Method for performing amplification on surface of solid support, and solid support having amplification product immobilized on its surface

Abstract

Provided is a method for performing amplification on the surface of a solid support. In an embodiment, the method comprises: a. providing a solid support, the surface of the solid support having a metal compound for immobilizing a nucleic acid template to be sequenced; b. loading said nucleic acid template onto the solid support, said nucleic acid template being immobilized on the surface of the metal compound; and c. performing a strand-displacement amplification reaction to obtain an amplification product, wherein a non-ionic surfactant is added in step b and step c, and the strand-displacement amplification reaction is performed in the presence of a polymerase having strand displacement activity. The method in the embodiment can improve the stability and efficiency of nucleic acid amplification.

Description

A method for amplification on the surface of a solid support and a solid support on which amplification products are immobilized. Technical Field

[0001] This invention relates to the field of sequencing, and more specifically, to a method for amplification on the surface of a solid support and a solid support on which amplification products are immobilized. Further, it relates to sequencing methods and amplification kits. Background Technology

[0002] Traditional methods of amplification on solid-phase support surfaces utilize specific amplification and immobilization techniques to achieve high-throughput, low-cost, and high-quality gene sequencing. However, existing technologies suffer from issues such as low gene sequencing quality and insufficient gene sequencing stability.

[0003] For example, commonly used regular microarray sequencing chips (APTMS (3-aminopropyltrimethoxysilane) sequencing chips) generally have the following defects: 1) The sequencing chip fabrication process is relatively complex, including plasma, coating, photolithography, CVD, and debinding steps, which has a long fabrication cycle and high process requirements, inevitably leading to higher costs and higher quality control requirements; 2) Due to the complex fabrication process and long cycle, batch-to-batch stability is difficult to control; 3) APTMS is a nitrogen-containing organic compound, which is easily oxidized and degraded, and contaminated by other materials used in chip fabrication. Therefore, it requires relatively strict storage and transportation conditions, and has certain requirements for gas, temperature, humidity, etc.; 4) The surface of APTMS chips is relatively hydrophobic before sequencing. When loading nucleic acid nanospheres (such as DNB) or sequencing nucleic acid nanospheres on an open platform, the hydrophobic surface can easily cause abnormal loading or insufficient sequencing reaction.

[0004] Metal oxides offer advantages such as easy availability, good stability, and strong adsorption for biomolecules like DNA and proteins. Furthermore, they can be obtained in large quantities and stably through techniques like nanoimprinting, reducing costs and batch-to-batch variations. The surface of the metal oxide layer can also be cleaned through treatment. Therefore, metal oxides (or nitrides) are a good choice for gene sequencing chips. However, metal oxides and APTMS differ in their DNA adsorption mechanisms; therefore, DNBSEQ is used directly. TM Reagents and procedures in the technology may cause abnormalities in DNB loading and sequencing.

[0005] Therefore, there is an urgent need to develop new methods for amplification on the surface of solid supports to improve gene sequencing quality and stability.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0008] This invention is based on the following discoveries of the inventors:

[0009] Traditional DNA nanosphere sequencing technology (DNBSEQ) TM DNA nanosphere sequencing technology utilizes rolling circle amplification (RCA) and combined probe-anchored polymerization (cPAS) for gene sequencing. However, this technology suffers from high non-specific adsorption of nucleic acid nanospheres (DNBs) during chip loading or sequencing. To overcome this issue, the inventors have developed a method for amplification on a solid support surface. By adding a nonionic surfactant, the specific adsorption of DNBs onto the chip is enhanced, thereby improving sequencing quality.

[0010] In a first aspect, the present invention provides a method for amplification on the surface of a solid support. According to an embodiment of the invention, the method includes: a. providing a solid support having a metal compound on its surface for adsorbing a nucleic acid template to be tested; b. loading the nucleic acid template to be tested onto the solid support, wherein the nucleic acid template is adsorbed onto the surface of the metal compound; c. performing a strand displacement amplification reaction to obtain an amplification product; wherein a nonionic surfactant is added in steps b and c, and the strand displacement amplification reaction is carried out in the presence of a polymerase with strand displacement activity. The method according to embodiments of the present invention can improve the stability and efficiency of nucleic acid amplification.

[0011] In a second aspect, the present invention provides a solid-phase support with amplification products immobilized on its surface. According to embodiments of the present invention, the amplification products are obtained based on the amplification method described in the first aspect of the present invention. The solid-phase support according to embodiments of the present invention can improve the stability and efficiency of nucleic acid amplification.

[0012] In a third aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the method includes: sequencing an amplification product immobilized on the surface of a solid support as described in the second aspect of the present invention; further comprising, after step c, hybridizing a one-stranded sequencing primer onto the amplification product, and performing multiple cycles of sequencing reactions. The method according to the embodiments of the present invention is low-cost, simple to operate, and can obtain sequencing data with high sequencing quality and high stability.

[0013] In a fourth aspect, the present invention provides an amplification reagent combination. According to embodiments of the present invention, the amplification reagent combination comprises: I. a solid support having a metal compound on its surface; II. a loading reagent including a nucleic acid template to be tested; III. a multiple displacement amplification reagent including a polymerase with strand displacement activity; wherein the loading reagent and the multiple displacement amplification reagent contain a nonionic surfactant. The amplification kit according to embodiments of the present invention can improve the efficiency and quality of nucleic acid amplification.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0016] Figure 1 is a schematic diagram of the HP chip.

[0017] Figure 2 is a schematic diagram of the MLG loading technology process.

[0018] Figure 3 is a comparison of the SE50 sequencing results (signal, background, Q30) of the HP chip using conventional loading technology and MLG loading technology in Example 1.

[0019] Figure 4 shows the sequencing results of Example 2 using MLG loading technology and with the addition of 0.05% Tween-20 SE100 loading reagent.

[0020] Figure 5 shows the sequencing results of Example 3 using MLG loading technology and loading reagents with different concentrations of Tween-20 SE100.

[0021] Figure 6 shows the sequencing results of the optimized MLG loading technology and the addition of 0.5% Tween-20 SE100 loading reagent in Example 4.

[0022] Figure 7 shows the sequencing results of different types of surfactant PE100 tested in Example 5.

[0023] Figure 8 shows the sequencing results of the test reagent with added Pluronic F-127 in Example 6.

[0024] Figure 9 shows the sequencing results of the test reagent with added Nonidet P40 in Example 7.

[0025] Figure 10 shows the sequencing results of adding Tween-20 PE100 to the MDA reagent in Example 9.

[0026] Figure 11 shows the AFM results of adding Tween-20 PE100 to the MDA reagent in Example 9 (the left side is the control group, and the right side is the experimental group 1).

[0027] Figure 12 shows the sequencing results of HP chip test with added CTAB and SDS in Comparative Example 1. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0032] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0033] In this invention, 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 from, 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).

[0034] In this invention, 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.

[0035] In this invention, the term "nucleic acid nanosphere" generally refers to a complex or multiplex containing multiple copies of nucleic acid molecules. In some typical embodiments, 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 the nucleic acid (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 may also differ. Nucleic acid nanospheres include, but are not limited to, DNA nanospheres, which are also referred to herein as DNBs.

[0036] In this invention, the term "HP chip" refers to a Hard patterned chip (HP chip). As shown in Figure 1, it is formed by depositing an adsorbent material layer such as titanium nitride and a silicon oxide layer on a silicon substrate, and then performing nanoimprinting and etching on the silicon oxide layer on the adsorbent material layer to form a nanopore array. The adsorbent material layer such as titanium nitride at the bottom of the pores is used to adsorb biological samples. It is consistent with or similar to the chips in patent applications CN118056894A and US20210384031A1.

[0037] Typically, an HP chip may contain an array of sites arranged in a predetermined pattern, where each site can be attached to or accommodate 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 accommodate 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.

[0038] In this invention, 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 the polymerization function, 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.

[0039] In this invention, the terms "chip" and "carrier" have the same meaning and can be used interchangeably.

[0040] In this invention, the term "MLG (Make DNB, Load DNB, Grow DNB) loading technology" refers to first obtaining a batch of small, uniform nucleic acid nanospheres using short-duration rolling circle amplification (RCA) technology. Once these small nucleic acid nanospheres have "occupied" their positions on the HP slide, appropriate biochemical reagents are added to activate them, allowing them to continue rolling circle amplification and growth (i.e., Grow DNB) at the slide sites until they reach the required copy number. This results in sufficiently large nucleic acid nanospheres to meet the strong sequencing signal required for sequencing, thereby achieving good sequencing quality. A schematic diagram of the MLG loading technology is shown in Figure 2. See also the description in patent application WO2022021163A1.

[0041] In this invention, the term "MDA (Multiple Displacement Amplification) technology" refers to the use of the strand displacement activity of phi29 DNA polymerase to extend the first sequencing strand (Read 1, first strand) to form a sequencing template for the second sequencing strand (Read 2, second strand), as described in patent application "201680010807.0", and all strand displacement amplification reactions using DNA single strands as templates and DNA polymerases with strand displacement activity.

[0042] In this invention, the term "nonionic surfactant" refers to a class of surfactants that do not generate ions in aqueous solutions. They have a polar head and a nonpolar tail structure, but unlike ionic surfactants (such as anionic and cationic surfactants), the polar head of nonionic surfactants does not dissociate into charged ions in water. The following are some characteristics of nonionic surfactants: Nonionic properties: They do not dissociate in water and do not generate ions, therefore they do not change the pH of the solution; Solubility: They generally dissolve well under various pH values ​​and electrolyte conditions; Stability: Nonionic surfactants remain stable even in hard water and do not precipitate due to the presence of calcium or magnesium ions; Foaming properties: Compared to ionic surfactants, nonionic surfactants produce less foam; Detergent properties: Although their detergency may not be as strong as ionic surfactants, they are very effective in specific applications; Biodegradability: Many nonionic surfactants are biodegradable and environmentally friendly.

[0043] This invention proposes a method for amplification on the surface of a solid support, a solid support on which amplification products are immobilized, a sequencing method, and an amplification kit, which will be described in detail below.

[0044] Methods for amplification on the surface of a solid support

[0045] In a first aspect, the present invention provides a method for amplification on the surface of a solid support. According to an embodiment of the invention, the method includes: a. providing a solid support having a metal compound on its surface for adsorbing a nucleic acid template to be tested; b. loading the nucleic acid template to be tested onto the solid support, wherein the nucleic acid template is adsorbed onto the surface of the metal compound; c. performing a chain displacement amplification reaction to obtain an amplification product; wherein a nonionic surfactant is added in steps b and c, and the chain displacement amplification reaction is carried out in the presence of a polymerase with chain displacement activity. According to the method of the embodiment of the invention, the interaction forces between DNB and the active sites (TiN metal nitride) and the upper surface of the chip channels (SiO2 oxide) can be modified by adjusting van der Waals forces, hydrogen bonding forces, electrostatic interactions, etc., thereby improving adsorption specificity and enhancing the stability and efficiency of nucleic acid amplification.

[0046] According to embodiments of the present invention, the metal compound is a metal oxide or a metal nitride.

[0047] According to embodiments of the present invention, the metal oxide is selected from at least one of silicon oxide (SiO2), titanium oxide (TiO2), zinc oxide (ZnO), nickel oxide (NiO), and cerium oxide (CeO2).

[0048] According to an embodiment of the present invention, the metal nitride is selected from at least one of titanium nitride (TiN) and silicon nitride (Si3N4).

[0049] According to an embodiment of the present invention, the surface of the solid support has a plurality of regularly distributed depressions or protrusions, and a metal compound is present at the bottom of the depressions or the top of the protrusions. Furthermore, the surface of the solid support has a spacer region for isolating the depressions or protrusions, wherein the surface of the spacer region is silicon dioxide.

[0050] According to an embodiment of the present invention, the nucleic acid template to be tested is a single-stranded circular nucleic acid library, and the amplification product is a single-stranded linear nucleic acid nanosphere.

[0051] According to an embodiment of the present invention, the nucleic acid template to be tested is a single-stranded linear nucleic acid nanosphere containing a small number of nucleic acid molecule copies, and the amplification product is a single-stranded linear nucleic acid nanosphere containing more nucleic acid molecule copies.

[0052] According to an embodiment of the present invention, the nucleic acid template to be tested is a single-stranded linear nucleic acid library, and the surface of the metal compound of the solid support is immobilized with oligonucleotide sequences.

[0053] In this process, the oligonucleotide sequences immobilized on the surface of the solid support are used for hybridization to capture single-stranded circular nucleic acid libraries, followed by rolling circle amplification to generate single-stranded linear nucleic acid nanospheres; or,

[0054] The oligonucleotide sequences immobilized on the surface of the solid support are used to capture the nucleic acid library to be tested, and a ligation reaction is performed to obtain a single-stranded circular nucleic acid library immobilized on the surface of the solid support; or,

[0055] Based on the single-stranded circular nucleic acid library obtained above, a solid-phase surface amplification reaction is performed to obtain single-stranded linear nucleic acid nanospheres containing multiple copies of nucleic acid molecules; or,

[0056] The amplification reaction on the solid surface described above is rolling ring amplification. A nonionic surfactant is added to the amplification reaction system to enhance the adsorption of the amplification product by the metal compound.

[0057] According to an embodiment of the present invention, the method further includes: d. using the amplification product obtained in step c as a template, performing a two-chain generation reaction in the presence of a polymerase with chain displacement activity; wherein a nonionic surfactant is added in step d.

[0058] According to embodiments of the present invention, the concentration of the nonionic surfactant in the strand displacement amplification reaction system is 0.01% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or 0.05% to 5% and 0.1% to 5%. A suitable concentration of nonionic surfactant can effectively improve the adsorption of DNA nanospheres and substances such as phi29 DNA polymerase by altering van der Waals forces, thereby resulting in more uniform specific adsorption of DNA nanospheres by the sequencing chip, increased sequencing signal rise and Q30 rise during two-strand sequencing, and improved sequencing quality.

[0059] According to embodiments of the present invention, the term "chain displacement amplification reaction system" refers to the sum of all components involved in the reaction and their environmental conditions in a chemical or biochemical experiment. This includes reactants, solvents, catalysts, and buffer solutions in a chain displacement amplification reaction.

[0060] According to embodiments of the present invention, the nonionic surfactant is selected from at least one of Tween-20, Triton X-100, Pluronic F-127, Nonidet P40 and Pluronic F68, preferably Tween-20 and Triton X-100.

[0061] According to embodiments of the present invention, the concentration of Tween-20 in the strand displacement amplification reaction system is 0.01% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or 0.05% to 5% and 0.1% to 5%. A suitable concentration of Tween-20 can effectively improve the adsorption of DNA nanospheres and substances such as phi29 DNA polymerase by altering van der Waals forces, thereby resulting in more uniform specific adsorption of DNA nanospheres by the sequencing chip, increased sequencing signal rise and Q30 rise during two-strand sequencing, and improved sequencing quality.

[0062] According to an embodiment of the present invention, the concentration of Tween-20 in the strand displacement amplification reaction system is 0.05% to 2%. A suitable concentration of Tween-20 can effectively improve the adsorption of DNA nanospheres and substances such as phi29 DNA polymerase by altering van der Waals forces, thereby resulting in more uniform specific adsorption of DNA nanospheres by the sequencing chip, increased sequencing signal rise and Q30 rise during two-strand sequencing, and improved sequencing quality.

[0063] According to embodiments of the present invention, the concentration of Triton X-100 in the strand displacement amplification reaction system is 0.01% to 5%, for example, it can be 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 0.1% to 5% and 0.5% to 5%. A suitable concentration of Triton X-100 can effectively improve the adsorption of DNA nanospheres and substances such as phi29 DNA polymerase by altering van der Waals forces, thereby resulting in more uniform specific adsorption of DNA nanospheres by the sequencing chip, increased sequencing signal rise and Q30 rise during two-strand sequencing, and improved sequencing quality.

[0064] According to embodiments of the present invention, the concentration of Triton X-100 in the strand displacement amplification reaction system is 0.05% to 2%, for example, it can be 0.05%, 0.1%, 0.5%, 1%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or a range between 0.05% to 1.9% and 0.05% to 1.8%. A suitable concentration of Triton X-100 can effectively improve the adsorption of DNA nanospheres and substances such as phi29 DNA polymerase by altering van der Waals forces, thereby resulting in more uniform specific adsorption of DNA nanospheres by the sequencing chip, increased sequencing signal rise and Q30 rise during two-strand sequencing, and improved sequencing quality.

[0065] Solid support with amplification products immobilized on its surface

[0066] In a second aspect, the present invention provides a solid-phase support with amplification products immobilized on its surface. According to embodiments of the present invention, the amplification products are obtained based on the amplification method described in the first aspect of the present invention. The solid-phase support according to embodiments of the present invention can improve the stability and efficiency of nucleic acid amplification.

[0067] sequencing methods

[0068] In a third aspect, the present invention provides a sequencing method. According to an embodiment of the present invention, the method includes: sequencing amplification products immobilized on the surface of a solid support as described in the second aspect of the present invention; further comprising, after step c, hybridizing the amplification products with one-stranded sequencing primers to perform multiple cycles of sequencing reactions. The method according to the embodiments of the present invention is low-cost, simple to operate, and can obtain sequencing data with high sequencing quality and high stability.

[0069] According to an embodiment of the present invention, the sequencing method includes, after step d, first hybridizing with second-strand sequencing primers and performing multiple rounds of cyclic second-strand sequencing reactions to obtain second-strand sequencing data, and then hybridizing with first-strand sequencing primers and performing multiple rounds of cyclic first-strand sequencing reactions to obtain first-strand sequencing data.

[0070] Amplification reagent combination

[0071] In a fourth aspect, the present invention provides an amplification reagent combination. According to embodiments of the present invention, the amplification reagent combination comprises: I. a solid support having a metal compound on its surface; II. a loading reagent including a nucleic acid template to be tested; III. a multiple displacement amplification reagent including a polymerase with chain displacement activity; wherein the loading reagent and the multiple displacement amplification reagent contain a nonionic surfactant. The amplification reagent combination according to embodiments of the present invention can improve the stability and efficiency of nucleic acid amplification.

[0072] According to embodiments of the present invention, the metal compound is a metal oxide or a metal nitride.

[0073] According to embodiments of the present invention, the metal oxide is selected from at least one of silicon oxide (SiO2), titanium oxide (TiO2), zinc oxide (ZnO), nickel oxide (NiO), and cerium oxide (CeO2).

[0074] According to an embodiment of the present invention, the metal nitride is selected from at least one of titanium nitride (TiN) and silicon nitride (Si3N4).

[0075] According to an embodiment of the present invention, the loading reagent is selected from at least one of DLBI, DLBII, and DLBIV.

[0076] According to an embodiment of the present invention, DLBI (BGI Genomics, 530-002796-00).

[0077] According to an embodiment of the present invention, DLBII (BGI Genomics, 1000004688).

[0078] According to an embodiment of the present invention, DLBIV (BGI Genomics, 530-000075-00).

[0079] According to an embodiment of the present invention, the surface of the solid support has a plurality of regularly distributed depressions or protrusions, and a metal compound is present at the bottom of the depressions or the top of the protrusions. Furthermore, the surface of the solid support has a spacer region for isolating the depressions or protrusions, wherein the surface of the spacer region is silicon dioxide.

[0080] According to an embodiment of the present invention, the concentration of the nonionic surfactant in the loading reagent is 0.01% to 5%, and the concentration of the nonionic surfactant in the multiple displacement amplification reagent is 0.01% to 5%.

[0081] According to embodiments of the present invention, the nonionic surfactant is selected from at least one of Tween-20, Triton X-100, Pluronic F-127, Nonidet P40 and Pluronic F68, preferably Tween-20 and Triton X-100.

[0082] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0083] In this embodiment, the APTMS chip used was manufactured and supplied by Wuhan BGI Genomics (item number: 930-000015-00), and the novel sequencing chip (HP chip) used was purchased from Guangdong Chip Semiconductor Technology Co., Ltd. (not in mass production) or Goertek Optics (not in mass production).

[0084] In this embodiment, the *E. coli* library used was provided by BGI Genomics (catalog number: 1000005033). When using the MGISEQ-2000RS sequencer, the sequencer and handheld loader were manufactured and provided by Wuhan BGI Genomics. The sequencing primers, sequencing reagents, Make DNB enzyme MIXⅠ and Make DNB enzymeⅡ, loading reagents DLBII / IV, and stop run buffer were all from the MGISEQ-2000RS high-throughput sequencing reagent kit (SE100) (catalog number: 1000012552) / (PE100) (catalog number: 1000012554). The sequencing slides were provided by Wuhan BGI Genomics (catalog number: 930-000015-00).

[0085] In this embodiment, a Plasma cleaning machine (SINDIN, SPV-100) was purchased from Dongguan Shengding.

[0086] In the examples, the Tween-20 (Sangon Biotech, A600560-0500), Triton X-100 (SIGMA, 1002194227), Pluronic F68 (SIGMA, P5556-100ML), Pluronic F-127 (SIGMA, P2443-250G), Nonidet P40 (SIGMA, 1003352681), and cetyltrimethylammonium bromide (CTAB, Sangon Biotech, A600108-0100) were purchased from Shanghai Sangon Biotech and Sigma-Aldrich, respectively. Sodium dodecyl sulfate (SDS, Myriel, M37156-25G) was purchased from Shanghai Myriel.

[0087] Example 1:

[0088] (1) Using a single-stranded circularized library of Escherichia coli (E. coli), the amount of which was 40 fmol, nucleic acid nanospheres (DNB) were prepared according to the MGISEQ-2000RS DNA NanoBalls (DNB) sample preparation and loading kit and instructions for use. The preparation time of DNB using rolling circle amplification (RCA) technology was 25 min.

[0089] (2) HP sequencing wafers (Hard pattern chips, size consistent with the MGISEQ-2000RS standard wafers, marked as Yuexin Process 5 chip: well depth 60nm, well diameter 220nm) provided by Guangdong Chip Semiconductor Technology Co., Ltd. were used. The control group used conventional loading technology (for details, please refer to the product manual: High-throughput (rapid) sequencing reagent kit, MGISEQ-2000RS version 12.0, instrument number: R2100400190181), that is, nucleic acid nanospheres (DNB) were prepared under rolling circle amplification for 25 min and loading reagent DLBII was added; the experimental group used MLG (Make DNB, Load DNB, Grow DNB) loading technology, that is, nucleic acid nanospheres (DNB) were prepared under rolling circle amplification for 25 min and loading reagent DLBIV was added. Before use, the chips were treated on a Plasma ion instrument with air as the gas source and power set to 200W for 10 min.

[0090] (3) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader. Let it stand at room temperature for 60 minutes, then place the slide in the sequencing chamber and insert the MGISEQ-2000RS standard single-end 100 (SE100) sequencing kit. Perform sequencing using the MGISEQ-2000RS. (The sequencing procedure for this experiment follows the instruction manual for the gene sequencer (MGISEQ-2000RS). Single-end SE50 sequencing verification was performed on the MGISEQ-2000RS platform.)

[0091] The results are shown in Table 1 and Figure 3. Compared with the control group, the MLG loading technology yielded higher sequencing signals, resulting in higher sequencing data. This indicates that the MLG loading technology allows for better loading of nucleic acid nanospheres onto the HP chip, which is beneficial for sequencing.

[0092] Table 1: Comparison of SE50 sequencing results for HP microarrays using conventional loading and MLG loading techniques.

[0093] Example 2:

[0094] (1) Using E. coli single-stranded circularized library, the amount of DNB was 40 fmol. DNB was prepared according to the MGISEQ-2000RS DNB sample preparation and loading kit and the instructions for use. The preparation time of DNB using rolling circle amplification (RCA) technology was 20 min.

[0095] (2) The HP sequencing carrier provided by Guangdong Semiconductor Technology Co., Ltd. (the size is consistent with the MGISEQ-2000RS standard carrier, marked as Guangdong Semiconductor Process 11 chip: the hole depth is 30nm and the hole diameter is 200nm) was used. Before the chip was used, it was treated on the Plasma ion instrument with air as the source and the power set to 200W for 10 minutes.

[0096] (3) In the control group, nucleic acid nanospheres (DNB) were prepared using MLG loading technology, i.e., rolling circle amplification for 20 min and loading reagent DLBIV was added. In the experimental group, nucleic acid nanospheres (DNB) were prepared using MLG loading technology, i.e., rolling circle amplification for 20 min and loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.05%) was added.

[0097] (4) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader, let it stand at room temperature for 60 minutes (the nanospheres continue to generate and react), place the slide in the sequencing chamber, and insert the MGISEQ-2000RS standard SE100 sequencing kit. Use the MGISEQ-2000RS for sequencing (the sequencing procedure in this experiment follows the instruction manual for the gene sequencer (MGISEQ-2000RS), and performs paired-end SE100 sequencing verification on the MGISEQ-2000RS platform).

[0098] The results are shown in Table 2 and Figure 4. When the experimental group added Tween-20 to the loading reagent DLBIV at a final concentration of 0.05%, the sequencing quality was further improved.

[0099] Table 2: SE100 sequencing results of HP microarrays using MLG loading technology and Tween-20 loading reagent.

[0100] Example 3

[0101] (1) Use E. coli single-stranded circularized library, add 40 fmol, and prepare DNB according to MGISEQ-2000RS DNB sample preparation and loading kit and instructions. The preparation time of DNB using rolling circle amplification (RCA) technology is 20 min or 25 min.

[0102] (2) The HP sequencing carrier provided by Guangdong Semiconductor Technology Co., Ltd. (the size is consistent with the MGISEQ-2000RS standard carrier, marked as Guangdong Semiconductor Process 11 chip: the hole depth is 30nm and the hole diameter is 200nm) was used. Before the chip was used, it was treated on the Plasma ion instrument with air as the source and the power set to 200W for 10 minutes.

[0103] (3) Among them, the control group used MLG loading technology, that is, rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding loading reagent DLBIV. The experimental groups used MLG loading technology. Specifically, experimental group 1 used rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.05%); experimental group 2 used rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.15%); and experimental group 3 used rolling circle amplification for 25 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.05%).

[0104] (4) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader, let it stand at room temperature for 60 minutes (the nanospheres continue to form), place the slide in the sequencing chamber, and insert the MGISEQ-2000RS standard SE100 sequencing kit. Use the MGISEQ-2000RS for sequencing. (The sequencing procedure in this experiment follows the instruction manual for the gene sequencer (MGISEQ-2000RS), and paired-end SE100 sequencing verification is performed on the MGISEQ-2000RS platform).

[0105] The results are shown in Table 3 and Figure 5. The experimental group's results were better than the control group's, further demonstrating that adding Tween-20 to the loading reagent DLBIV can improve the sequencing quality of nucleic acid nanospheres loaded onto the HP chip for sequencing. Experimental group 2 showed better sequencing quality than experimental group 1 across all sequencing metrics, further indicating that increasing the final concentration of Tween-20 to 0.15% is beneficial for improving sequencing quality and stability. Experimental group 3 used nucleic acid nanospheres amplified by rolling circles for 25 minutes for loading and sequencing; even with a Tween-20 concentration of 0.05%, higher sequencing quality was achieved, indicating that when using MLG loading technology, the rolling circle amplification time for nucleic acid nanospheres can be further optimized to improve sequencing quality. This also further demonstrates that different amounts of surfactant have different effects on the adsorption of nucleic acid nanospheres onto the HP chip, which can be further optimized.

[0106] Table 3: Sequencing results of HP chips using SE100 with different concentrations of Tween-20 and different amplification times

[0107] Example 4

[0108] (1) Use E. coli single-stranded circularized library, add 40 fmol, and prepare DNB according to MGISEQ-2000RS DNB sample preparation and loading kit and instructions. The preparation time of DNB using rolling circle amplification (RCA) is 15 min, 20 min or 25 min.

[0109] (2) The HP sequencing carrier provided by Guangdong Semiconductor Technology Co., Ltd. (the size is consistent with the MGISEQ-2000RS standard carrier, marked as Guangdong Semiconductor Process 11 chip: the hole depth is 30nm and the hole diameter is 200nm) was used. Before the chip was used, it was treated on the Plasma ion instrument with air as the source and the power set to 200W for 10 minutes.

[0110] (3) The control group used MLG loading technology, i.e., rolling circle amplification for 15 min to prepare nucleic acid nanospheres (DNB) and adding loading reagent DLBIV. The experimental groups used MLG loading technology. Specifically, experimental group 1 used rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV; experimental group 2 used rolling circle amplification for 25 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV; and experimental group 3 used rolling circle amplification for 25 min to prepare nucleic acid nanospheres (DNB) and added loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.5%).

[0111] (4) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader, let it stand at room temperature for 60 minutes, place the slide in the sequencing chamber, and insert the MGISEQ-2000RS standard SE100 sequencing kit. Use the MGISEQ-2000RS for sequencing (the sequencing procedure in this experiment follows the instruction manual for the gene sequencer (MGISEQ-2000RS), and performs paired-end SE100 sequencing verification on the MGISEQ-2000RS platform).

[0112] The results are shown in Table 4 and Figure 6. Compared with experimental groups 1 and 2, the sequencing indicators improved with increasing rolling circle amplification time in the control group, indicating that optimizing the rolling circle amplification time of MLG loading technology can improve the loading of nucleic acid nanospheres onto the HP chip, thereby obtaining better sequencing quality. Comparing experimental groups 1 and 2, when preparing nucleic acid nanospheres (DNB) using MLG loading technology, loading them onto the HP chip after 25 minutes of rolling circle amplification yielded better sequencing quality. Compared with experimental group 2, adding 0.5% Tween-20 to the loading reagent DLBIV during loading further improved sequencing quality and stability in experimental group 3.

[0113] Table 4: HP chip MLG loading time optimization and sequencing results with 0.5% Tween-20 SE100.

[0114] Example 5

[0115] (1) Using E. coli single-stranded circularized library, the amount of DNB was 40 fmol. DNB was prepared according to the MGISEQ-2000RS DNB sample preparation and loading kit and the instructions for use. The preparation time of DNB using rolling circle amplification (RCA) was 20 min.

[0116] (2) Use the HP sequencing slides provided by Goertek (the size is the same as the MGISEQ-2000RS standard slides, marked as nanoimprint chips: pore depth is 30nm, pore diameter is 220nm). Before use, the chips are treated on a Plasma instrument with air as the source and power set to 200W for 10 minutes.

[0117] (3) The control group used MLG loading technology, which involved rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding loading reagent DLBIV. The experimental groups used MLG loading technology. Specifically, experimental group 1 involved rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding loading reagent DLBIV (which contained Tween-20 at a final concentration of 0.5%). Experimental group 2 involved rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding loading reagent DLBIV (which contained Triton X-100 at a final concentration of 0.5%).

[0118] (4) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader, let it stand at room temperature for 60 minutes, place the slide in the sequencing chamber, and insert the MGISEQ-2000RS standard PE100 sequencing kit. Use the MGISEQ-2000RS for sequencing. (The sequencing procedure in this experiment is based on the instruction manual of the gene sequencer (MGISEQ-2000RS), and the paired-end PE100+10 sequencing is verified on the MGISEQ-2000RS platform.)

[0119] The results are shown in Table 5 and Figure 7. Compared with the conventional loading reagent DLBIV (control group), the addition of 0.5% Tween-20 to the loading reagent DLBIV in experimental group 1 and the addition of 0.5% Triton X-100 to the loading reagent DLBIV in experimental group 2 both resulted in a decrease in sequencing background and achieved better sequencing quality than the control group. This indicates that adding multiple types of surfactants to the loading reagent DLBIV can improve adsorption and enhance sequencing quality and stability. The comparison between experimental group 2 and experimental group 3 shows that different surfactants have different effects.

[0120] Table 5: Sequencing results of different types of surfactant PE100 tested using HP chip

[0121] Example 6

[0122] The experimental procedures in Example 6 were basically the same as those in Example 5, except that the HP sequencing slides provided by Goertek Optics (with dimensions consistent with the MGISEQ-2000RS standard slides, labeled as nanoimprint chips: pore depth 45nm, pore diameter 220nm) were used. Before use, the chips were pre-treated on a Plasma instrument with air as the source and a power setting of 450W for 10 minutes. The control group used MLG loading technology, i.e., nucleic acid nanospheres (DNB) were prepared using rolling circle amplification for 20 minutes and then loaded with the loading reagent DLBIV. The experimental groups used MLG loading technology. Experimental group 1 used rolling circle amplification for 20 minutes to prepare nucleic acid nanospheres (DNB) and then added the loading reagent DLBIV (which contained a final concentration of 0.5% Pluronic F-127); Experimental group 2 used rolling circle amplification for 20 minutes to prepare nucleic acid nanospheres (DNB) and then added the loading reagent DLBIV (which contained a final concentration of 2% Pluronic F-127). Other procedures were the same as in Example 5. The results are shown in Table 6 and Figure 8. When the experimental group added Pluronic F-127 at final concentrations of 0.5% and 2% to the loading reagent DLBIV, the sequencing quality was improved, and the higher the concentration, the better the quality.

[0123] Table 6: SE100 sequencing results of HP chip assays with added Pluronic F-127 reagents

[0124] Example 7

[0125] The experimental procedures in Example 7 were basically the same as those in Example 5, except that the HP sequencing slides provided by Goertek Optics (with dimensions consistent with the MGISEQ-2000RS standard slides, labeled as nanoimprint chips: pore depth 45nm, pore diameter 220nm) were used. Before use, the chips were pre-treated on a Plasma instrument with air as the source and a power setting of 450W for 10 minutes. The control group used MLG loading technology, preparing nucleic acid nanospheres (DNB) using rolling circle amplification for 20 minutes and adding the loading reagent DLBIV. The experimental groups used MLG loading technology. Experimental group 1 prepared nucleic acid nanospheres (DNB) using rolling circle amplification for 20 minutes and added the loading reagent DLBIV (which contained a final concentration of 0.5% Nonidet P40); Experimental group 2 prepared nucleic acid nanospheres (DNB) using rolling circle amplification for 20 minutes and added the loading reagent DLBIV (which contained a final concentration of 2% Nonidet P40). Other procedures were the same as in Example 5. The results are shown in Table 7 and Figure 9. When the experimental group added Nonidet P40 at final concentrations of 0.5% and 2% to the loading reagent DLBIV, the sequencing quality was improved, and the higher the concentration, the better the quality.

[0126] Table 7: SE100 sequencing results using HP chip assays with reagents loaded with Nonidet P40

[0127] Example 8

[0128] The experimental procedures in Example 8 were basically the same as those in Example 5, except that HP sequencing wafers (with dimensions identical to the MGISEQ-2000RS standard wafer, labeled as Yuexin Process 9 chip: 30nm well depth, 220nm well diameter) provided by Guangdong Chip Semiconductor Technology Co., Ltd. were used. The control group used MLG loading technology, i.e., rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding the loading reagent DLBIV. The experimental group used MLG loading technology, i.e., rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and adding the loading reagent DLBIV (which contained a final concentration of 2% Pluronic F68). Other procedures were the same as in Example 5. The results showed that when the experimental group added a final concentration of 2% Pluronic F68 to the loading reagent DLBIV, the sequencing quality did not improve further.

[0129] Table 8: SE100 sequencing results with Pluronic F68 added using HP chip assay reagents

[0130] Example 9

[0131] (1) Using E. coli single-stranded circularized library, 40 fmol was added. Nucleic acid nanospheres (DNB) were prepared according to the MGISEQ-2000RS DNB sample preparation and loading kit and instructions for use. The preparation time of DNB using rolling circle amplification (RCA) technique was 25 min.

[0132] (2) Use the HP sequencing slides provided by Goertek (the size is the same as the MGISEQ-2000RS standard slides, marked as nanoimprint chips: pore depth is 30nm, pore diameter is 220nm). Before use, the chips are treated on the Plasma ion instrument with air as the gas source and power set to 200W for 10 minutes.

[0133] (3) The control group used conventional loading techniques, i.e., nucleic acid nanospheres (DNB) were prepared and loading reagent DLBII was added after rolling circle amplification for 25 min, and conventional MDA reagent was used for MDA. The experimental group used conventional loading techniques, i.e., nucleic acid nanospheres (DNB) were prepared and loading reagent DLBII was added after rolling circle amplification for 25 min, the difference being that Tween-20 with a final concentration of 2% was added to the MDA reagent during MDA (i.e., the MDA reagent contained a final concentration of 2% Tween-20).

[0134] (4) Load the prepared nucleic acid nanospheres onto the sequencing slide using a handheld loader, let it stand at room temperature for 30 minutes, place the slide in the sequencing chamber, and insert the MGISEQ-2000RS standard PE100 sequencing kit. Use the MGISEQ-2000RS for sequencing (the sequencing procedure in this experiment follows the instruction manual for the gene sequencer (MGISEQ-2000RS), and the paired-end PE100+10 sequencing verification is performed on the MGISEQ-2000RS platform).

[0135] The results are shown in Table 6 and Figures 10 and 11. Under consistent loading conditions onto the HP chip, experimental group 1, with only 2% Tween-20 added to the MDA reagent, showed a significant improvement in the average second-strand signal recovery, increasing from 1.48 times to 3.4 times. Q30 recovery also improved significantly, resulting in better overall sequencing quality. Furthermore, the AFM scan revealed fewer large particles of impurities on the chip, a cleaner chip surface, and less "masking" of the concave pore structure, indicating that adding surfactants to the MDA reagent can also improve the adsorption of phi29 DNA polymerase and other enzymes onto the HP chip.

[0136] Table 9: Sequencing results of adding Tween-20 PE100 to the MDA test reagent

[0137] Comparative Example 1

[0138] The experimental procedures for Comparative Example 1 and Example 5 were basically the same, except that the HP sequencing slides provided by Goertek Optics (with dimensions identical to the MGISEQ-2000RS standard slides, labeled as nanoimprint chips: pore depth 45 nm, pore diameter 220 nm) were used. Furthermore, before use, the chips were pre-treated on a Plasma instrument using air as the source and a power setting of 450 W for 10 minutes. The control group used MLG loading technology, employing rolling circle amplification for 20 minutes to prepare nucleic acid nanospheres (DNB) and adding the loading reagent DLBIV. The experimental groups used MLG loading technology. Experimental group 1 involved preparing nucleic acid nanospheres (DNB) using rolling circle amplification for 20 min and adding the loading reagent DLBIV (which contained 0.5% CTAB, a cationic surfactant). Experimental group 2 involved preparing nucleic acid nanospheres (DNB) using rolling circle amplification for 20 min and adding the loading reagent DLBIV (which contained 2% CTAB). Experimental group 3 involved preparing nucleic acid nanospheres (DNB) using rolling circle amplification for 20 min and adding the loading reagent DLBIV. BIV (the DLBIV contained 0.5% SDS (sodium dodecyl sulfate, an anionic surfactant) at a final concentration); Experimental group 4 used rolling circle amplification for 20 min to prepare nucleic acid nanospheres (DNB) and added the loading reagent DLBIV (the DLBIV contained 2% SDS at a final concentration). Other procedures were the same as in Example 5. The results are shown in Figure 12. When 0.5% or 2% CTAB or SDS was added to the loading reagent DLBIV in the experimental groups, it caused DNB abnormalities; the DNB aggregated and could not be loaded onto the chip, resulting in abnormal sequencing results.

[0139] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0140] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for amplification on the surface of a solid support, characterized in that, include: a. Provide a solid support having a metal compound on its surface for adsorbing nucleic acid templates to be tested; b. The nucleic acid template to be tested is loaded onto a solid support, and the nucleic acid template to be tested is adsorbed onto the surface of the metal compound; c. Perform a chain substitution amplification reaction to obtain the amplification product; In step b and step c, a nonionic surfactant is added, and the chain displacement amplification reaction is carried out in the presence of a polymerase with chain displacement activity.

2. The method according to claim 1, characterized in that, The metal compound is a metal oxide or a metal nitride.

3. The method according to claim 1, characterized in that, The solid support surface has multiple regularly distributed depressions or protrusions, with a metal compound present at the bottom of the depressions or the top of the protrusions. Furthermore, the solid support surface has spacer regions for isolating the depressions or protrusions, wherein the surface of the spacer regions is silicon dioxide.

4. The method according to claim 1, characterized in that, The nucleic acid template to be tested is a single-stranded circular nucleic acid library, and the amplification product is a single-stranded linear nucleic acid nanosphere.

5. The method according to claim 4, characterized in that, The method further includes: d. Using the amplification product obtained in step c as a template, a two-strand formation reaction is carried out in the presence of a polymerase with chain displacement activity; In step d, a nonionic surfactant is added.

6. The method according to claim 1, characterized in that, The concentration of the nonionic surfactant in the chain displacement amplification reaction system is 0.01% to 5%.

7. The method according to claim 1, characterized in that, The nonionic surfactant is selected from at least one of Tween-20, Triton X-100, Pluronic F-127, Nonidet P40 and Pluronic F68, preferably Tween-20 and Triton X-100.

8. The method according to claim 7, characterized in that, The concentration of Tween-20 in the chain displacement amplification reaction system is 0.01% to 5%.

9. The method according to claim 8, characterized in that, The concentration of Tween-20 in the chain displacement amplification reaction system is 0.05% to 2%.

10. The method according to claim 7, characterized in that, The concentration of Triton X-100 in the chain displacement amplification reaction system is 0.01% to 5%.

11. The method according to claim 10, characterized in that, The concentration of Triton X-100 in the chain displacement amplification reaction system is 0.05% to 2%.

12. A solid support with amplified products immobilized on its surface, characterized in that, The amplification product is obtained based on the amplification method according to any one of claims 1 to 11.

13. A sequencing method, characterized in that, include: Based on the solid support as described in claim 12, sequencing of the amplification product immobilized on the surface of the solid support further includes hybridizing a one-stranded sequencing primer onto the amplification product after step c, and performing multiple cycles of sequencing reactions.

14. The sequencing method according to claim 13, characterized in that, Further, it includes paired-end sequencing reactions, including multiple cycles of sequencing reactions after step d using the generated two-stranded hybridization two-stranded sequencing primers.

15. An amplification reagent combination, characterized in that, include: I. A solid support having a metal compound on its surface; II. Load reagents, including the nucleic acid template to be tested; III. Multiple displacement amplification reagents, including polymerases with chain displacement activity; The loading reagent and the multiple displacement amplification reagent contain nonionic surfactants.

16. The amplification reagent combination according to claim 15, characterized in that, The metal compound is a metal oxide or a metal nitride.

17. The amplification reagent combination according to claim 15, characterized in that, The solid support surface has multiple regularly distributed depressions or protrusions, with a metal compound present at the bottom of the depressions or the top of the protrusions. Furthermore, the solid support surface has spacer regions for isolating the depressions or protrusions, wherein the surface of the spacer regions is silicon dioxide.

18. The amplification reagent combination according to claim 15, characterized in that, The concentration of the nonionic surfactant in the loading reagent is 0.01% to 5%, and the concentration of the nonionic surfactant in the multiple displacement amplification reagent is 0.01% to 5%.

19. The amplification reagent combination according to claim 18, characterized in that, The nonionic surfactant is selected from at least one of Tween-20, Triton X-100, Pluronic F-127, Nonidet P40 and Pluronic F68, preferably Tween-20 and Triton X-100.

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