切割所需的光不影响修饰的核苷酸的其他组分。例如，如果使用荧光基团作为标记，则优选其吸收与切割接头分子所需的光不同波长的光。
在某些实施方案中，待掺入的核苷酸是携带阻断基团且不带标记的核苷酸。此类核苷酸的掺入将有利于补充掺入反应不完全的部分，保障合成效率，降低反应不充分的风险；并且由于此类核苷酸没有标记，也减低了标记切除不干净，或者切除的标记洗脱不干净导致的信号干扰，提高了切除的效率，降低了测序错误率。
在某些实施方案中，待掺入的核苷酸是未标记的核苷酸。当掺入的携带标记的核苷酸不形成磷酸二酯键时，去除标记的步骤将同时去除该掺入核苷酸，在此类实施方案中，需要重新加入未标记的核苷酸，以允许下一轮测序反应的进行。在某些实施方案中，所述未标记的核苷酸是指不携带标记但具有阻断基团的核苷酸。
在某些实施方案中，待掺入的核苷酸可以形成磷酸二酯键。
在某些实施方案中，待掺入的核苷酸不能形成磷酸二酯键。
固相支持物
在某些实施方案中，所述靶核酸分子固定于固相支持物。
在本文中，术语“固定”当提及核酸使用时，意指经由共价键或非共价键直接或间接附接至固体支持物。在某些实施方案中，核酸共价附接在固体支持物上。在某些实施方案中，将核酸固定在固体支持物上可以包括将待用作捕获引物或扩增引物的寡核苷酸固定在固体支持物上，使得3'末端对于酶促延伸是可利用的并且该引物序列的至少一部分能够杂交至互补核酸序列；然后将待固定的核酸杂交至所述寡核苷酸，在这种情况下固定的寡核苷酸或多核苷酸可以为3'-5'方向。在某些实施方案中，将核酸固定在固体支持物上可以包括通过氨基化修饰将核酸结合蛋白质结合在固体支持物上，并通过核酸结合蛋白质捕获核酸分子。核酸与固体支持物附接方式的非限制性示例包括核酸杂交、生 物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等。
在本文中，所述支持物可以由各种合适的材料制成。此类材料包括例如：无机物、天然聚合物、合成聚合物，以及其任何组合。具体的例子包括但不限于：纤维素、纤维素衍生物(例如硝化纤维素)、丙烯酸树脂、玻璃、硅胶、二氧化硅、聚苯乙烯、明胶、聚乙烯吡咯烷酮、乙烯基和丙烯酰胺的共聚物、与二乙烯基苯等交联的聚苯乙缔(参见例如，Merrifield Biochemistry 1964,3,1385-1390)、聚丙烯酰胺、乳胶、葡聚糖、橡胶、硅、塑料、天然海绵、金属塑料、交联的葡聚糖(例如，SephadexTM)、琼脂糖凝胶(SepharoseTM)，以及本领域技术人员已知的其他支持物。
在某些实施方案中，用于固定待测序的核酸分子的支持物可以是包括惰性基底或基质(例如，载玻片、聚合物珠等)的固体支持物，所述惰性基底或基质已例如通过应用含有活性基团的中间材料而被功能化，所述活性基团允许共价连接诸如多核苷酸的生物分子。在某些实施方案中，所述支持物为表面修饰了一层亲和素、氨基、丙烯酰胺硅烷或醛基化学基团的玻片或硅片。
在本文中，所述固体支持物不受限于其大小、形状和构造。在一些实施方案中，固体支持物是平面结构，例如载片、芯片、微芯片和/或阵列。此类支持物的表面可以是平面层的形式。
在某些实施方案中，用于固定待测序的核酸分子的支持物为芯片，例如高通量测序芯片。
多个拷贝的靶核酸分子
为增强所述标记的检测信号，本领域技术人员可选择形成多个拷贝的待测靶核酸分子，其拷贝数不受限制。在这类实施方案中，所述靶核酸分子的多个拷贝以核酸簇的形式被连接于所述固相支持物。在某些实施方案中，通过桥式扩增(bridge amplification)的固相扩增方法制备所述核酸簇。在某些实施方案中，通过滚环扩增来制备所述核酸簇，从而形成DNA纳米球(DNB)。
在某些实施方案中，所述靶核酸分子存在于核酸阵列。在某些实施方案中，所述阵列上的每个位点可以包括单个靶核酸分子的多个拷贝。
在某些实施方案中，所述靶核酸分子可以是单个靶核酸分子的多个拷贝形成的DNB。
DNA纳米球(DNB)是包含多拷贝的靶核酸分子的多联体。这些核酸拷贝典型地一个 接一个地布置在核苷酸的连续线型链中，该串联重复结构连同DNA的单链性质引起纳米球折叠(folding)配置。一般而言，DNB中的多拷贝的靶核酸分子各自包含序列已知的接头序列，以便于对其进行扩增或测序。各靶核酸分子的接头序列通常是相同的，但也可以不同。DNB可以使用例如滚环复制(RCA)来产生。
DNB可以装载在如本文所述的固体支持物的表面上。DNB可以通过任何合适的方法附着到固体支持物的表面，这样的方法的非限制性示例包括核酸杂交、生物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等，或它们的组合。在某些实施方案中，固体支持物表面可能带有反应性官能团，所述反应性官能团与多核苷酸分子上的互补官能团反应形成共价键，例如采用与附着cDNA到微阵列上所用的技术相同的方式进行。DNB还可以有效地附着到疏水性表面，例如带有低浓度的各种反应官能团(例如-OH基团)的干净的玻璃表面。在其他实施方案中，DNB可以吸附到表面上。在这种实施方式中，多核苷酸通过与表面的非特异性相互作用，或者通过诸如氢键、范德华力等的非共价相互作用被固定。
试剂盒
在另一方面，本发明提供了一种试剂盒，其包含：
(a)包含携带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液；和/或，
(b)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。
在一些实施方案中，(a)中所述携带标记的核苷酸还包含独立的阻断基团，并且能够形成磷酸二酯键。在此类实施方案中，(b)中所述去除试剂包括能够去除标记的试剂以及去除阻断基团的试剂，两种试剂可以相同或不同。
在另一些实施方案中，(a)中所述携带标记的核苷酸所包含的标记可用作阻断基团，并且能够形成磷酸二酯键。在此类实施方案中，(b)中所述去除试剂包括能够去除标记的试剂，本领域技术人员理解所述标记的去除将同时去除其阻断作用。
在某些实施方案中，所述试剂盒还包括：(c)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液。在某些实施方案中，(c)所述携带阻断基团且不带标记的核苷酸能够形成磷酸二酯键。
在某些实施方案中，所述试剂盒还包括：(d)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。
在某些实施方案中，(a)-(d)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同。
在某些实施方案中，所述试剂盒包含：(a)和(b)；(a)和(c)；(b)和(c)；(a)和(d)；(b)和(d)；(a)、(b)和(c)；(a)、(b)和(d)；或(a)、(b)、(c)和(d)。在某些实施方案中，所述试剂盒用于测序，例如本文中所述的测序方法。在某些实施方案中，所述试剂盒还包含用于测序的其他试剂。在某些实施方案中，当前述任一项的试剂盒用于生物发光测序技术时，所述试剂盒还可以包含：(e)包含携带亲和标记的荧光素酶以及具有可逆热致成胶性质的聚合物的组合物，和/或(f)包含特异性底物以及具有可逆热致成胶性质的聚合物的组合物。
在某些实施方案中，所述试剂盒中包含的任一组合物由凝胶状态转化为液态的温度为约0～30℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约0～10℃，例如约2～8℃，例如约4℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约20～30℃，例如约20～25℃，例如约20℃或约25℃。
在某些实施方案中，(a)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(b)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(c)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(d)所述的组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。在某些实施方案中，(e)所述的组合物转化为凝胶的预设温度为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃。在某些实施方案中，(f)所述的组合物转化为凝胶的预设温度为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃。
在某些实施方案中，上述任意实施方案中所述的试剂盒还可以包含洗涤溶液。
在另一方面，本发明还提供了一种试剂盒，其包含：
(1)包含携带阻断基团的核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液；和/或，
(2)包含携带荧光标记的亲和试剂(例如识别核苷酸的抗体)以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。
在某些实施方案中，(1)所述携带阻断基团的核苷酸能够形成磷酸二酯键。
在某些实施方案中，(2)所述亲和试剂为带有阻断基团的核苷酸A\T\C\G\U的抗体，所述抗体可以特异性识别核苷酸，并与之发生亲和反应。
在某些实施方案中，所述试剂盒还包括：(3)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。所述去除试剂包括能够去除阻断基团的试剂。
在某些实施方案中，所述试剂盒还包括：(4)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。
在某些实施方案中，所述试剂盒包含：(1)和(2)；(1)和(3)；(1)和(4)；(2)和(3)；(2)和(4)；(1)、(2)和(3)；(1)、(2)和(4)；或(1)、(2)、(3)和(4)。
在某些实施方案中，所述试剂盒中包含的任一组合物由凝胶状态转化为液态的温度为约0～30℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约0～10℃，例如约2～8℃，例如约4℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约20～30℃，例如约20～25℃，例如约20℃或约25℃。
在某些实施方案中，(1)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(2)所述的组合物转化为凝胶的预设温度为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃。在某些实施方案中，(3)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(4)所述的组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
在另一方面，本发明还提供了一种试剂盒，其包含：
(i)包含携带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液；
(ii)包含洗脱试剂以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液；和/或，
(iii)包含未标记核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液。
在一些实施方案中，(i)中所述携带标记的核苷酸还包含独立的阻断基团，并且不形成磷酸二酯键。在另一些实施方案中，(i)中所述携带标记的核苷酸所包含的标记可用作阻断基团，并且不形成磷酸二酯键。
在某些实施方案中，(iii)中所述未标记核苷酸能够形成磷酸二酯键。在某些实施方案中，(iii)中所述未标记核苷酸带有阻断基团。
在某些实施方案中，所述试剂盒还包括：(iv)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地还包含酶(例如聚合酶，例如DNA聚合酶)、引物和/或缓冲液。在某些实施方案中，(iv)所述携带阻断基团且不带标记的核苷酸能够形成磷酸二酯键。
在某些实施方案中，所述试剂盒还包括：(v)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物，所述组合物任选地包含缓冲液。
在某些实施方案中，(i)-(v)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同。
在某些实施方案中，所述试剂盒包含：(i)以及任选的(iv)和/或(v)；(ii)以及任选的(iv)和/或(v)；(iii)以及任选的(iv)和/或(v)；(i)和(ii)以及任选的(iv)和/或(v)；(i)和(iii)以及任选的(iv)和/或(v)；(ii)和(iii)以及任选的(iv)和/或(v)；(i)、(ii)和(ii)以及任选的(iv)和/或(v)。
在某些实施方案中，所述试剂盒用于测序，例如本文中所述的测序方法。在某些实施方案中，所述试剂盒还包含用于测序的其他试剂。在某些实施方案中，当前述任一项的试剂盒用于生物发光测序技术时，所述试剂盒还可以包含：包含携带亲和标记的荧光素酶以及具有可逆热致成胶性质的聚合物的组合物，和/或包含特异性底物以及具有可逆热致成胶性质的聚合物的组合物。
在某些实施方案中，所述试剂盒中包含的任一组合物由凝胶状态转化为液态的温度为约0～30℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约0～10℃，例如约2～8℃，例如约4℃。在某些实施方案中，所述组合物由凝胶状态转化为液态的温度为约20～30℃，例如约20～25℃，例如约20℃或约25℃。
在某些实施方案中，(i)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(ii)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(iii)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(iv)所述的组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃。在某些实施方案中，(v)所述的组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
在某些实施方案中，上述任一方面中，所述的试剂盒还可以包含洗涤溶液。
在某些实施方案中，上述任一方面中，所述具有可逆热致成胶性质的聚合物在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应。在某些实施方案中，所述聚合物的浓度为约0.5～30％(w/w)，例如约10～30％(w/w)，约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)。在某些实施方案中，所述聚合物具有约0.5％-25％的成胶浓度。在某些实施方案中，所述聚合物具有约10-65℃的成胶温度。在某些实施方案中，所述聚合物是嵌段共聚物、接枝共聚物和均聚物。在某些实施方案中，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸)(pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)。
本发明还涉及上述任一方面所述的试剂盒或者所述试剂盒中的任一种组合物用于测序的用途。本发明还涉及一种测序方法，其包括使用上述任一方面所述的试剂盒或者所述试剂盒中的任一种组合物。
发明的有益效果
本申请提供了基于水凝胶的测序方法，该方法不仅支持基于流动池的传统测序体系，也支持开放式测序体系，可支持大尺寸测序芯片。该方法可减少试剂损失，以及避免干燥、结晶损伤载片表面的靶核酸分子；并且无需复杂流体和温控；还可以有效降低试剂使用量，从而实现单次使用，并降低成本。本申请的方法具有广阔的应用前景。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
制备例：
I.试剂：

II.试剂配制步骤：
1、RR-gel试剂的配置：称取37.5g Pluronic F-127，溶解在150mL再生试剂(RR)中，得到RR-gel试剂(含20％(w/w)PF127)，4℃保存。或者，称取1.515g Methocel A15C，溶解在150mL再生试剂(RR)中，得到RR-gel试剂(含1％(w/w)Methocel A15C)，4℃保存。
2、hot-gel试剂和cold-gel试剂的配置：称取37.5g Pluronic F-127，溶解在150mL测序试剂中，4℃保存。将3mL dNTP混合液(带荧光标记)和3mL测序酶加到144mL上述制备的测序试剂(含20％(w/w)PF127)中，得到hot-gel试剂，4℃下保存。将6mL dNTP混合液II(不带荧光标记)和3mL测序酶加到141mL上述制备的测序试剂(含20％(w/w)PF127)中，得到cold-gel试剂，4℃保存。或者，
称取1.515g Methocel A15C，溶解在150mL测序试剂中。将3mL dNTP混合液(带荧光标记)和3mL测序酶加到144mL上述制备的测序试剂(含1％(w/w)Methocel A15C)中，得到hot-gel试剂，4℃下保存。将6mL dNTP混合液II(不带荧光标记)和3mL测序酶加到141mL上述制备的测序试剂(含1％(w/w)Methocel A15C)中，得到cold-gel试剂，4℃保存。
3、hot试剂和cold试剂的配置：将3mL dNTP混合液(带荧光标记)和3mL测序酶添加到144mL测序试剂(不含PF127和Methocel A15C)中，得到hot试剂，4℃保存。将6mL dNTP混合液II(不带荧光标记)和3mL测序酶添加到141mL测序试剂(不含PF127和Methocel A15C)中，得到cold试剂，4℃保存。
4、结果分析：如图1所示，4℃条件下含20％(w/w)PF127的试剂为溶液状态，室温下为水凝胶状态。将含20％(w/w)PF127的试剂滴加在载片上，加热后形成水凝胶，倾斜载片时水凝胶不会流动。类似地，将含1％(w/w)Methocel A15C的试剂滴加在载片上，加热后形成水凝胶，该水凝胶具有一定弹性。
III.测序循环步骤:
1.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，完成加载的测序载片保存在Wash buffer 2中。
2.用测序试剂冲洗测序载片，然后低温条件下滴加hot-gel，放置在55℃加热板上形成水凝胶。反应1-3min或5-10min后降温到20℃或水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
3.低温条件下滴加cold-gel，放置在55℃加热板上形成水凝胶。反应1-3min或5-10min后降温到20℃或水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
4.使用测序仪对测序载片拍照和basecall，完成cycle1测序。
5.用再生缓冲液冲洗测序载片，然后低温条件下滴加RR-gel，放置在55℃加热板上形成水凝胶。反应1-3min或5-10min后降温到20℃或水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
6.重复步骤2、3、4、5，完成多个测序循环。
实施例1：RR-gel试剂Barcode测序
1.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，杂交一链barcode测序引物，完成加载的测序载片保存在Wash buffer 2中。
2.用测序试剂冲洗测序载片，然后低温条件下滴加3mL hot试剂，放置在55℃加热板上，反应3min后倒掉试剂，用Wash buffer 2冲洗测序载片3次。
3.低温条件下滴加3mL cold试剂，放置在55℃加热板上，反应3min后倒掉试剂，用Wash buffer 2冲洗测序载片3次。
4.设置测序类型为SE1+barcode10。使用测序仪对测序载片拍照和basecall，完成cycle1测序。
5.对步骤4的测序载片再次拍照和basecall，完成cycle2测序。
6.用再生缓冲液冲洗测序载片，然后低温条件下滴加RR-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应10min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
7.重复步骤2、3、4、6，继续完成cycle3到cycle11。总共完成11个测序循环，结束测序。
8.结果分析：如图2所示，RR-gel(含20％(w/w)PF127)反应5min，然后再次滴加RR-gel(含20％(w/w)PF127)反应5min，切除完全；单次RR-gel(含20％(w/w)PF127)反应10min的切除效果较好，与两次RR-gel反应的效率相近。如图3所示，使用RR-gel可完成SE1+barcode10测序，441个fov的拆分率为79％，单个fov的拆分率为81％。
实施例2：Hot-gel试剂Read1测序
1.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，杂交一链测序引物1，完成加载的测序载片保存在Wash buffer 2中。
2.用测序试剂冲洗测序载片，然后低温条件下滴加hot-gel(含1％(w/w)Methocel A15C)，放置在55℃加热板上形成水凝胶。反应3min后降温到20℃并水平放置或浸泡在Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
3.低温条件下滴加3mL cold试剂，放置在55℃加热板上，反应3min后倒掉试剂，用Wash buffer 2冲洗测序载片3次。
4.设置测序类型为SE7。使用测序仪对测序载片拍照和basecall，完成cycle1测序。
5.用再生缓冲液冲洗测序载片，然后低温条件下滴加RR，放置在55℃加热板上，反应5min后倒掉试剂，用Wash buffer 2冲洗3次。
6.重复步骤2、3、4、5，继续完成cycle2到cycle8。总共完成8个测序循环(第8个cycle为校正cycle)，结束测序。
7.结果分析：如图4所示，使用hot-gel可完成SE7测序，信号强度较稳定，Unfilter Q30保持在80％左右。
实施例3：Hot-gel、Cold-gel和RR-gel试剂Barcode测序
1.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，杂交一链barcode测序引物，完成加载的测序载片保存在Wash buffer 2中。
2.用测序试剂冲洗测序载片，然后低温条件下滴加hot-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应8min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
3.低温条件下滴加cold-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应8min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
4.设置测序类型为SE1+barcode10。使用测序仪对测序载片拍照和basecall，完成cycle1测序。
5.对步骤4的测序载片再次拍照和basecall，完成cycle2测序。
6.用再生缓冲液冲洗测序载片，然后低温条件下滴加RR-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应5min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
7.重复步骤2、3、4、6，继续完成cycle3到cycle11。总共完成11个测序循环，结束测序。
8.结果分析：如图5所示，使用hot-gel、cold-gel和RR-gel可完成SE1+barcode10测序，36个fov的拆分率为72.25％，单个fov的拆分率为75.53％。
实施例4：Hot-gel、Cold-gel和RR-gel试剂Read1测序
1.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，杂交一链测序引物，完成加载的测序载片保存在Wash buffer 2中。
2.用测序试剂冲洗测序载片，然后低温条件下滴加hot-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应8min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
3.低温条件下滴加cold-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应8min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
4.设置测序类型为SE6。使用测序仪对测序载片拍照和basecall，完成cycle1测序。
5.用再生缓冲液冲洗测序载片，然后低温条件下滴加RR-gel(含20％(w/w)PF127)，放置在55℃加热板上形成水凝胶。反应5min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
6.重复步骤2、3、4、5，继续完成cycle2到cycle7。总共完成7个测序循环(第7个cycle为校正cycle)，结束测序。
7.结果分析：如图6所示，使用hot-gel、cold-gel和RR-gel可完成SE6测序，信号强度较稳定，Unfilter Q30保持在80％左右。
实施例5：Primer-gel引物杂交反应
1.称取5g Pluronic F-127，溶解在20mL测序引物1溶液中，得到Primer-gel试剂，4℃下保存。
2.使用DNB制备试剂盒制备DNB，使用DIPSEQ-T1测序载片和DNB加载试剂进行DNB加载，杂交一链测序引物，完成加载的测序载片保存在Wash buffer 2中。
3.使用测序仪对测序载片拍照和basecall。
4.用Wash buffer 2冲洗测序载片，然后滴加3mL去离子甲酰胺，放置在37℃加热板上反应10min，去除一链测序引物。倒掉甲酰胺，用Wash buffer 2冲洗3次，低温条件下滴加Primer-gel试剂，放置在37℃加热板上形成水凝胶，反应20min后水平放置在4℃环境或浸泡在4℃ Wash buffer 2中，待水凝胶转变为溶液后，倒掉试剂，用Wash buffer 2冲洗3次。
5.再次使用测序仪对测序载片拍照和basecall。
6.结果分析：如图7所示，形成水凝胶的试剂中的引物可以杂交到DNB。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解，根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (28)

  1. 对靶核酸分子测序的方法，其包括以下步骤：
    (1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
    (2)对掺入核苷酸的标记进行检测；
    (3)去除标记；
    其中，步骤(1)和(3)中的至少一个步骤在凝胶状态下进行；
    任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列；
    优选地，步骤(1)中所述的核苷酸掺入形成磷酸二酯键；
    优选地，步骤(3)将允许所述靶核酸分子互补链进行延伸反应。
  2. 根据权利要求1所述的方法，其中，所述标记为荧光标记。
  3. 根据权利要求1所述的方法，其中，所述标记为亲和标记，所述步骤(2)包括：步骤(2a)，加入携带亲和标记的荧光素酶，与步骤(1)的产物进行亲和反应；步骤(2b)，加入特异性底物，与步骤(2a)的产物进行催化反应；步骤(2c)，对步骤(2b)的产物进行检测。
  4. 根据权利要求3所述的方法，其中，所述步骤(2a)和(2b)在凝胶状态下反应。
  5. 对靶核酸分子测序的方法，其包括以下步骤：
    (1)将携带阻断基团的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
    (2)对掺入核苷酸进行检测；
    (3)去除阻断基团；
    其中，步骤(1)和(2)中的至少一个步骤在凝胶状态下进行；
    任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列；
    优选地，步骤(1)中所述的核苷酸掺入形成磷酸二酯键；
    优选地，步骤(3)将允许所述靶核酸分子互补链进行延伸反应。
  6. 根据权利要求5所述的方法，其中，所述步骤(2)包括：步骤(2a)，加入携带荧光标记的亲和试剂，与步骤(1)的产物进行亲和反应；步骤(2b)，对步骤(2a)的产物进行检测。
  7. 根据权利要求6所述的方法，其中，所述步骤(2a)在凝胶状态下反应。
  8. 对靶核酸分子测序的方法，其包括以下步骤：
    (1)将携带标记的核苷酸与所述靶核酸分子接触，以允许核苷酸的掺入；
    (2)对掺入核苷酸的标记进行检测；
    (3)去除掺入核苷酸及其携带的标记；
    (4)将未标记核苷酸与所述靶核酸分子接触，以允许所述未标记核苷酸的掺入；
    其中，步骤(1)、(3)和(4)中的至少一个步骤在凝胶状态下进行；
    任选地，所述方法包括依次重复上述步骤，以确定所述靶核酸分子的序列；
    优选地，步骤(1)中所述的核苷酸掺入不形成磷酸二酯键；
    优选地，步骤(4)中所述的核苷酸掺入形成磷酸二酯键；
    优选地，步骤(4)中所述掺入的核苷酸具有阻断基团；
    优选地，步骤(3)还包括去除所述靶核酸互补链的3’末端遗留的阻断基团，将允许所述靶核酸分子互补链的延伸反应。
  9. 根据权利要求1-8任一项所述的方法，其中，当步骤(1)在凝胶状态下进行时包括以下步骤：
    (1a)将包含携带标记或阻断基团的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶(thermogel)性质的聚合物，并且所述反应组合物为液态；
    (1b)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；
    (1c)改变温度使所述反应组合物转化为液态。
  10. 根据权利要求9所述的方法，其中步骤(1c)中使所述反应组合物转化为液态后，进一步包括：去除反应组合物或直接进入步骤(2)的检测反应。
  11. 根据权利要求1-7任一项所述的方法，其中，当步骤(3)在凝胶状态下进行时包括以下步骤：
    (3a)将包含去除试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；
    (3b)提供预设温度使所述反应组合物转化为凝胶，并进行切除反应以允许核苷酸上标记或阻断基团的去除；
    (3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
  12. 根据权利要求8所述的方法，其中，当步骤(3)在溶液状态下进行时包括：利用洗脱试剂在合适条件下将掺入的带有标记的核苷酸去除；优选地还包括将遗留的阻断基团去除。
  13. 根据权利要求8所述的方法，其中，当步骤(3)在凝胶状态下进行时包括以下步骤：
    (3a)将包含洗脱试剂的反应组合物与上一步骤的产物接触，所述反应组合物还包含具有可逆热致成胶性质的聚合物，所述反应组合物为液态；
    (3b)提供预设温度使所述反应组合物转化为凝胶，并进行洗脱反应以允许掺入核苷酸的去除以及遗留的阻断基团的去除；
    (3c)改变温度使所述反应组合物转化为液态，并去除反应组合物。
  14. 根据权利要求8所述的方法，其中，当步骤(4)在凝胶状态下进行时包括以下步骤：
    (4a)将包含未标记核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶(thermogel)性质的聚合物，并且所述反应组合物为液态；优选地，所述未标记的核苷酸具有阻断基团；
    (4b)提供预设温度使所述反应组合物转化为凝胶，并进行聚合反应以允许核苷酸的掺入；
    (4c)改变温度使所述反应组合物转化为液态，任选地去除反应组合物。
  15. 根据权利要求1-4、8任一项所述的方法，其中，步骤(1)中所述的携带标记的核苷酸还包含独立的阻断基团；或者，所述携带标记的核苷酸所包含的标记可用作阻断基团。
  16. 根据权利要求1-4、8任一项所述的方法，其中，在步骤(3)之前的任意步骤之前、之中或之后(例如，在步骤(1)之后且步骤(2)之前)，所述方法还包括：将携带阻断基团且 不带标记的核苷酸与所述靶核酸分子接触的步骤；所述步骤任选地在凝胶状态下进行。
  17. 根据权利要求16所述的方法，其中，在步骤(1)之后且步骤(2)之前，所述方法包括以下步骤：
    (i)将包含携带阻断基团且不带标记的核苷酸的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
    (ii)提供预设温度使所述反应组合物转化为凝胶，并进行掺入反应以允许核苷酸的掺入；
    (iii)改变温度使所述反应组合物转化为液态，去除反应组合物。
  18. 根据权利要求1-17任一项所述的方法，其中，在步骤(1)之前，所述方法还包括：将测序引物与所述靶核酸分子接触以允许杂交的步骤；所述步骤任选地在凝胶状态下进行；
    优选地，在步骤(1)之前所述方法包括以下步骤：
    (i)将包含测序引物的反应组合物与所述靶核酸分子接触，所述反应组合物包含具有可逆热致成胶性质的聚合物，并且所述反应组合物为液态；
    (ii)提供预设温度使所述反应组合物转化为凝胶，以使得所述测序引物杂交至靶核酸分子；优选地，所述预设温度允许引物杂交的发生；
    (iii)改变温度使所述反应组合物转化为液态，并去除反应组合物。
  19. 根据权利要求1-18任一项所述的方法，其中，所述具有可逆热致成胶性质的聚合物在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应；
    优选地，所述聚合物的浓度为约0.5～30％(w/w)，例如约10～30％(w/w)，约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)；
    优选地，所述聚合物具有约0.5％-25％(w/w)的成胶浓度；
    优选地，所述聚合物具有约10-65℃的成胶温度；
    优选地，所述聚合物是嵌段共聚物、接枝共聚物和均聚物；
    优选地，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸) (pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)。
  20. 根据权利要求1-19任一项所述的方法，其中：
    (a)凝胶状态转化为液态的温度为约0～30℃(例如约0～10℃，例如约2～8℃，例如约4℃；例如约20～30℃，例如约20～25℃，例如约20℃或约25℃)；
    (b)包含核苷酸(例如携带标记的核苷酸、未标记核苷酸、或携带阻断基团且不带标记的核苷酸)的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；
    (c)包含去除试剂或洗脱试剂的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；和/或，
    (d)包含测序引物的反应组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
  21. 根据权利要求1-20任一项所述的方法，其中，所述靶核酸分子固定于固相支持物，例如芯片。
  22. 根据权利要求1-21任一项所述的方法，其中，所述方法是开放式测序；
    优选地，所述靶核酸分子固定于开放式测序载片表面；
    优选地，所述方法包括将固定有靶核酸分子的开放式测序载片分别与测序反应所需的一种或多种测序试剂接触或浸泡于其中，所述一种或多种测序试剂各自置于分开的反应容器中。
  23. 根据权利要求1-21任一项所述的方法，其中，所述方法是流道式(Flowcell)测序；
    优选地，所述靶核酸分子固定于流动池(flow cell)表面；
    优选地，所述方法包括将测序反应所需的一种或多种测序试剂引入固定有靶核酸分子的流动池。
  24. 试剂盒，其包括：
    (a)包含携带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；和/或，
    (b)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，所述试剂盒还包括：(c)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，所述试剂盒还包括：(d)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，(a)-(d)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同；
    优选地，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
  25. 试剂盒，其包含：
    (1)包含携带阻断基团的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；和/或，
    (2)包含携带荧光标记的亲和试剂以及具有可逆热致成胶性质的聚合物的组合物。
    优选地，所述试剂盒还包括：(3)包含去除试剂以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，所述试剂盒还包括：(4)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，(1)-(4)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同；
    优选地，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
  26. 试剂盒，其包括：
    (i)包含携带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；
    (ii)包含洗脱试剂以及具有可逆热致成胶性质的聚合物的组合物；和/或，
    (iii)包含未标记核苷酸以及具有可逆热致成胶性质的聚合物的组合物；优选地，所述未标记核苷酸带有阻断基团；
    优选地，所述试剂盒还包括：(iv)包含携带阻断基团且不带标记的核苷酸以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，所述试剂盒还包括：(v)包含测序引物以及具有可逆热致成胶性质的聚合物的组合物；
    优选地，(i)-(v)任一项中所述的具有可逆热致成胶性质的聚合物可以相同或不同；
    优选地，所述试剂盒进一步包含测序所需的其他试剂，例如洗涤溶液。
  27. 根据权利要求24-26任一项所述的试剂盒，其中，所述具有可逆热致成胶性质的聚合物在温度升高的情况下会通过从液体变为凝胶而对温度变化做出反应；
    优选地，所述聚合物的浓度为约0.5～30％(w/w)，例如约10～30％(w/w)，约15～25％(w/w)，约18～22％(w/w)，如约20％(w/w)；
    优选地，所述聚合物具有约0.5％-25％的成胶浓度；
    优选地，所述聚合物具有约10-65℃的成胶温度；
    优选地，所述聚合物是嵌段共聚物、接枝共聚物和均聚物；
    优选地，所述聚合物选自Pluronic嵌段聚合物(例如Pluronic F127)、Tetronic嵌段聚合物、羟丙基甲基纤维素、甲基纤维素(例如Methocel A15C、Methocel A15 LV)、甲氧基聚乙二醇-嵌段-聚(ε-己内酯)(mPEG-PCL)、聚(N-异丙基丙烯酰胺-co-甲基丙烯酸)(pNIPAm-co-AA)、聚(乳酸-co-羟基乳酸)-聚乙二醇-聚(乳酸-co-羟基乳酸)(PLGA-PEG-PLGA)。
  28. 根据权利要求24-27任一项所述的试剂盒，其中：
    (a)-(d)、(1)-(4)、(i)-(v)任一项中所述的组合物由凝胶状态转化为液态的温度为约0～30℃(例如约0～10℃，例如约2～8℃，例如约4℃；例如约20～30℃，例如约20～25℃，例如约20℃或约25℃)；
    (a)、(1)或(i)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；
    (2)所述的反应组合物转化为凝胶的预设温度为约30～40℃，例如约30～37℃，约32～37℃，约35～37℃，例如约35℃；
    (b)、(3)或(ii)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；
    (iii)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；
    (c)或(iv)所述的反应组合物转化为凝胶的预设温度为约50～60℃，例如约55～60℃，例如约55℃；和/或，
    (d)、(4)或(v)所述的反应组合物转化为凝胶的预设温度为约35～45℃，例如约37～45℃，约37～42℃，例如约37℃。
PCT/CN2023/121926 2022-09-28 2023-09-27 测序方法 WO2024067674A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112368397A (zh) * 2018-03-13 2021-02-12 萨摩尔股份有限公司 用于单分子测序的方法
CN112384632A (zh) * 2018-10-11 2021-02-19 深圳华大智造极创科技有限公司 对多核苷酸进行测序的方法
CN112840035A (zh) * 2018-11-07 2021-05-25 深圳华大智造极创科技有限公司 对多核苷酸进行测序的方法
CN113748216A (zh) * 2019-05-15 2021-12-03 深圳华大智造极创科技有限公司 一种基于自发光的单通道测序方法
CN114286867A (zh) * 2019-08-20 2022-04-05 深圳华大智造极创科技有限公司 一种基于发光标记物光信号动力学及二次发光信号对多核苷酸进行测序的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112368397A (zh) * 2018-03-13 2021-02-12 萨摩尔股份有限公司 用于单分子测序的方法
CN112384632A (zh) * 2018-10-11 2021-02-19 深圳华大智造极创科技有限公司 对多核苷酸进行测序的方法
CN112840035A (zh) * 2018-11-07 2021-05-25 深圳华大智造极创科技有限公司 对多核苷酸进行测序的方法
CN113748216A (zh) * 2019-05-15 2021-12-03 深圳华大智造极创科技有限公司 一种基于自发光的单通道测序方法
CN114286867A (zh) * 2019-08-20 2022-04-05 深圳华大智造极创科技有限公司 一种基于发光标记物光信号动力学及二次发光信号对多核苷酸进行测序的方法

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