Method for loading nucleic acid molecules on a solid support

Abstract

Provided are methods of loading nucleic acid molecules, such as nucleic acid nanoballs (e.g., DNA nanoballs (DNBs)), on a solid support and kits for use in the methods.

Description

Technical Field

[0001] This application relates to the field of nucleic acid detection. Specifically, this application relates to a method for loading nucleic acid molecules, such as nucleic acid nanospheres (e.g., DNA nanospheres (DNB)), onto a solid support, and a kit for said method. Background Technology

[0002] Currently, gene chips or DNA microarrays are widely used in high-throughput sequencing. They consist of regular array slides or a single special glass slide coated with DNA microarrays. Thousands or tens of thousands of nucleic acid probes are mounted on an area of ​​a few square centimeters, providing a large amount of gene sequence-related information in a single test. They are important tools for genomics and genetics research.

[0003] High-throughput sequencing technologies include DNA and RNA sequencing. Generally, the DNA or RNA sample to be sequenced needs to be constructed into a library, such as a single-stranded circular library or a linear library. This library is then loaded onto a gene chip, either directly or in a specific manner (e.g., prepared as DNA molecules), for sequencing. This allows for the simultaneous sequencing of hundreds of thousands to millions of DNA molecules. Loading technology is crucial for both sequencing throughput and quality; efficient and stable loading techniques can significantly improve both. Furthermore, the sufficiency of the signal generated by the loaded DNA or DNA is also critical. A sufficiently high signal-to-noise ratio allows for higher resolution, facilitating signal extraction and improving sequencing quality.

[0004] When using DNB as a template for sequencing or research, the DNB needs to be loaded onto a corresponding vector. For example, DNB sequencing generally requires loading the DNB onto a sequencing slide, followed by sequencing analysis. Currently, the general procedure involves preparing a single-stranded circular library, performing rolling circle amplification to form DNB containing a certain number of copies of nucleic acid molecules, then loading the DNB directly onto a sequencing slide, and finally performing sequencing and data acquisition and analysis. However, the copy number of each DNB varies; some may have a low copy number, while others may have a high copy number, resulting in many DNBs of varying sizes. During loading, these DNBs of different sizes are affected by Brownian / thermal motion and shearing, leading to situations where large DNBs may be loaded onto two adjacent sites on the slide (i.e., occupying two sites), multiple small DNBs may be loaded onto the same site simultaneously, and some DNBs may even be lost into the solution, resulting in empty vectors. These phenomena are particularly pronounced on ultra-high-density slides, reducing slide utilization and causing data waste to some extent.

[0005] In high-throughput sequencing or detection processes, the demand for accurate loading is even greater for certain precious samples or libraries with limited availability (such as PCR-free libraries, stLFR libraries, libraries with large inserts, tumors, plasma, etc.). This requires considering not only the utilization rate of the slide but also the uniformity and strength of the signal generated by each DNB, especially when encountering low laser power, to ensure easy detection. Therefore, it is necessary to develop a method that can efficiently and stably load DNBs onto slides or other carriers, and ensure that the loaded DNBs generate sufficiently uniform and strong signals for detection. Summary of the Invention

[0006] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein are all standard procedures widely used in the relevant fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0007] Terminology Definition

[0008] As used herein, the term "nucleic acid" can refer to any type of nucleic acid. For example, a nucleic acid can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a DNA or RNA analog made of, for example, nucleotide analogs. Nucleic acids can be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. Nucleic acid molecules can be derived from double-stranded DNA (dsDNA) (e.g., genomic DNA, PCR and amplification products, etc.) or from single-stranded forms such as DNA (ssDNA) or RNA. Furthermore, nucleic acid molecules can be linear or circular. In some embodiments, nucleic acid molecules can be single-stranded circular nucleic acid molecules (e.g., single-stranded circular DNA).

[0009] As used herein, the term "nanosphere" generally refers to a macromolecule or complex having a compact (approximately) spherical shape with an inner diameter typically ranging from about 1 nm to about 1000 nm, such as 1-10 nm, 10-20 nm, 20-50 nm, 50-100 nm, 100-200 nm, 200-500 nm, 500-1000 nm, preferably between about 50 nm and about 500 nm.

[0010] As used herein, the term "nucleic acid nanosphere" generally refers to a complex or multiplex containing multiple copies of nucleic acid molecules. In some typical schemes, these nucleic acid copies may be arranged one after another in a continuous linear strand of nucleotides. This tandem repeat structure, along with the single-stranded nature of nucleic acids (e.g., DNA), causes the nanosphere to fold. It is readily understood that each of the multiple copies of nucleic acid molecules in the nucleic acid nanosphere may contain a known adapter sequence to facilitate amplification or sequencing. The adapter sequences of the individual nucleic acid molecules are typically identical, but can also differ. Nucleic acid nanospheres include, but are not limited to, DNA nanospheres, which are also referred to herein as DNBs.

[0011] As used herein, the term "solid support" means any insoluble substrate or matrix to which nucleic acids can be attached, such as, for example, latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, and silicon wafers. The surface of a solid support can be any desired shape, including, for example, planar, spherical, or porous shapes suitable for a particular application. For example, a solid support can be a planar glass surface. Solid supports can be fixed, for example, mounted inside a flow cell to allow interaction with a solution containing reagents. Alternatively, solid supports can also be movable to facilitate contact with solutions in different reaction vessels.

[0012] Typically, solid supports may comprise an array of sites arranged in a predetermined pattern, where each site can attach to or contain a nucleic acid molecule (e.g., a nucleic acid nanosphere). When the size of the attached nucleic acid molecule (e.g., a nucleic acid nanosphere) exceeds the maximum size that a single site can contain or exceeds the spacing between adjacent sites in the array, the nucleic acid molecule (e.g., a nucleic acid nanosphere) may occupy two or more sites. This can lead to a decrease in the utilization rate of the array sites and may affect sequencing efficiency and sequencing quality.

[0013] As used herein, the term "nucleic acid polymerase" has the meaning commonly understood by those skilled in the art, including DNA polymerase and RNA polymerase. In some preferred embodiments, the nucleic acid polymerase is a DNA polymerase.

[0014] As used herein, the term "DNA polymerase with strand displacement activity" refers to a DNA polymerase that, during downstream extension / synthesis of a nucleic acid chain, if a double-stranded nucleic acid exists downstream, displaces the existing downstream single-stranded nucleic acid with its newly synthesized single-stranded nucleic acid, thereby stripping the downstream single-stranded nucleic acid and producing a free single strand. DNA polymerases with strand displacement activity have been used in various amplification techniques, such as strand displacement amplification, rolling circle amplification (RCA), multiple displacement amplification, and loop-mediated isothermal amplification. DNA polymerases with strand displacement activity include, but are not limited to, Bst DNA polymerase, phi29 DNA polymerase, and exo-Klenow.

[0015] As used herein, the term "phi29 DNA polymerase" refers to a DNA polymerase with strand displacement activity cloned from the phage phi29 of Bacillus subtilis. When used for rolling circle amplification, the C-terminal domain of phi29 DNA polymerase performs polymerization, while the N-terminal domain performs 3' to 5' exonuclease reading and strand displacement functions. For specific applications of phi29 DNA polymerase, see Lieberman, KR, GMCherf. Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. J Am Chem Soc. 2010. 132(50):17961-72. phi29 DNA polymerase is commercially available; see, for example, New England Biolabs, catalog number M0269L.

[0016] As used herein, the term "Bst DNA polymerase" refers to a DNA polymerase with strand displacement activity cloned from *Bacillus stearothermophilus*, possessing 5' to 3' DNA polymerase activity but lacking 5' to 3' exonuclease activity, and therefore without error correction capabilities. Bst DNA polymerases are commercially available; see, for example, the Bst DNA polymerase series from New England Biolabs, such as Bst1.0, Bst2.0, and Bst3.0.

[0017] As used herein, the terms “loading,” “fixing,” and “attaching,” when referring to the use of nucleic acids, mean direct or indirect attachment to a solid support via covalent or non-covalent bonds. In some embodiments of this disclosure, the method of the invention includes fixing nucleic acids to a solid support via covalent attachment. However, typically, it is only necessary for nucleic acids to remain fixed or attached to a solid support when the use of the solid support is desired (e.g., in applications requiring nucleic acid amplification and / or sequencing). Optionally, loading can occur via base-pairing hybridization or other methods, such as covalent attachment as described above. Non-limiting examples of nucleic acid attachment to a solid support include nucleic acid hybridization, biotin-streptavidin binding, thiol binding, photoactivated binding, covalent binding, antibody-antigen, physical confinement via hydrogels or other porous polymers, etc. Various exemplary methods for immobilizing nucleic acids on solid supports are available, for example, G. Steinberg-Tatman et al., Bioconjugate Chemistry 2006, 17, 841-848; Xu X. et al., Journal of the American Chemical Society 128(2006)9286-9287; U.S. patents or patent applications US 5639603, US 5641658, US2010248991; and international patent applications WO 2001062982, WO 2001012862, WO 2007111937, WO0006770. For all purposes, and especially for the purposes of all teachings relating to the preparation of solid supports on which nucleic acids are immobilized, the above references are incorporated herein by reference in their entirety.

[0018] As used in this article, the terms "chip" and "carrier" have the same meaning and are used interchangeably.

[0019] As used herein, the term "primer" refers to a polynucleotide that, in a suitable buffer and at a suitable temperature, under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and reagents for polymerization, such as DNA or RNA polymerase or reverse transcriptase), can serve as the starting point for nucleic acid synthesis. Therefore, a primer includes a target-binding region that hybridizes with the target nucleic acid (template). Primers are generally oligonucleotides and are single-stranded; however, a primer can refer to a polynucleotide with a double-stranded segment (e.g., a partially double-stranded oligonucleotide). The appropriate length of the target-binding region of a primer depends on its intended use. Short primer molecules typically require lower temperatures to form a sufficiently stable hybridization complex with the template. Primers do not need to reflect the exact sequence of the nucleic acid template, but they must be sufficiently complementary to hybridize with the template.

[0020] In a first aspect, the present invention provides a method for loading nucleic acid molecules onto a solid support or for constructing a nucleic acid library immobilized on a solid support, comprising:

[0021] (1) Provides a composition containing nucleic acid molecules, and a solid support;

[0022] (2) Perform a first amplification on the nucleic acid molecules in the composition to obtain an amplification product containing nucleic acid molecules;

[0023] (3) Stop or slow down the first amplification;

[0024] (4) Load the amplification product containing nucleic acid molecules into a solid support; and

[0025] (5) Perform a second amplification on the nucleic acid molecules in the amplification products loaded onto the solid support.

[0026] In some embodiments, the amplification product containing nucleic acid molecules is a nucleic acid nanosphere.

[0027] In some embodiments, the solid support has an array of sites for loading amplification products (e.g., nucleic acid nanospheres). In some embodiments, the sites are arranged on the solid support in a predetermined pattern.

[0028] In some embodiments, the nucleic acid molecule can be any type of nucleic acid. For example, the nucleic acid molecule can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a DNA or RNA analog made from nucleotide analogs. The nucleic acid molecule can be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. The nucleic acid molecule can be derived from double-stranded DNA (dsDNA) (e.g., genomic DNA, PCR and amplification products, etc.) or from single-stranded forms such as DNA (ssDNA) or RNA. Furthermore, the nucleic acid molecule can be linear or circular. In some embodiments, the nucleic acid molecule can be a single-stranded circular nucleic acid molecule (e.g., single-stranded circular DNA). In some embodiments, the composition comprises a single-stranded circular nucleic acid molecule. In some embodiments, the composition comprises a single-stranded circular DNA molecule.

[0029] In some embodiments, the composition comprises one or more nucleic acid molecules, such as at least 10, at least 20, at least 50, at least 100, at least 1000, or at least 10 4 At least 10 species 5 At least 10 species 6 At least 10 species 7One or more nucleic acid molecules. In some embodiments, the nucleic acid molecule is single-stranded circular DNA. In some embodiments, the nucleic acid molecule contains a target nucleic acid fragment with a length of at least 100 bp, at least 200 bp, at least 500 bp, at least 800 bp, at least 1000 bp, or at least 2000 bp.

[0030] In some embodiments, the size of the amplification product (e.g., nucleic acid nanospheres) obtained in step (2) does not exceed a predetermined size. In some embodiments, the amplification product (e.g., nucleic acid nanospheres) obtained in step (2) is a linear nucleic acid molecule, such as linear DNA.

[0031] In some embodiments, the predetermined size is the maximum size of a nucleic acid molecule (e.g., a nucleic acid nanosphere) that a single site in the solid support can accommodate or attach to, or the spacing between adjacent sites in the solid support.

[0032] In some embodiments, the size of the obtained nucleic acid nanospheres does not exceed 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the maximum size or the spacing between adjacent sites.

[0033] In some embodiments, in step (2), the nucleic acid molecules in the composition are first amplified by rolling circle amplification. In some embodiments, the amplification time (e.g., rolling circle amplification) is controlled so that the size of the amplification product (e.g., nucleic acid nanospheres) does not exceed a predetermined size.

[0034] In some implementations, in step (3), the first amplification is stopped or slowed down by one or more methods selected from the following:

[0035] (a) Controlling or reducing the amount of dNTPs in the reaction mixture used for the first amplification; and / or

[0036] (b) Inhibit the activity of the nucleic acid polymerase used in the first amplification or inactivate the nucleic acid polymerase.

[0037] In some implementations, the first amplification is stopped or slowed down by inhibiting the activity of the nucleic acid polymerase used in the first amplification or by inactivating the nucleic acid polymerase.

[0038] In some embodiments, the activity of the nucleic acid polymerase used in the first amplification is inhibited, reducing the activity of the nucleic acid polymerase by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0039] In some embodiments, the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by one or more methods selected from the following:

[0040] (a) Add an activity inhibitor or denaturant to the said nucleic acid polymerase;

[0041] (b) Remove the cofactors required for the nucleic acid polymerase to function;

[0042] (c) Adjust the temperature of the reaction mixture for the first amplification to deviate (e.g., above or below) the operating temperature range of the nucleic acid polymerase;

[0043] (d) Adjust the pH of the reaction mixture for the first amplification to deviate (e.g., above or below) the working pH range of the nucleic acid polymerase;

[0044] In some embodiments, the nucleic acid polymerase is a DNA polymerase, such as a DNA polymerase with strand displacement activity, for example, Bst DNA polymerase, phi29 DNA polymerase, and exo-Klenow.

[0045] It is readily understood that the biological activity and stability of enzymes (nucleic acid polymerases) are affected by many factors, such as temperature, pH, protein concentration, salt conditions, solvents, or other factors. Those skilled in the art can modify these factors as needed to influence (e.g., reduce or restore) the activity of the enzyme (nucleic acid polymerase). Therefore, the methods of the present invention can influence enzyme activity (e.g., reduce or restore enzyme activity) by various suitable means, and are not limited to the exemplary schemes or means described above. For example, the activity of a nucleic acid polymerase can be influenced (e.g., reduce or restore) by changing the operating conditions of the nucleic acid polymerase (e.g., operating temperature and operating pH). Suitable operating conditions for a nucleic acid polymerase can be readily determined by those skilled in the art. For example, suitable operating conditions for phi29 DNA polymerase can be found in Nelson JRphi29 DNA polymerase-based methods for genomics applications[J]. Journal of Clinical Ligand Assay, 2002, 25(3):276-279, which is incorporated herein by reference in its entirety.

[0046] In some embodiments, the activity of the nucleic acid polymerase can be inhibited or the polymerase can be inactivated by removing a cofactor required for the polymerase to function. In some embodiments, the cofactor is a cation (e.g., magnesium ions). In some embodiments, the cofactor is removed by adding a cation chelating agent (e.g., a magnesium ion chelating agent), thereby inhibiting or inactivating the nucleic acid polymerase. In some embodiments, the magnesium ion chelating agent is selected from NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium gluconate, and sodium metasilicate. In some embodiments, the reagent for chelating magnesium ions is EDTA.

[0047] In some embodiments, the activity of the nucleic acid polymerase can be inhibited or the polymerase can be inactivated by adjusting the temperature of the reaction mixture used for the first amplification to deviate from (e.g., above or below) the operating temperature range of the nucleic acid polymerase. In some embodiments, the temperature of the reaction mixture is set above the upper limit of the operating temperature of the nucleic acid polymerase, for example, at least 5°C, at least 10°C, at least 15°C, or more; or the temperature of the reaction mixture is set below the lower limit of the operating temperature of the nucleic acid polymerase, for example, at least 5°C, at least 10°C, at least 15°C, or more. In some embodiments, the temperature of the reaction mixture is lowered to ≤20°C, ≤10°C, ≤4°C, or lower; or the temperature of the reaction mixture is raised to ≥50°C, ≥60°C, ≥70°C, ≥80°C, or higher.

[0048] In some embodiments, the pH of the reaction mixture for the first amplification can be adjusted by adding an acidic or alkaline buffer to deviate (e.g., above or below) the working pH range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the polymerase. In some embodiments, the pH of the reaction mixture is made higher than the upper limit of the working pH of the nucleic acid polymerase, for example, at least one, at least two, at least three or more pH units higher; or, the pH of the reaction mixture is made lower than the lower limit of the working pH of the nucleic acid polymerase, for example, at least one, at least two, at least three or more pH units lower. In some embodiments, the acidic or alkaline buffer is selected from citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, and Tris hydrochloride buffer.

[0049] In some embodiments, the activity of the nucleic acid polymerase can be inhibited or the polymerase can be inactivated by adding an activity inhibitor or denaturant. In some embodiments, the activity inhibitor or denaturant of the nucleic acid polymerase is a surfactant, such as sodium deoxycholate, sodium dodecyl sarcosinate, sodium dodecyl sulfate, Tween 20, NP-40, and Tritox-100.

[0050] In some embodiments, the activity of the nucleic acid polymerase or the inactivation of the nucleic acid polymerase can be achieved through one or more of the methods described above.

[0051] In some embodiments, the solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers. In some embodiments, the solid support is planar, spherical, or porous. In some embodiments, the maximum size of nucleic acid molecules (e.g., nucleic acid nanospheres) that can be accommodated or attached to a single site in the solid support is ≤5000 nm, ≤2000 nm, ≤1000 nm, ≤700 nm, ≤500 nm, ≤300 nm, or ≤100 nm. In some embodiments, the spacing between adjacent sites in the solid support is ≤5000 nm, ≤2000 nm, ≤1000 nm, ≤700 nm, ≤500 nm, ≤300 nm, or ≤100 nm.

[0052] In some embodiments, in step (2), the nucleic acid molecules in the composition are first amplified by rolling circle amplification. In some embodiments, in step (5), the nucleic acid molecules in the amplification product are second amplified by rolling circle amplification. In some embodiments, the first and second amplifications use the same or different (preferably the same) nucleic acid polymerases (e.g., DNA polymerase).

[0053] In some embodiments, the solid support is pretreated prior to step (4); for example, the solid support is pretreated with poloxamer, an enzyme (e.g., DNA polymerase (e.g., phi29 DNA polymerase), T4 ligase, BSA), or any combination thereof.

[0054] In some embodiments, the amplification product obtained in step (5) is a linear nucleic acid molecule, such as linear DNA. In some embodiments, the product obtained in step (5) is used as a nucleic acid sequencing library. In some embodiments, after step (5), the nucleic acid molecule loaded onto a solid support is sequenced.

[0055] In some embodiments, in step (5), the nucleic acid molecules in the amplification product loaded onto the solid support are amplified a second time by adding reagents required for nucleic acid amplification. In some embodiments, the reagents are selected from nucleic acid polymerases (e.g., DNA polymerase), dNTPs, working buffers for nucleic acid polymerases, and any combination thereof.

[0056] In some implementations, in step (5), the nucleic acid molecules in the amplification product loaded onto the solid support are amplified a second time by eliminating the conditions in step (3) that cause the first amplification to stop or slow down.

[0057] In some embodiments, in step (3), the first amplification is stopped or slowed down by inhibiting the activity of the nucleic acid polymerase used in the first amplification, and in step (5), a second amplification is performed by restoring the activity of the nucleic acid polymerase (e.g., by eliminating the conditions that inhibit the activity of the nucleic acid polymerase). In some embodiments, in step (5), the activity of the nucleic acid polymerase is restored to at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or higher of its normal activity.

[0058] In some implementations, in step (3), the first amplification is stopped or slowed down by adding an activity inhibitor of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by removing the activity inhibitor, and a second amplification is performed.

[0059] In some implementations, in step (3), the first amplification is stopped or slowed down by removing the cofactor required for the nucleic acid polymerase to function, and in step (5), the activity of the nucleic acid polymerase is restored by adding the cofactor, and a second amplification is performed.

[0060] In some embodiments, in step (3), the first amplification is stopped or slowed down by adjusting the temperature of the reaction mixture to deviate (e.g., above or below) the operating temperature range of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by adjusting the temperature of the reaction mixture to the operating temperature range of the nucleic acid polymerase, and a second amplification is performed.

[0061] In some embodiments, in step (3), the first amplification is stopped or slowed down by adjusting the pH of the reaction mixture to deviate (e.g., above or below) the working pH range of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by adjusting the pH of the reaction mixture to the working pH range of the nucleic acid polymerase, and a second amplification is performed.

[0062] In some implementations, in step (3), the first amplification is stopped or slowed down by denaturing the nucleic acid polymerase, and in step (5), the second amplification is performed by adding the nucleic acid polymerase.

[0063] In a second aspect, the present invention provides a kit comprising: reagents for amplifying nucleic acids, reagents for terminating or inhibiting nucleic acid amplification, and reagents for loading nucleic acid molecules onto a solid support. It is readily understood that the kit can be used to implement the methods of the present invention as described above. For example, the kit can be used to load nucleic acid molecules onto a solid support or to construct a nucleic acid library immobilized on a solid support.

[0064] In some embodiments, the reagents for amplifying nucleic acids may include a nucleic acid polymerase, a working buffer (concentrated or unconcentrated) for the nucleic acid polymerase, cofactors required for the function of the nucleic acid polymerase, dNTPs, or any combination thereof. In some embodiments, the nucleic acid polymerase is a DNA polymerase, such as a DNA polymerase with strand displacement activity, for example, Bst DNA polymerase, phi29 DNA polymerase, and exo-Klenow. In some embodiments, the cofactor is a cation (e.g., magnesium ions). In some embodiments, the working buffer is concentrated. For example, the working buffer may be concentrated at least 2-fold, at least 5-fold, or at least 10-fold.

[0065] In some implementations, the reagent used to stop or inhibit nucleic acid amplification may include one or more selected from the following:

[0066] (1) Inhibitors or denaturants of nucleic acid polymerase activity;

[0067] (2) A reagent that can remove the cofactors required for nucleic acid polymerases to function;

[0068] (3) An acidic or alkaline buffer solution that can adjust the pH of the solution.

[0069] In some embodiments, the reagent used to stop or inhibit nucleic acid amplification may include an activity inhibitor or denaturant of the nucleic acid polymerase. In some embodiments, the activity inhibitor or denaturant of the nucleic acid polymerase is a surfactant, such as sodium deoxycholate, sodium dodecyl sarcosinate, sodium dodecyl sulfate, Tween 20, NP-40, and Tritox-100.

[0070] In some embodiments, the reagent for terminating or inhibiting nucleic acid amplification may include a reagent capable of removing cofactors required for the function of nucleic acid polymerases. In some embodiments, the cofactor is a cation (e.g., magnesium ion). In some embodiments, the reagent capable of removing cofactors required for the function of nucleic acid polymerases is a cationic chelating agent (e.g., a magnesium ion chelating agent). In some embodiments, the cationic chelating agent (e.g., a magnesium ion chelating agent) is selected from NTA, EDTA, HEDP, EDTMPS, DTPMPA, EDDHA, STPP, sodium gluconate, and sodium metasilicate. In some embodiments, the cationic chelating agent (e.g., a magnesium ion chelating agent) is EDTA.

[0071] In some embodiments, the reagent used to stop or inhibit nucleic acid amplification may include an acidic or alkaline buffer capable of adjusting the pH of the solution. In some embodiments, the acidic or alkaline buffer is selected from citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, and Tris hydrochloride buffer.

[0072] In some implementations, reagents used to load nucleic acid molecules into a solid support include tripotassium citrate, citrate, phi29 polymerase, and Pluronic F68.

[0073] In some embodiments, the kit further comprises reagents for pretreating the solid support. In some embodiments, the reagents for pretreating the solid support are selected from poloxamer, enzymes (e.g., DNA polymerases (e.g., Phi29 DNA polymerase), T4 ligase, BSA), or any combination thereof.

[0074] Beneficial effects

[0075] Compared with existing technologies, the method for loading nucleic acid nanospheres (e.g., DNB) onto a solid support provided by this invention has higher loading density and loading stability. Furthermore, the method of this invention amplifies the target nucleic acid molecules in the nucleic acid nanospheres (e.g., DNB) twice, forming more copies of the target nucleic acid molecules on the slide, which can generate a stronger and more stable signal, facilitating detection.

[0076] The method of this invention can improve the quality of DNB loaded on high-density arrays, increase the target copy number of sequencing sites, improve the signal-to-noise ratio of array sites, and allow for longer read lengths, shorter imaging exposure times, and better overall data quality. This is particularly advantageous for high-throughput sequencing. Attached Figure Description

[0077] Figure 1This illustrates a general procedure for loading DNA molecules (DNBs) onto a sequencing slide. In an exemplary embodiment, a single-stranded circularized DNA library is used, and rolling circle amplification is performed under appropriate conditions using primers, dNTPs, and phi29 DNA polymerase or other polymerases with strand displacement activity to obtain DNBs. After rolling circle amplification, the DNBs are directly loaded onto a sequencing slide. Because the copy number of nucleic acid molecules contained in each DNB may vary (some DNBs contain high copy numbers, some contain low copy numbers), the sizes of different DNBs vary. During loading, due to the effects of Brownian / thermal motion and shearing, large DNBs may be loaded onto two or more adjacent sites on the slide, while multiple small DNBs may be loaded onto the same site simultaneously. Some DNBs may even be lost into the solution, resulting in empty vectors. This significantly affects subsequent sequencing results (sequencing efficiency and sequencing quality).

[0078] Figure 2 The process of loading DNB onto a slide using the method of the present invention is illustrated. In an exemplary embodiment, a single-stranded circularized DNA library is used, and a first rolling circle amplification is performed under suitable conditions using primers, dNTPs, and phi29 DNA polymerase or other polymerases with strand displacement activity to obtain DNB. The conditions and time of the first rolling circle amplification are controlled to obtain DNB of suitable size. In a preferred embodiment, the size of the DNB does not exceed the spacing between adjacent sites on the slide, thereby allowing the DNB to be loaded onto the slide efficiently, avoiding multiple DNBs being loaded onto the same site simultaneously, or one DNB being loaded onto two or more adjacent sites on the slide. For example, when the site spacing of the chip is 700 nm, a rolling circle amplification time of no more than 20 min can be used; when the site spacing of the chip is 500 nm, a rolling circle amplification time of no more than 10 min can be used; when the site spacing of the chip is further reduced, a shorter rolling circle amplification time can be used to obtain DNB of suitable size.

[0079] After rolling circle amplification for a predetermined time, various methods can be used to stop or slow down the first rolling circle amplification. For example, methods that can be used include, but are not limited to: (1) adding a certain concentration of EDTA to the reaction mixture to chelate magnesium ions, thereby weakening or terminating the polymerization ability of phi29 DNA polymerase, so that DNB growth stops or slows down; (2) adjusting the temperature of the reaction mixture to a low temperature (e.g., below 20°C, such as 4°C) to weaken or terminate the polymerization ability of phi29 DNA polymerase, so that DNB growth stops or slows down; (3) adjusting the pH of the reaction mixture to acidic (e.g., using a mixture of tripotassium citrate and citric acid at pH = 4.7) to weaken or terminate the polymerization ability of phi29 DNA polymerase, so that DNB growth stops or slows down.

[0080] Subsequently, the DNB is loaded onto a sequencing slide, and a second rolling circle amplification is performed on the slide to increase the copy number of nucleic acid molecules contained in the DNB. Various methods can be used to perform the second rolling circle amplification. In some embodiments, rolling circle amplification can be restarted by eliminating conditions that inhibit phi29 DNA polymerase activity. For example, when EDTA is used in the previous step to chelate magnesium ions and weaken or terminate the polymerization ability of phi29 DNA polymerase, an appropriate concentration of magnesium ion buffer can be added after loading the DNB onto the sequencing slide to restore the polymerization ability of phi29 DNA polymerase, allowing it to continue rolling circle amplification on the slide. For example, when low temperature (e.g., below 20°C, e.g., 4°C) is used in the previous step to weaken or terminate the polymerization ability of phi29 DNA polymerase, the temperature of the reaction system can be increased (e.g., 30-37°C) after loading the DNB onto the sequencing slide to restore the polymerization ability of phi29 DNA polymerase, allowing it to continue rolling circle amplification on the slide. For example, when the polymerization ability of phi29 DNA polymerase is weakened or terminated in the previous step using an acidic buffer (e.g., a mixture of tripotassium citrate and citric acid at pH 4.7), the pH of the reaction system can be increased (e.g., by using the working buffer for phi29 DNA polymerase or by adding an alkaline buffer) after loading DNB onto the sequencing slide to restore the polymerization ability of phi29 DNA polymerase and allow it to continue rolling circle amplification on the slide.

[0081] By using the loading scheme described above in this invention, the problem of low loading efficiency caused by unsuitable DNB size (e.g., excessively large size) during the loading process is effectively avoided, ensuring that each site corresponds to one DNB. Furthermore, the DNB loaded at each site contains a higher copy number of nucleic acid molecules, resulting in a stronger signal. Therefore, the method of this invention can improve the DNB loading efficiency and quality at each site of the chip (high-density array), improve the signal-to-noise ratio at each site, and allow for longer readout lengths, shorter imaging exposure times, and better overall data quality.

[0082] Figure 3 The base signals of the first sequencing cycle of the experimental group 1 chip, the experimental group 2 chip, and the control group chip are shown.

[0083] Figure 4 The control group chip was shown. Figure 4 A) and the chip in experimental group 1 ( Figure 4 B), Experimental group 2 chip ( Figure 4 C) The repetition rate of DNBs at adjacent sites (i.e., the probability that a DNB occupies two well sites on the slide). Detailed Implementation

[0084] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0085] The main equipment used in this embodiment is as follows: DNBSEQ-T7RS sequencer (purchased from Shenzhen BGI Technology Co., Ltd.), MGIDL-T7RS (purchased from MGI), DNBSEQ-T7RS chip (700nm, purchased from MGI). 3.0 fluorescence quantitative instrument (purchased from Thermo), PCR instrument (purchased from Trobot), high-speed centrifuge (purchased from Eppendorf).

[0086] The key reagents used in the embodiments of this application are shown in Table 1 below:

[0087] Table 1. Reagents used

[0088] One-strand sequencing primers MGI DNBSEQ-T7RS High-Throughput Sequencing Kit MGI DNBSEQ-T7RS Cleaning Kit MGI phi29 DNA polymerase MGI DNB Rapid Preparation Kit MGI DNBSEQ-T7RS DNB Rapid Loading Kit MGI 25mM dNTP Mix Biotechnology 10% Pluronic F68 Sigma 1X TE buffer MGI Citric acid Sigma Tripotassium citrate Sigma 10X phi29 buffer MGI DNA nanosphere loading buffer II MGI E. coli library MGI

[0089] Example 1: Amplifying and Loading DNB

[0090] This embodiment is divided into experimental group 1, experimental group 2 and control group.

[0091] (1) The implementation plan for experimental group 1 is as follows: Figure 2 As shown, the specific experimental procedure is as follows:

[0092] 1.1 First amplification

[0093] According to the manufacturer's instructions, use MGIEasy TMA DNA library preparation kit (Shenzhen BGI Genomics Co., Ltd.) was used to prepare a 300 bp E. coli single-stranded circular DNA library with the insert fragment as the master band. The sequences of the two nucleic acid strands of the library adapter used were: 5'-AGT CGG AGG CCA AGC GGT CTT AGG AAG ACA ATC CTT TTG TACAAC TCC TTG GCT CAC A-3' (SEQ ID NO:1); and 5'-TTG TCT TCC TAA GGA ACG ACA TGGCTA CGA TCC GAC TT-3' (SEQ ID NO:2). Then, according to the instructions of the DNB rapid preparation kit (Shenzhen BGI Genomics Co., Ltd.), 2 fmol / uL of the single-stranded circular DNA library was taken, and 20uL of DNB primer mixture was added (the specific formula is shown in Table 2), wherein the RCA primer sequence was 5'-GCC ATG TCG TTC TGT GAG CCA AGG-3' (SEQ ID NO:3). Primer hybridization was performed on a PCR instrument (95℃, 1 min → 65℃, 1 min → 40℃, 1 min → 4℃, 1 min); then 40 μL of DNA polymerase mixture (specific formulation shown in Table 3) and 4 μL of phi29 DNA polymerase (Shenzhen BGI Genomics Co., Ltd.) were added, and rolling circle amplification was performed on a PCR instrument (30℃, 20 min). The prepared DNB was used for subsequent loading onto the DNBSEQ-T7 chip (the spacing between adjacent sites on this chip is 700 nm, and the maximum size of the nucleic acid nanospheres that can be accommodated or attached to a single site is 220 nm).

[0094] Table 2: DNB primer mixture

[0095] 10X phi29 buffer 1X RCA primers 1uM <![CDATA[H2O]]> Add volume to 1ml

[0096] Table 3: DNB polymerase mixture

[0097] 10X phi29 buffer 1X 25mM dNTP Mix 400uM 10% Pluronic F68 1％ 0.1 U / ul pyrophosphatase 0.005U <![CDATA[H2O]]> Add volume to 1ml

[0098] After performing rolling circle amplification according to the above time, immediately place the amplification reaction mixture on an ice box, add 8 μL of 0.5 mM EDTA, and slowly pipette 5-8 times to mix, avoiding shaking and vigorous pipetting. Then, take 2 μL of LDNB and use... ssDNA Assay Kit (purchased from Thermo Fisher, item number Q10212) and Concentration was detected using a 3.0 fluorescence quantitative PCR instrument. The results showed that the nucleic acid concentration was 15 ng / µl. After 20 minutes of rolling circle amplification, the size of the resulting DNB was approximately 200 nm. Based on the loading volume of the DNBSEQ-T7 chip (Shenzhen BGI Genomics Co., Ltd.), a total of 5 tubes of DNB were prepared.

[0099] 1.2 Configuring the DNB loading system:

[0100] According to the instructions of the DNBSEQ-T7RS DNB Rapid Loading Kit (Shenzhen BGI Genomics Co., Ltd.), take 400 μL of the prepared DNB mixture, add 100 μL of PBS, and slowly mix 5-8 times with a wide-mouth pipette tip. Avoid centrifugation, shaking, and vigorous pipetting.

[0101] 1.3 Preprocessing and loading sequencing slides

[0102] First, pump slide pretreatment reagent 1 (specific formulation shown in Table 4) into the chip and let it stand at room temperature for 5 minutes. Then, pump slide pretreatment reagent 2 (specific formulation shown in Table 5). Pretreatment of the chip with these two reagents helps to efficiently load DNB onto the sequencing slide. After chip pretreatment, add the DNB loading system using the MGIDL-T7RS device and load DNB. Load at 4°C for 30 minutes.

[0103] Table 4: Slide Pretreatment Reagents 1

[0104] 1M Tripotassium Citrate 0.3M 1M Citric Acid 0.3M 1X TE buffer Add to 1ml 10 U / ul Phy29 polymerase 10ul

[0105] Table 5: Slide Pretreatment Reagents 2

[0106] 1M Tripotassium Citrate 0.03M 1M Citric Acid 0.03M 10% Pluronic F68 6ul 1XTE buffer Add to 1ml

[0107] 1.4 Second amplification

[0108] After loading was complete, 740 μL of a mixture of magnesium ions and DNB polymerase at a final concentration of 5 mM, and 1 μL of a mixture of phi29 DNA polymerase were pumped in to reactivate the polymerase. The temperature was set to 30 °C, and a second amplification was performed on the sequencing slide for 30 min. After amplification, the sequencing chip for experimental group 1 was obtained.

[0109] (2) The implementation plan for Experimental Group 2 is as follows: Figure 2 As shown, the specific experimental procedure is as follows:

[0110] 2.1 First amplification

[0111] According to the manufacturer's instructions, use MGIEasy TMA DNA library preparation kit (Shenzhen BGI Genomics Co., Ltd.) was used to prepare a 300 bp E. coli single-stranded circular DNA library with the insert fragment as the master band. The sequences of the two nucleic acid strands of the library adapter used were: 5'-AGT CGG AGG CCA AGC GGT CTT AGG AAG ACA ATC CTT TTG TACAAC TCC TTG GCT CAC A-3' (SEQ ID NO:1); and 5'-TTG TCT TCC TAA GGA ACG ACA TGGCTA CGA TCC GAC TT-3' (SEQ ID NO:2). Then, according to the instructions of the DNB rapid preparation kit (Shenzhen BGI Genomics Co., Ltd.), 2 fmol / uL of the single-stranded circular DNA library was taken, and 20uL of DNB primer mixture was added (the specific formula is shown in Table 2), wherein the RCA primer sequence was 5'-GCC ATG TCG TTC TGT GAG CCA AGG-3' (SEQ ID NO:3). Primer hybridization was performed on a PCR instrument (95℃, 1 min → 65℃, 1 min → 40℃, 1 min → 4℃, 1 min); then 40 μL of DNA polymerase mixture (specific formulation shown in Table 3) and 4 μL of phi29 DNA polymerase (Shenzhen BGI Genomics Co., Ltd.) were added, and rolling circle amplification was performed on a PCR instrument (30℃, 20 min). The prepared DNB was used for subsequent loading onto the DNBSEQ-T7 chip (the spacing between adjacent sites on this chip is 700 nm, and the maximum size of the nucleic acid nanospheres that can be accommodated or attached to a single site is 220 nm).

[0112] Table 2: DNB primer mixture

[0113] 10X phi29 buffer 1X RCA primers 1uM <![CDATA[H2O]]> Add volume to 1ml

[0114] Table 3: DNB polymerase mixture

[0115] 10X phi29 buffer 1X 25mM dNTP Mix 400uM 10% Pluronic F68 1％ 0.1 U / ul pyrophosphatase 0.005U <![CDATA[H2O]]> Add volume to 1ml

[0116] After performing rolling circle amplification according to the above timeframes, immediately place the amplification reaction mixture on an ice pack and gently pipette it 5-8 times using a wide-mouth pipette tip, avoiding shaking or vigorous pipetting. Then, take 2 μL of DNB and use... ssDNAAssay Kit (purchased from Thermo Fisher, item number Q10212) and Concentration was detected using a 3.0 fluorescence quantitative PCR instrument. The results showed that the nucleic acid concentration was 15 ng / µl. After 20 minutes of rolling circle amplification, the size of the resulting DNB was approximately 200 nm. Based on the loading volume of the DNBSEQ-T7 chip (Shenzhen BGI Genomics Co., Ltd.), a total of 5 tubes of DNB were prepared.

[0117] 2.2 Configuring the DNB loading system:

[0118] According to the instructions of the DNBSEQ-T7RS DNB Rapid Loading Kit (Shenzhen BGI Genomics Co., Ltd.), take 500 μL of the prepared DNB mixture and place it in a DNB loading tube, and immediately place it on ice for later use.

[0119] 2.3 Preprocessing and loading sequencing slides

[0120] First, pump slide pretreatment reagent 1 (specific formulation shown in Table 4) into the chip and let it stand at room temperature for 5 minutes. Then, pump slide pretreatment reagent 2 (specific formulation shown in Table 5). Pretreatment of the chip with these two reagents helps to efficiently load DNB onto the sequencing slide. After chip pretreatment, add the DNB loading system using the MGIDL-T7RS device and load DNB. Load at 4°C for 30 minutes.

[0121] Table 4: Slide Pretreatment Reagents 1

[0122] 1M Tripotassium Citrate 0.3M 1M Citric Acid 0.3M 1X TE buffer Add to 1ml 10 U / ul Phi29 polymerase 10ul

[0123] Table 5: Slide Pretreatment Reagents 2

[0124] 1M Tripotassium Citrate 0.03M

[0125] 10% Pluronic F68 6ul 1XTE buffer Add to 1ml

[0126] 2.4 Second amplification

[0127] After loading at 4℃ for 30 min, the temperature was set to 30℃ to reactivate the phi29 enzyme, and a second amplification was performed on the sequencing slide for 30 min. After amplification, the sequencing chip for experimental group 2 was obtained.

[0128] (3) The implementation plan for the control group is as follows: Figure 1 As shown, the specific experimental procedure is as follows:

[0129] The control group used the same samples as the experimental group, specifically an E. coli single-stranded circular DNA library with the insert fragment main band located at 300 bp. Rolling circular amplification was performed on the samples using the same kits and protocols as the experimental group. Due to the requirements of the sequencing protocol on copy number and sequencing signal, the amplification time for the control group was set to 40 min. The nucleic acid concentration of the amplified product was 35 ng / µl, and the average size of the resulting DNA was approximately 300 nm.

[0130] Subsequently, using the same kit and protocol as the experimental group, the amplification product (DNB) was loaded onto the sequencing chip, and a second rolling circle amplification was not performed (i.e., step 1.4 was not performed), thus obtaining the control group sequencing chip.

[0131] Example 2: Sequencing

[0132] According to the instructions of the DNBSEQ-T7RS high-throughput sequencing kit (Shenzhen BGI Genomics Co., Ltd.) and the DNBSEQ-T7RS cleaning kit (Shenzhen BGI Genomics Co., Ltd.), the sequencing chips obtained from experimental group 1, experimental group 2 and control group were placed on the DNBSEQ-T7RS sequencer and PE100 (paired-end 100bp) sequencing was performed. The sequence of the single-strand sequencing primer (Shenzhen BGI Genomics Co., Ltd.) used in the sequencing process was 5'-GC TCA CAG AAC GACATG GCT ACG ATC CGA CTT-3' (SEQ ID NO:4).

[0133] Figure 3 The results show a comparison of base signals from the experimental group 1 chip, experimental group 2 chip, and control group chip during the first sequencing cycle. The results indicate that the signals from experimental groups 1 and 2 are higher than those from the control group. Table 6 shows the sequencing reports of the PE100 chips from experimental groups 1, 2, and the control group. The results show that experimental groups 1 and 2 using the proposed method have better total data volume (total number of reads) and sequencing quality (Q30) than the control group. Figure 4 This diagram shows the repetition rate of adjacent DNBs in experimental groups 1, 2, and the control group of this invention. A higher value indicates a more severe phenomenon where one DNB occupies two well sites. Figure 4 It can be seen that the control group ( Figure 4 The duplication rate (dup-rate) of group A was 10.09%, while that of experimental group 1 ( Figure 4 The duplication rate (dup-rate) of experimental group B was 1.14%, and that of experimental group 2 ( Figure 4 The duplication rate (dup-rate) of C) was 1.6%. The results indicate that the duplication rate of the experimental group in this invention was significantly lower than that of the control group.

[0134] Table 6. Sequencing data of PE100 in the examples and control group

[0135] sequencing cycle number 210 210 210

[0136] Q30 87.35 90.72 90.5 Removal rate (%) 99.01 99.01 99.00

[0137] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for loading nucleic acid molecules onto a solid support or for constructing a nucleic acid library immobilized on a solid support, comprising: (1) Provides a composition comprising nucleic acid molecules, and a solid support; (2) The nucleic acid molecules in the composition are amplified for the first time, wherein the amplification is rolling circle amplification, and nucleic acid nanospheres are obtained; Furthermore, the size of the obtained nucleic acid nanospheres does not exceed a predetermined size, which is the maximum size of the nucleic acid nanospheres that can be accommodated or attached to a single site in the solid support; (3) Stop or slow down the first amplification; (4) Loading nucleic acid nanospheres onto a solid support; and (5) Perform a second amplification on the nucleic acid nanospheres loaded onto the solid support; The solid support has an array of sites for loading nucleic acid nanospheres, and the sites are arranged on the solid support in a predetermined pattern.

2. The method of claim 1, wherein the nucleic acid molecule is selected from deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

3. The method of claim 1, wherein the composition comprises at least 10 nucleic acid molecules.

4. The method of claim 1, wherein the nucleic acid molecule contains a target nucleic acid fragment with a length of at least 100 bp.

5. The method of claim 1, wherein the size of the nucleic acid nanospheres is not greater than a predetermined size by controlling the amplification time.

6. The method of claim 1, wherein, In step (3), the first amplification is stopped or slowed down by one or more of the following methods: (a) Controlling or reducing the amount of dNTPs in the reaction mixture used for the first amplification; and / or (b) Inhibit the activity of the nucleic acid polymerase used in the first amplification or inactivate the nucleic acid polymerase.

7. The method of claim 6, wherein the first amplification is stopped or slowed down by inhibiting the activity of the nucleic acid polymerase used in the first amplification or by inactivating the nucleic acid polymerase.

8. The method of claim 6, wherein the activity of the nucleic acid polymerase used in the first amplification is inhibited, thereby reducing the activity of the nucleic acid polymerase by at least 40%.

9. The method of claim 6, wherein the activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by one or more methods selected from the following: (a) Add an activity inhibitor or denaturant to the said nucleic acid polymerase; (b) Remove the cofactors required for the nucleic acid polymerase to function; (c) Adjust the temperature of the reaction mixture for the first amplification to deviate from the operating temperature range of the nucleic acid polymerase; (d) Adjust the pH of the reaction mixture for the first amplification to deviate from the working pH range of the nucleic acid polymerase.

10. The method of claim 9, wherein the nucleic acid polymerase is a DNA polymerase.

11. The method of claim 9, wherein the nucleic acid polymerase is a DNA polymerase with strand displacement activity.

12. The method of claim 10, wherein the nucleic acid polymerase is Bst DNA polymerase and phi29 DNA polymerase.

13. The method of claim 9, wherein, The activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by removing the cofactor required for the function of the nucleic acid polymerase; the cofactor is a cation.

14. The method of claim 13, wherein the cofactor is magnesium ions.

15. The method of claim 13, wherein the cofactor is removed by adding a magnesium ion chelating agent, thereby inhibiting or inactivating the nucleic acid polymerase activity.

16. The method of claim 15, wherein the magnesium ion chelating agent is selected from NTA, EDTA, HEDP, EDTMPS, DTPMPA, STPP, sodium gluconate and sodium metasilicate.

17. The method of claim 9, wherein, The temperature of the reaction mixture used for the first amplification is adjusted to deviate from the operating temperature range of the nucleic acid polymerase, thereby inhibiting the activity of the nucleic acid polymerase or inactivating the nucleic acid polymerase.

18. The method of claim 17, wherein the temperature of the reaction mixture is above the upper limit of the operating temperature of the nucleic acid polymerase; or, the temperature of the reaction mixture is below the lower limit of the operating temperature of the nucleic acid polymerase.

19. The method of claim 17, wherein the temperature of the reaction mixture is reduced to a temperature of ≤20°C or lower; or the temperature of the reaction mixture is increased to a temperature of ≥50°C or higher.

20. The method of claim 9, wherein, The pH of the reaction mixture used for the first amplification is adjusted by adding acidic or alkaline buffers to deviate it from the working pH range of the nucleic acid polymerase, thereby inhibiting or inactivating the activity of the nucleic acid polymerase.

21. The method of claim 20, wherein the pH of the reaction mixture is higher than the upper limit of the working pH of the nucleic acid polymerase; or, the pH of the reaction mixture is lower than the lower limit of the working pH of the nucleic acid polymerase.

22. The method of claim 20, wherein the acidic or alkaline buffer is selected from citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, and Tris hydrochloride buffer.

23. The method of claim 9, wherein, The activity of the nucleic acid polymerase is inhibited or the nucleic acid polymerase is inactivated by adding an activity inhibitor or denaturant of the nucleic acid polymerase.

24. The method of claim 23, wherein the activity inhibitor or denaturant of the nucleic acid polymerase is a surfactant.

25. The method of claim 24, wherein the activity inhibitor or denaturant of the nucleic acid polymerase is selected from sodium deoxycholate, sodium dodecyl sarcosinate, sodium dodecyl sulfate, Tween 20, NP-40, and Tritox-100.

26. The method of any one of claims 1-25, wherein, The method has one or more features selected from the following: (a) The solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes and silicon wafers; (b) The solid support is planar, spherical, or porous; (c) The maximum size of the nucleic acid nanospheres that can be accommodated or attached at a single site in the solid support is ≤5000 nm; (d) The first and second amplifications may use the same or different nucleases; (e) The solid support is pretreated before step (4); and (f) The product obtained in step (5) is used as a nucleic acid sequencing library; (g) The method further includes, after step (5), sequencing the nucleic acid molecules loaded onto the solid support.

27. The method of claim 1, wherein, In step (5), the nucleic acid molecules in the amplification product loaded onto the solid support are amplified a second time by adding the reagents required for nucleic acid amplification.

28. The method of claim 27, wherein the reagent is selected from nucleic acid polymerases, dNTPs, working buffers for nucleic acid polymerases, and any combination thereof.

29. The method of claim 1, wherein, In step (5), the nucleic acid molecules in the amplification product loaded onto the solid support are amplified a second time by eliminating the conditions in step (3) that caused the first amplification to stop or slow down.

30. The method of claim 1, wherein in step (3), the first amplification is stopped or slowed down by inhibiting the activity of the nucleic acid polymerase used in the first amplification, and in step (5), the second amplification is performed by restoring the activity of the nucleic acid polymerase.

31. The method of claim 1, having any one of the following features: (1) In step (3), the first amplification is stopped or slowed down by adding an activity inhibitor of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by removing the activity inhibitor, and a second amplification is performed; (2) In step (3), the first amplification is stopped or slowed down by removing the cofactor required for the nucleic acid polymerase to function, and in step (5), the activity of the nucleic acid polymerase is restored by adding the cofactor, and a second amplification is performed; (3) In step (3), the first amplification is stopped or slowed down by adjusting the temperature of the reaction mixture to deviate from the working temperature range of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by adjusting the temperature of the reaction mixture to the working temperature range of the nucleic acid polymerase, and a second amplification is performed. (4) In step (3), the first amplification is stopped or slowed down by adjusting the pH of the reaction mixture to deviate from the working pH range of the nucleic acid polymerase, and in step (5), the activity of the nucleic acid polymerase is restored by adjusting the pH of the reaction mixture to the working pH range of the nucleic acid polymerase, and a second amplification is performed; or (5) In step (3), the first amplification is stopped or slowed down by denaturing the nucleic acid polymerase, and in step (5), the second amplification is performed by adding the nucleic acid polymerase.

Images