Amplification method and kit

Abstract

The invention discloses a plurality of amplification methods and kits.The amplification method comprises the steps that first-round amplification is conducted on target polynucleotide through the steps S20-S40, and an extension chain of a first amplification primer is generated on the surface of a solid-phase carrier; amplifying the extension chain of the first amplification primer through the steps S60-S100, and generating an extension chain of a second amplification primer on the surface of the solid-phase carrier; and performing cyclic amplification according to the step S120-160 by taking the extension chain of the first amplification primer and the extension chain of the second amplification primer as templates to obtain a molecular cluster of the target polynucleotide, so as to amplify a signal generated by doping nucleotide or nucleotide analogues into the target polynucleotide in the sequencing process of the target polynucleotide, therefore, the signal can be detected more easily, and the efficiency and accuracy of signal detection are improved. Meanwhile, according to the amplification method provided by the invention, at least one of betaine with the concentration being greater than 0 mol / L and smaller than or equal to 4 mol / L, Mg < 2 + > with the concentration being 0.5-4 mmol / L, NH4 < + > with the concentration being greater than 0 mmol / L and smaller than or equal to 100 mmol / L and formamide with the volume percent concentration being smaller than or equal to 15% is prepared in a first solution, a second solution and a fourth solution, so that formation of secondary structures in template molecules can be reduced; the amplification efficiency is improved, and the combination of the template molecule and the amplification primer and the extension of the amplification primer are facilitated, so that the GC preference is reduced.

Description

Technical Field

[0001] The present application relates to the technical field of high-throughput sequencing, and in particular to an amplification method and a kit. Background Art

[0002] Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has become a mature and standard molecular biology technology, and has been applied to various fields of biomedicine, such as molecular diagnosis, medical testing, high-throughput sequencing, etc. Through the hybridization pairing of forward primers and reverse primers with template DNA molecules, under the action of polymerase, the free deoxyribonucleic acid (dNTP) in the system is captured to replicate the template chain, so as to achieve the effect of enriching template DNA molecules.

[0003] Due to the diversity of template DNA molecular sequences, the replication efficiency of DNA templates with different sequences usually varies under the same PCR system. The main reason is that according to the principle of base complementary pairing, single-stranded DNA can form intramolecular secondary structures, especially DNA molecules with unbalanced bases (such as extreme GC sequences), which are more likely to form complex secondary structures. The presence of complex secondary structures affects the extension of polymerases, and the amplification efficiency of DNA molecules will be greatly reduced, ultimately resulting in their relative abundance being much lower than other DNA molecules. This is the preference for amplification.

[0004] In high-throughput sequencing, DNA templates are amplified by PCR on the surface of a solid phase carrier. Due to the amplification preference, DNA molecules with high GC content (e.g., GC content greater than 50%) tend to have lower amplification efficiency, and the generated clusters are smaller and darker, with lower detection efficiency; while those larger and brighter clusters are easier to detect, such as AT-rich clusters; this results in the final sequencing results, GC-rich DNA templates will be less abundant, less representative, and have poorer sequence accuracy. We call this effect GC bias.

[0005] GC bias can lead to a series of problems, such as (1) when detecting copy number, the coverage of regions with high GC content is lower than that of regions with low GC content, but it does not mean that the copy number of regions with low GC content is higher than that of regions with high GC content based solely on the sequencing coverage; (2) when doing RNA sequencing analysis, the small number of sequencing reads in regions with high GC content does not necessarily mean that the expression level of this gene is low; (3) when assembling the genome, due to the existence of GC bias, regions with high GC content are rarely measured, and the assembly of these regions is more difficult; (4) it is difficult to determine the sequences in certain genomes, especially some regions with important functions, such as GC-rich CpG islands in promoter regions; and so on.

[0006] Therefore, how to reduce GC preference is an issue worthy of attention. Summary of the invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application provides an amplification method and a kit to reduce GC preference.

[0008] In one aspect, the present application provides an amplification method, comprising:

[0009] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0010] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer;

[0011] S60, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0012] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0013] S100, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0014] S120, replacing the first solution or the second solution with the third solution;

[0015] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0016] S160, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0017] S180, repeat S120-S160 at least once;

[0018] Among them, the first solution, the second solution, and the fourth solution all contain betaine, Mg 2+ NH 4 + , formamide, wherein the concentration of betaine is greater than 0 mol / L and less than or equal to 4 mol / L, Mg 2+ The concentration of NH 4+ The concentration is greater than 0 mmol / L and less than or equal to 100 mmol / L, and the volume percentage concentration of formamide is less than or equal to 15%.

[0019] The amplification method provided by the present application performs a first round of amplification on the target polynucleotide through steps S20 to S40 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S60 to S100 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates to perform cyclic amplification according to steps S120 to 160 to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by incorporating nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, thereby making it easier to detect the signal and improving the efficiency and accuracy of signal detection. At the same time, the amplification method provided by the present application configures betaine with a concentration greater than 0 mol / L and less than or equal to 4 mol / L, Mg with a concentration of 0.5 to 4 mmol / L in the first solution, the second solution, and the fourth solution. 2+ , NH with a concentration greater than 0mmol / L and less than or equal to 100mmol / L 4 + At least one of the following: , formamide with a volume percentage concentration less than or equal to 15% can reduce the formation of secondary structure in the template molecule, improve amplification efficiency, facilitate the binding of the template molecule to the amplification primer and the extension of the amplification primer, thereby reducing GC preference.

[0020] On the other hand, the present application provides another amplification method, comprising:

[0021] S200, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0022] S400, using the target nucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer;

[0023] S600, replacing the first solution or the second solution with a third solution to remove the target nucleotide, wherein the third solution contains a denaturing agent;

[0024] S800, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer is combined with the second amplification primer;

[0025] S1000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer, thereby obtaining the extended chain of the second amplification primer;

[0026] S1200, replacing the first solution or the second solution with a third solution;

[0027] S1400, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0028] S1600, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0029] S1800, repeat S1200-S1600 at least once;

[0030] Among them, S1600 is carried out under variable temperature conditions, S1200 and S1400 are carried out under constant temperature conditions, and the reaction temperature of S1600 is not lower than the reaction temperatures of S1200 and S1400.

[0031] The amplification method provided by the present application performs a first round of amplification on the target polynucleotide through steps S20 to S40 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S60 to S100 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates to perform cyclic amplification according to steps S120 to 160 to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by incorporating nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, thereby making it easier to detect the signal and improving the efficiency and accuracy of signal detection. At the same time, the amplification method of the present application can promote the melting of the secondary structure formed in the template molecule by introducing variable temperature amplification during the amplification process, which is beneficial to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0032] In another aspect of the present application, another amplification method is provided, comprising:

[0033] S2000, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0034] S4000, using the target polynucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer;

[0035] S6000, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0036] S8000, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0037] S10000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer, thereby obtaining the extended chain of the second amplification primer;

[0038] S12000, replacing the first solution or the second solution with a third solution;

[0039] S14000, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0040] S16000, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0041] S18000, repeat S12000-S16000 at least once;

[0042] Wherein, in S10000 and S16000, the first solution or the second solution contains dATP, dTTP, dCTP and ddTP, and the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP.

[0043] The amplification method provided by the present application performs a first round of amplification on the target polynucleotide through steps S20 to S40 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S60 to S100 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates according to steps S120 to 160 to perform cyclic amplification to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by incorporating nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, so that the signal is easier to detect, thereby improving the efficiency and accuracy of signal detection. At the same time, the amplification method of the present application introduces an unbalanced base ratio, that is, the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP, thereby increasing the probability of complementary pairing of dGTP and dCTP with GC bases on the template molecule, preferentially amplifying sequences with high GC content, thereby enhancing the amplification of GC bases in the template molecule, improving amplification efficiency, and reducing GC preference.

[0044] In another aspect of the present application, a kit is provided, comprising the first solution, the second solution, the third solution and the fourth solution mentioned in any one of the above aspects.

[0045] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A schematic diagram of an amplification method according to an embodiment of the present application;

[0047] Figure 2 This is a flow chart of the amplification method of an embodiment of the present application;

[0048] Figure 3 This is a GC preference scatter plot of an embodiment of the present application;

[0049] Figure 4 This is the GC preference scatter plot in Comparative Example 1-1 of the present application;

[0050] Figure 5 This is a GC preference scatter plot in Comparative Example 1-2 of the present application;

[0051] Figure 6 It is the GC preference scatter plot in Comparative Examples 1-3 of the present application;

[0052] Figure 7 This is the GC preference scatter plot in Comparative Example 2-1 of this application;

[0053] Figure 8 This is the GC preference scatter plot in Comparative Example 3-1 of this application;

[0054] Fig. 9 It is the GC preference scatter plot in Example 4 of the present application;

[0055] Fig.10 This is the GC preference scatter plot in Comparative Example 5-1 of the present application;

[0056] Fig.11 This is a comparison curve between the constant temperature amplification and the pulse heating amplification in Example 6 of the present application;

[0057] Fig.12 This is a GC preference scatter plot in Comparative Example 6-1 of the present application. DETAILED DESCRIPTION

[0058] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0059] In this application, the terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The term "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "and" relationship.

[0060] The term "at least one" means one or more, and "plurality" means two or more. "At least one" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be a single item or multiple items, respectively.

[0061] The singular forms of "a", "said", and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise. The terms "first" and "second" are used only for descriptive purposes to distinguish purposes such as substances, locations, interfaces, messages, requests, and terminals from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the feature.

[0062] In the description of the present application, the concentration of the relevant components mentioned may not only refer to the specific content of each component, but also indicate the proportional relationship between the contents of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application.

[0063] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.Chemical structures and formulae herein are constructed according to the standard rules of chemical valency known in the chemical art.

[0064] It should be noted that the term "solid phase carrier" can be any solid support that can be used to fix nucleic acid sequences, such as nylon membranes, glass slides, plastics, silicon wafers, magnetic beads, etc.

[0065] The term "target polynucleotide" may refer to a single-stranded nucleic acid molecule or a nucleic acid analog, such as single-stranded DNA, RNA, PNA, LNA or 2'-O-methRNA. The target polynucleotide may be derived from, for example, animals, plants, microorganisms, and the like.

[0066] The term "amplification primer" can be an oligonucleotide or nucleic acid molecule hybridized with a target polynucleotide of interest. In an embodiment, the amplification primer acts as a substrate, and nucleotides or nucleotide analogs can be polymerized onto the substrate by a polymerase. For example, the amplification primer can be used as a starting point for DNA or RNA synthesis. For example, the amplification primer can hybridize with the target polynucleotide to form a hybridization complex, so as to initiate the synthesis of a new chain complementary to the target polynucleotide. The amplification primer can include any combination of nucleotides or their analogs. Exemplarily, the amplification primer can be a single-stranded oligonucleotide or a polynucleotide.

[0067] The term "denaturing agent" may refer to chemical substances that can destroy the double-stranded structure in nucleic acid molecules and change them from double-stranded to single-stranded. These chemical substances can be used to denature nucleic acid molecules by destroying the hydrogen bonds between the double-stranded nucleic acid molecules. Exemplarily, the denaturing agent may be, for example, a strong acid (e.g., HCl), a strong base (e.g., NaOH), urea, methanol, ethanol, amide compounds, guanidine hydrochloride, guanidine isothiocyanate, etc.

[0068] The term "dNTPs" refers to deoxyribonucleoside triphosphates, which contain a phosphate group, deoxyribose and a nitrogenous base (one of A, T, C, G), and usually include dATP, dGTP, dTTP and dCTP, which stand for deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxycytidine triphosphate, respectively.

[0069] Generally, clones of template molecules can be generated by amplifying template molecules. Template molecules amplified multiple times can generate multiple clones and form clusters, i.e., amplification clusters. Amplification clusters can amplify the signal generated when nucleotides or nucleotide analogs are incorporated into template molecules during sequencing, thereby making it easier for the detection system to detect the signal, thereby improving the efficiency and accuracy of signal detection. However, during the amplification process, since the template molecule may form an intramolecular secondary structure due to its own folding, especially the template molecule with high GC content, it is easier to form a complex secondary structure during the amplification process, and since there are three hydrogen bonds between the paired GCs and two hydrogen bonds between the paired ATs, the secondary structure formed in the template molecule with high GC content is more difficult to unwind relative to the secondary structure formed in the template molecule with low GC content. The secondary structure in the template molecule has an adverse effect on the binding between the template molecule and the amplification primer and the extension of the amplification primer, thereby reducing the amplification efficiency, resulting in a small number of clones generated and a small amplification cluster. Since the amplification cluster is small, the signal generated when the nucleotide or nucleotide analog is incorporated into the template molecule during sequencing is weak, making it difficult for the detection system to detect the signal, resulting in low efficiency and accuracy of signal detection. As a result, in the final sequencing results, the abundance of the template molecule with a high GC content is low relative to the abundance of the template molecule with a low GC content. This effect is usually called GC preference.

[0070] In order to improve amplification efficiency and reduce GC preference, the inventors of the present application have conducted a long period of exploration and research and developed a variety of amplification methods. Figure 1 and Figure 2 As shown, an amplification method provided in an embodiment of the present application comprises:

[0071] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer.

[0072] Wherein, the surface of the solid phase carrier can be the surface of a sequencing chip. Exemplarily, the surface of the solid phase carrier includes a number of nanopores arranged in an array. The size of the nanopore and the distance between the holes can be determined according to actual conditions. The first amplification primer and the second amplification primer are fixed on the inner surface of the nanopore by surface chemical modification. The first amplification primer and the second amplification primer are, for example, single-stranded oligonucleotides. The first amplification primer and the second amplification primer can be fixed on the surface of the solid phase carrier by means known in the art, such as covalent bonding or physical adsorption. The target polynucleotide can be, for example, a single-stranded nucleic acid molecule or a nucleic acid analog, and at least a portion of one end of the target polynucleotide is complementary hybridized with the first amplification primer in a base complementary pairing manner to form a hybrid complex, thereby fixing the target polynucleotide on the surface of the solid phase carrier. The first amplification primer complementary hybridized with the target polynucleotide can be used as the starting point for synthesizing the complementary chain of the target polynucleotide, and the synthesis of the complementary chain of the target polynucleotide is initiated under the action of DNA polymerase and under conditions suitable for polymerase chain reaction.

[0073] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0074] S40, using the target polynucleotide as a template, using the first solution or the second solution to extend the first amplification primer to obtain an extended chain of the first amplification primer.

[0075] Wherein, the first solution or the second solution contains the components required for extension, such as DNA polymerase, nucleotides (such as A, T, C, G) or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, with the target polynucleotide as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer, thereby extending the first amplification primer, and the extended chain obtained by the first amplification primer is the complementary chain of the target polynucleotide. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST and BSU. Preferably, the concentration of the DNA polymerase is 4U / ml.

[0076] like Figure 1 As shown, the amplification method provided in the embodiment of the present application further includes:

[0077] S60, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent.

[0078] By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the target polynucleotide and its complementary chain, for example, to break the hydrogen bond between the target polynucleotide and its complementary chain, so that the double-stranded molecule is unzipped to form a single-stranded molecule to remove the target polynucleotide. Exemplarily, the denaturing agent can be, for example, a strong acid (such as HCl), a strong base (such as NaOH), urea, methanol, ethanol, an amide compound, guanidine hydrochloride, guanidine isothiocyanate, etc. Preferably, the denaturing agent is an amide compound, such as formamide.

[0079] like Figure 1 As shown, the amplification method provided in the embodiment of the present application further includes:

[0080] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer.

[0081] Wherein, the free end of the extended chain of the newly synthesized first amplification primer contains a part that can be complementary hybridized with the second amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize with the second amplification primer on the surface of the solid phase carrier, so as to extend the second amplification primer using the extended chain of the first amplification primer as a template to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer.

[0082] like Figure 1 As shown, the amplification method provided in the embodiment of the present application further includes:

[0083] S100, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer to obtain the extended chain of the second amplification primer.

[0084] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, the nucleotides or nucleotide analogs are bound to the 3' end of the second amplification primer using the extended chain of the first amplification primer as a template, thereby extending the second amplification primer to obtain the extended chain of the second amplification primer, which is the complementary chain of the extended chain of the first amplification primer.

[0085] like Figure 1 As shown, the amplification method provided in the embodiment of the present application further includes:

[0086] S120, replacing the first solution or the second solution with the third solution.

[0087] The third solution contains a denaturing agent. By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the extended chain of the first amplification primer and the extended chain of the second amplification primer, for example, to break the hydrogen bond between the extended chain of the first amplification primer and the extended chain of the second amplification primer, so that the double-stranded molecule is unzipped to form a single-stranded molecule.

[0088] The amplification method provided in the embodiment of the present application further includes:

[0089] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively.

[0090] Wherein, the free end of the extended chain of the first amplification primer contains a part that can be complementary hybridized with the second amplification primer, and the free end of the extended chain of the second amplification primer contains a part that can be complementary hybridized with the first amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize and bind with the second amplification primer on the surface of the solid phase carrier, and the free end of the extended chain of the second amplification primer hybridize and bind with the first amplification primer on the surface of the solid phase carrier.

[0091] The amplification method provided in the embodiment of the present application further includes:

[0092] S160, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates.

[0093] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, respectively:

[0094] Using the extended chain of the first amplification primer as a template, a nucleotide or a nucleotide analog is bound to the 3' end of the second amplification primer, and the second amplification primer is extended to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer, i.e., the target polynucleotide; and,

[0095] Using the extended chain of the second amplification primer as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer to extend the first amplification primer, thereby obtaining the extended chain of the first amplification primer, i.e., the complementary chain of the extended chain of the second amplification primer, i.e., the complementary chain of the target polynucleotide.

[0096] The amplification method provided in the embodiment of the present application further includes:

[0097] S180, repeat S120-S160 at least once.

[0098] In this way, multiple target polynucleotides can be obtained, that is, a target polynucleotide molecular cluster is formed.

[0099] The amplification method provided in the present application performs a first round of amplification on the target polynucleotide through steps S20 to S40 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S60 to S100 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates to perform cyclic amplification according to steps S120 to 160 to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by the incorporation of nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, thereby making it easier to detect the signal and improving the efficiency and accuracy of signal detection.

[0100] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain betaine, Mg 2+ NH 4 + , formamide, wherein the concentration of betaine is greater than 0 mol / L and less than or equal to 4 mol / L, Mg 2+ The concentration of NH 4 + The concentration of is greater than 0 mmol / L and less than or equal to 100 mmol / L, and the volume percentage concentration of formamide is less than or equal to 15%. The amplification method provided by the present application comprises configuring betaine with a concentration greater than 0 mol / L and less than or equal to 4 mol / L, Mg with a concentration of 0.5 to 4 mmol / L in the first solution, the second solution, and the fourth solution. 2+ , NH with a concentration greater than 0mmol / L and less than or equal to 100mmol / L 4 + , at least one of formamide with a volume percentage concentration less than or equal to 15%, so that the formation of secondary structure in the template molecule can be reduced, the amplification efficiency can be improved, and it is beneficial to the combination of the template molecule and the amplification primer and the extension of the amplification primer, thereby reducing the GC preference.

[0101] Specifically, the inventors of the present application have found through research that betaine can reduce the formation of secondary structures caused by GC enrichment in template molecules, improve the amplification efficiency of GC-rich sequences, and thus reduce GC preference. The inventors believe that this may be due to the fact that betaine can increase the hydration of guanine- and cytosine-rich regions in template molecules, affect the structure of template molecules, change their flexibility, and help DNA polymerases extend along template molecules, thereby reducing the formation of secondary structures within template molecules, improving amplification efficiency, and reducing GC preference. In addition, betaine can also reduce the melting temperature of sequences rich in GC content, which is conducive to more uniform denaturation of double-stranded molecules, reducing the melting stability differences caused by different GC contents, thereby reducing GC preference. Furthermore, betaine can stabilize the template molecule-protein complex, help maintain the stability of the template molecule during amplification, and reduce the polymerase dissociation caused by GC-rich regions. The inventors of the present application have also found through research that betaine plays the above-mentioned role or has the above-mentioned effect within a certain concentration range. If the concentration of betaine exceeds this range, amplification will be inhibited.

[0102] In some embodiments, the concentration of betaine in the first solution is greater than 0 mol / l and less than or equal to 3 mol / l. For example, the concentration of betaine in the first solution can be 0.5 mol / l, 1 mol / l, 1.5 mol / l, 2 mol / l, 2.5 mol / l, 3 mol / l, or a concentration between any two values.

[0103] In some embodiments, the concentration of betaine in the second solution is 1.5-2.5 mol / l. For example, the concentration of betaine in the second solution can be 1.5 mol / l, 1.6 mol / l, 1.7 mol / l, 1.8 mol / l, 1.9 mol / l, 2.0 mol / l, 2.1 mol / l, 2.2 mol / l, 2.3 mol / l, 2.4 mol / l, 2.5 mol / l, or a concentration between any two values.

[0104] In some embodiments, the concentration of betaine in the fourth solution is 1.5-2.5 mol / l. For example, the concentration of betaine in the fourth solution can be 1.5 mol / l, 1.6 mol / l, 1.7 mol / l, 1.8 mol / l, 1.9 mol / l, 2.0 mol / l, 2.1 mol / l, 2.2 mol / l, 2.3 mol / l, 2.4 mol / l, 2.5 mol / l, or a concentration between any two values.

[0105] The inventor of the present application has also found through research that Mg 2+It can be used as an active auxiliary agent of polymerase, which helps to activate the activity of polymerase, so that the polymerase catalyzes the formation of a phosphodiester bond between the 3'-OH of the amplification primer bound to the template molecule and the phosphate group of the nucleotide or nucleotide analog, thereby combining the nucleotide or nucleotide analog with the amplification primer, and making the nucleotide or nucleotide analog bind to the template molecule in a complementary base pairing manner, that is, incorporating the nucleotide or nucleotide analog into the template molecule, thereby improving the amplification efficiency and reducing the GC preference, thereby helping to reduce the intensity difference of the signal generated by the high AT and high GC target polynucleotide molecular clusters during the sequencing process, and improving the accuracy of sequencing. In addition, Mg 2+ The concentration of Mg is also critical to the amplification process. 2+ If the concentration is too low, amplification will not proceed, and if it is too high, base mismatches will increase during amplification. 2+ concentration.

[0106] In some embodiments, Mg 2+ The concentration can be 0.5mmol / L, 1mmol / L, 1.5mmol / L, 2mmol / L, 2.5mmol / L, 3mmol / L, 3.5mmol / L, 4mmol / L or a concentration between the above two values.

[0107] In some embodiments, in S40, the first amplification primer is extended using the first solution, Mg 2+ The concentration of Mg in the first solution is 2-4 mmol / l, and the concentration of betaine in the first solution is 0.1-2 mol / l. 2+ Cooperating with betaine at a lower concentration can improve amplification efficiency, reduce GC preference, and facilitate extension of the first amplification primer to generate an extended chain of the first amplification primer.

[0108] In S100 or S160, the fourth solution is replaced by the second solution, Mg 2+ The concentration of Mg in the second solution is 2-4 mmol / l, and the concentration of betaine in the second solution is 0.1-2 mol / l; or, in S100 or S160, the fourth solution is replaced by the second solution, Mg 2+ The concentration of betaine in the second solution is 1-1.5 mmol / l, and the concentration of betaine in the second solution is 2-3 mol / l.

[0109] The inventors of the present application have found that, during the extension process of the first amplification primer and / or the second amplification primer of S100 or S160, the use of Mg containing 2 to 4 mmol / l 2+and 0.1-2 mol / l of betaine as the second solution to replace the fourth solution, or using 1-1.5 mmol / l of Mg 2+ The invention speculates that this may be due to the fact that a higher concentration of Mg in S40 is used to replace the fourth solution with a second solution of 2-3 mol / l betaine, which can effectively extend the first amplification primer and / or the second amplification primer to generate an extended chain of the first amplification primer and / or an extended chain of the second amplification primer. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0110] In some embodiments, in S40, the first amplification primer is extended using the second solution, Mg 2+ The concentration of Mg in the second solution is 2-4 mmol / l, and the concentration of betaine in the second solution is 0.1-2 mol / l. 2+ Cooperating with betaine at a lower concentration can improve amplification efficiency, reduce GC preference, and facilitate extension of the first amplification primer to generate an extended chain of the first amplification primer.

[0111] In S100 or S160, the fourth solution is replaced by the first solution, Mg 2+ The concentration of Mg in the first solution is 2-4 mmol / l, and the concentration of betaine in the first solution is 0.1-2 mol / l; or, in S100 or S160, the fourth solution is replaced by the first solution, Mg 2+ The concentration of betaine in the first solution is 1-1.5 mmol / l, and the concentration of betaine in the first solution is 2-3 mol / l.

[0112] The inventors of the present application have found that, during the extension process of the first amplification primer and / or the second amplification primer of S100 or S160, the use of Mg containing 2 to 4 mmol / l 2+ and 0.1-2 mol / l of betaine in the first solution to replace the fourth solution, or use 1-1.5 mmol / l of Mg 2+ The inventors speculate that this may be due to the fact that a higher concentration of Mg in S40 is used to replace the fourth solution with the first solution of 2-3 mol / l betaine. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0113] In some embodiments, in S40, the first amplification primer is extended using the first solution, Mg 2+ The concentration of betaine in the first solution is 2-4 mmol / l, the concentration of betaine in the first solution is 0.1-2 mol / l, and in S100 or S160, the fourth solution is replaced by the second solution, Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; or,

[0114] In S40, the first amplification primer is extended using the second solution, Mg 2+ The concentration of betaine in the second solution is 2-4 mmol / l, the concentration of betaine in the second solution is 0.1-2 mol / l, and in S100 or S160, the fourth solution is replaced by the first solution, Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l;

[0115] The first solution or the second solution also contains dNTPs, and dNTPs includes dATP, dTTP, dCTP, and ddTP, and the sum of the contents of dATP and dTTP is equal to the sum of the contents of dGTP and dCTP. The amplification primer is extended using the first solution or the second solution, and in particular, in S40, a base solution having a balanced ratio, i.e., the sum of the contents of dATP and dTTP is equal to the sum of the contents of dGTP and dCTP, is configured in the first solution or the second solution, which is conducive to amplifying all target polynucleotide molecules as much as possible. Exemplarily, dATP, dTTP, dCTP, and ddTP can exist in equal concentrations, for example, the concentrations of dATP, dTTP, dCTP, and ddTP can all be 200 μmol / l, 100 μmol / l, 50 μmol / l, 10 μmol / l, 5 μmol / l, 1 μmol / l, etc.

[0116] In some embodiments, in S40, the first amplification primer is extended using the first solution, Mg 2+ The concentration of betaine in the first solution is 2-4 mmol / l, the concentration of betaine in the first solution is 0.1-2 mol / l, and in S100 or S160, the fourth solution is replaced by the second solution, Mg 2+ The concentration of the betaine in the second solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the second solution is 2 to 3 mol / l; or,

[0117] In S40, the first amplification primer is extended using the second solution, Mg 2+The concentration of betaine in the second solution is 2-4 mmol / l, the concentration of betaine in the second solution is 0.1-2 mol / l, and in S100 or S160, the fourth solution is replaced by the first solution, Mg 2+ The concentration of the betaine in the first solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the first solution is 2 to 3 mol / l;

[0118] The second solution or the first solution also contains dNTPs, and the dNTPs include dATP, dTTP, dCTP, and ddTP, and the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP. The first solution or the second solution is used to extend the amplification primer, especially in S100 or in S160, the first solution or the second solution is configured with a base solution with an unbalanced ratio, that is, the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP, which is conducive to increasing the probability of complementary pairing of dGTP and dCTP with GC bases on the template molecule, preferentially amplifying sequences with high GC content, thereby enhancing the amplification of GC bases in the template molecule, improving the amplification efficiency, and reducing GC preference. Exemplarily, the sum of the contents of dATP and dTTP is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, etc. of the sum of the contents of dGTP and dCTP.

[0119] The inventor of the present application has also found through research that NH 4 + It can improve the reaction activity of polymerase, thereby improving amplification efficiency and reducing GC preference. The inventors speculate that NH 4 + Improving the polymerase activity may be related to NH 4 + It is related to the ability to change the charge distribution on the surface of the polymerase and / or change the spatial morphology of the polymerase, making it easier for the polymerase to bind to the reaction site of the template molecule and / or the amplification primer, catalyzing the formation of a phosphodiester bond between the 3'-OH of the amplification primer and the phosphate group of the nucleotide or nucleotide analogue, thereby combining the nucleotide or nucleotide analogue with the amplification primer, and allowing the nucleotide or nucleotide analogue to bind to the nucleic acid template in a base complementary pairing manner, that is, incorporating the nucleotide or nucleotide analogue into the template molecule.

[0120] In some embodiments, NH 4 +The concentration can be 0.1mmol / L, 1mmol / L, 5mmol / L, 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or a concentration between any two values.

[0121] In some embodiments, NH 4 + NH provided by at least one of ammonium sulfate, ammonium chloride and ammonium acetate 4 + That is to say, the first solution, the second solution, and the fourth solution of the present application all contain at least one of ammonium sulfate, ammonium chloride, and ammonium acetate, or at least one of ammonium sulfate, ammonium chloride, and ammonium acetate is added when the first solution, the second solution, and the fourth solution are prepared.

[0122] In addition, the inventors of the present application have also found that formamide can not only be used as a denaturing agent, but also can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. This difference in effect is mainly related to the concentration of formamide in the solution. For example, when the volume percentage concentration of formamide reaches or exceeds 60%, such as 60-100%, it is used as a denaturing agent, and when the volume percentage concentration of formamide is greater than or equal to 3% and less than or equal to 15%, then formamide can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. Therefore, by controlling the concentration of formamide, formamide can be controlled to play different roles and achieve different purposes.

[0123] In some embodiments, the volume percentage concentration of formamide in the first solution, the second solution, and the fourth solution can be 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15% or a volume percentage concentration between any two values.

[0124] In the embodiments of the present application, the first solution, the second solution and the fourth solution all contain DMSO (dimethyl sulfoxide), and the volume percentage concentration of DMSO in the first solution, the second solution and the fourth solution is 1-5%.

[0125] Since sequences with high GC content in template molecules are often difficult to amplify due to their easy formation of secondary structures, DMSO can improve the spatial morphology of template molecules with high GC content, reduce the formation of secondary structures, and enable the polymerase to extend along the template molecules, thereby improving the amplification efficiency and reducing GC preference.

[0126] In some embodiments, the volume percentage concentration of DMSO in the first solution, the second solution, and the fourth solution can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a concentration between any two values.

[0127] In the embodiment of the present application, the first solution, the second solution and the fourth solution further contain Trisbase (tris(hydroxymethyl)aminomethane), and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10-500 mmol / l. Among them, Trisbase can provide a suitable buffer environment and ionic strength for amplification.

[0128] In some embodiments, the concentration of Trisbase in the first solution, the second solution, and the fourth solution can be 10 mmol / l, 15 mmol / l, 20 mmol / l, 25 mmol / l, 30 mmol / l, 35 mmol / l, 40 mmol / l, 45 mmol / l, 50 mmol / l, 55 mmol / l, 60 mmol / l, 65 mmol / l, 70 mmol / l, 75 mmol / l, 80 mmol / l, 85 mmol / l, 90 mmol / l, 95 mmol / l, 100 mmol / l, 110 mmol / l, 120 mmol / l, 130 mmol / l, 140 mmol / l, 150 mmol / l, 160 mmol / l, 170 mmol / l, 180 mmol / l, 190 mmol / l, 200 mmol / l, 210 mmol / l, 190mmol / l, 200mmol / l, 210mmol / l, 220mmol / l, 230mmol / l, 240mmol / l, 250mmol / l, 260mmol / l, 270mmol / l, 280mmol / l, 290mmol / l, 300mmol / l, 310mmol / l, 320mmol / l, 330mmol / l, 340mmol / l, 350mmol / l, 360mmol / l, 370mmol / l, 380mmol / l, 390mmol / l, 400mmol / l, 410mmol / l, 420mmol / l, 430mmol / l, 440mmol / l, 450mmol / l, 460mmol / l, 470mmol / l, 480mmol / l, 490mmol / l, 500mmol / l or a concentration between any two of the values.

[0129] In an embodiment of the present application, the first solution, the second solution, and the fourth solution further contain single-stranded binding protein and / or bovine serum albumin. Single-stranded binding protein and / or bovine serum albumin can protect DNA polymerase and effectively promote the amplification of template molecules and reduce GC preference. The concentration of single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; the volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution is 0.05 to 2%. Illustratively, the concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution can be 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.75 mg / ml, 0.8 mg / ml, 0.85 mg / ml, 0.9 mg / ml, 0.95 mg / ml, 1 mg / ml or a concentration between any two values. The volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0130] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain 10 to 50 mmol / l of tetramethylammonium chloride or tetraethylammonium chloride. Although the inventors do not know the reason, they have found through research that tetramethylammonium chloride or tetraethylammonium chloride can promote the amplification of template molecules, thereby reducing GC preference. Exemplarily, the concentration of tetramethylammonium chloride or tetraethylammonium chloride can be 10 mmol / l, 20 mmol / l, 30 mmol / l, 40 mmol / l, 50 mmol / l, 60 mmol / l, or a concentration between any two values.

[0131] In the embodiment of the present application, the first solution, the second solution, and the fourth solution further contain a surfactant with a volume percentage concentration of 0.01 to 2%. Exemplarily, the surfactant is a nonionic surfactant, and the nonionic surfactant is selected from one or more of Tween 20, DD (dodecyl dimethyl amine oxide), DDM (dodecyl-β-D-maltoside) and TritonX-100 (polyethylene glycol octylphenyl ether).

[0132] In some embodiments, the concentration of the surfactant can be 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0133] In the embodiment of the present application, S160 is performed under variable temperature conditions, S120 and S140 are performed under constant temperature conditions, and the reaction temperature of S160 is not lower than the reaction temperatures of S120 and S140.

[0134] The inventors of the present application have found through research that temperature can affect the stability of the secondary structure formed by high GC content in the template molecule, and the higher the temperature, the easier it is to cause the secondary structure in the template molecule to melt. Based on this discovery, the present application introduces variable temperature amplification during the isothermal amplification process to cause the secondary structure formed by high GC content in the template molecule to melt, thereby facilitating the binding of the template molecule to the amplification primer and the extension of the amplification primer, improving the amplification efficiency and reducing the GC preference.

[0135] Specifically, S160 is performed under variable temperature conditions, including:

[0136] In S160, the reaction system of S160 is heated for the first time, so that the temperature of the reaction system of S160 is raised to a first temperature, and the first temperature is maintained for a first preset time;

[0137] After the first preset time, the reaction system of S160 is heated for the second time, so that the temperature of the reaction system of S160 is increased from the first temperature to the second temperature, and the second temperature is maintained for a second preset time.

[0138] Since the temperature at which the polymerase exhibits the best activity may be different from the temperature at which the secondary structure melts, the amplification may not be able to take into account both the activity of the polymerase and the effect of the melting of the secondary structure by using a single temperature. Therefore, the present application creatively proposes to adopt the above gradient heating to the reaction system of S160, which can balance the activity of the polymerase in the reaction system and the effect of the melting of the secondary structure formed in the template molecule. For example, it is assumed that the polymerase exhibits the best activity at the first temperature, but the secondary structure cannot be caused to melt well at this temperature. Therefore, it can be considered that after the first preset duration, the temperature of the reaction system is raised to the second temperature on the basis of the first temperature and the second preset duration of the second temperature is maintained. In this way, not only can the polymerase maintain a good activity to continue to play its function, but the secondary structure in the template molecule can be fully caused to open its double-stranded structure well, which is beneficial to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0139] In some embodiments, the first temperature is 60°C to 65°C, and the first preset time is 1 to 10s; and / or, the second temperature is 80°C to 85°C, and the second preset time is 1 to 10s. Exemplarily, the first temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or a temperature between any two values. The first preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values. The second temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The second preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0140] In some embodiments, the first heating rate and the second heating rate are 0.1 to 5.0°C / s. Exemplarily, the first heating rate and the second heating rate can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5 ℃ / s, 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0141] In some embodiments, S160 is performed under variable temperature conditions, including:

[0142] In S160, the reaction system of S160 is pulse-heated at least once, so that the temperature of the reaction system of S160 is raised to a third temperature; wherein the duration of one pulse is the third preset duration.

[0143] By adopting pulse heating, the hydrogen bonds between GC in the secondary structure formed in the template molecule can be more effectively destroyed, making the secondary structure easier to melt, which is beneficial to the binding of the template molecule to the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0144] In some embodiments, the third temperature is 60°C to 85°C, and the third preset time is 1 to 10s. The third temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The third preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0145] In some embodiments, the heating rate of pulse heating is 0.1-5.0°C / s. The heating rate of pulse heating can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5°C / s , 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0146] In some embodiments, after one pulse ends and before the next pulse is applied to the reaction system of S160, the temperature of the reaction system of S160 is restored to the temperature before the pulse heating. In this way, the hydrogen bonds between GCs in the secondary structure formed in the template molecule can be repeatedly destroyed, so that the secondary structure in the template molecule is completely opened, which is beneficial to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0147] In addition to the above amplification methods, Figure 1 and Figure 2 As shown, the present application also provides another amplification method, which comprises:

[0148] S200, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer.

[0149] Wherein, the surface of the solid phase carrier can be, for example, the surface of a sequencing chip, and illustratively, the surface of the solid phase carrier includes a number of nanopores arranged in an array. The size of the nanopore and the inter-hole spacing can be determined according to actual conditions. The first amplification primer and the second amplification primer are fixed on the inner surface of the nanopore by surface chemical modification, and the first amplification primer and the second amplification primer are, for example, single-stranded oligonucleotides, and the first amplification primer and the second amplification primer can be fixed on the surface of the solid phase carrier by means known in the art, such as covalent bonding or physical adsorption. The target polynucleotide can be, for example, a single-stranded nucleic acid molecule or a nucleic acid analog, and at least a portion of one end of the target polynucleotide is complementary hybridized with the first amplification primer in a base complementary pairing manner to form a hybrid complex, thereby fixing the target polynucleotide on the surface of the solid phase carrier. The first amplification primer complementary hybridized with the target polynucleotide can be used as the starting point for synthesizing the complementary chain of the target polynucleotide, and the synthesis of the complementary chain of the target polynucleotide is initiated under the action of DNA polymerase and under conditions suitable for polymerase chain reaction.

[0150] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0151] S400, using the target polynucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer.

[0152] Wherein, the first solution or the second solution contains the components required for extension, such as DNA polymerase, nucleotides (such as A, T, C, G) or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, with the target polynucleotide as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer, thereby extending the first amplification primer, and the extended chain obtained by the first amplification primer is the complementary chain of the target polynucleotide. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST and BSU. Preferably, the concentration of the DNA polymerase is 4U / ml.

[0153] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0154] S600, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent.

[0155] By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the target polynucleotide and its complementary chain, for example, to break the hydrogen bond between the target polynucleotide and its complementary chain, so that the double-stranded molecule is unzipped to form a single-stranded molecule to remove the target polynucleotide. Exemplarily, the denaturing agent can be, for example, a strong acid (such as HCl), a strong base (such as NaOH), urea, methanol, ethanol, an amide compound, guanidine hydrochloride, guanidine isothiocyanate, etc. Preferably, the denaturing agent is an amide compound, such as formamide.

[0156] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0157] S800, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer.

[0158] Wherein, the free end of the extended chain of the newly synthesized first amplification primer contains a part that can be complementary hybridized with the second amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize with the second amplification primer on the surface of the solid phase carrier, so as to extend the second amplification primer using the extended chain of the first amplification primer as a template to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer.

[0159] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0160] S1000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer to obtain the extended chain of the second amplification primer.

[0161] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, the nucleotides or nucleotide analogs are bound to the 3' end of the second amplification primer using the extended chain of the first amplification primer as a template, thereby extending the second amplification primer to obtain the extended chain of the second amplification primer, which is the complementary chain of the extended chain of the first amplification primer.

[0162] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0163] S1200, replacing the first solution or the second solution with the third solution.

[0164] The third solution contains a denaturing agent. By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the extended chain of the first amplification primer and the extended chain of the second amplification primer, for example, to break the hydrogen bond between the extended chain of the first amplification primer and the extended chain of the second amplification primer, so that the double-stranded molecule is unzipped to form a single-stranded molecule.

[0165] The amplification method provided in the embodiment of the present application further includes:

[0166] S1400, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively.

[0167] Wherein, the free end of the extended chain of the first amplification primer contains a part that can be complementary hybridized with the second amplification primer, and the free end of the extended chain of the second amplification primer contains a part that can be complementary hybridized with the first amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize and bind with the second amplification primer on the surface of the solid phase carrier, and the free end of the extended chain of the second amplification primer hybridize and bind with the first amplification primer on the surface of the solid phase carrier.

[0168] The amplification method provided in the embodiment of the present application further includes:

[0169] S1600, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates.

[0170] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, respectively:

[0171] Using the extended chain of the first amplification primer as a template, a nucleotide or a nucleotide analog is bound to the 3' end of the second amplification primer, and the second amplification primer is extended to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer, i.e., the target polynucleotide; and,

[0172] Using the extended chain of the second amplification primer as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer to extend the first amplification primer, thereby obtaining the extended chain of the first amplification primer, i.e., the complementary chain of the extended chain of the second amplification primer, i.e., the complementary chain of the target polynucleotide.

[0173] The amplification method provided in the embodiment of the present application further includes:

[0174] S1800, repeat S1200-S1600 at least once.

[0175] In this way, multiple target polynucleotides can be obtained, that is, a target polynucleotide molecular cluster is formed.

[0176] The amplification method provided in the present application performs a first round of amplification on the target polynucleotide through steps S200 to S400 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S600 to S1000 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates to perform cyclic amplification according to steps S1200 to 1600 to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by the incorporation of nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, thereby making it easier to detect the signal and improving the efficiency and accuracy of signal detection.

[0177] In the embodiment of the present application, S1600 is performed under variable temperature conditions, S1200 and S1400 are performed under constant temperature conditions, and the reaction temperature of S1600 is not lower than the reaction temperatures of S1200 and S1400.

[0178] The inventors of the present application have found through research that temperature can affect the stability of the secondary structure formed by high GC content in the template molecule, and the higher the temperature, the easier it is to cause the secondary structure in the template molecule to melt. Based on this discovery, the present application introduces variable temperature amplification during the isothermal amplification process to cause the secondary structure formed by high GC content in the template molecule to melt, thereby facilitating the binding of the template molecule to the amplification primer and the extension of the amplification primer, improving the amplification efficiency and reducing the GC preference.

[0179] Specifically, S1600 is performed under variable temperature conditions, including:

[0180] In S1600, the reaction system of S1600 is heated for the first time, so that the temperature of the reaction system of S1600 is raised to a first temperature, and the first temperature is maintained for a first preset time;

[0181] After the first preset time, the reaction system of S1600 is heated for the second time, so that the temperature of the reaction system of S1600 is increased from the first temperature to the second temperature, and the second temperature is maintained for a second preset time.

[0182] Since the temperature at which the polymerase exhibits the best activity may be different from the temperature at which the secondary structure melts, the amplification may not be able to take into account both the activity of the polymerase and the effect of the secondary structure melting. Therefore, the present application creatively proposes to adopt the above gradient heating to the reaction system of S1600, which can balance the activity of the polymerase in the reaction system and the effect of the melting of the secondary structure formed in the template molecule. For example, it is assumed that the polymerase exhibits the best activity at the first temperature, but the secondary structure cannot be melted well at this temperature. Therefore, it can be considered that after the first preset duration, the temperature of the reaction system is raised to the second temperature on the basis of the first temperature and the second preset duration of the second temperature is maintained. In this way, not only can the polymerase maintain a good activity to continue to play its function, but the secondary structure in the template molecule can be fully prompted to open its double-stranded structure well, which is conducive to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0183] In some embodiments, the first temperature is 60°C to 65°C, and the first preset time is 1 to 10s; and / or, the second temperature is 80°C to 85°C, and the second preset time is 1 to 10s. Exemplarily, the first temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or a temperature between any two values. The first preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values. The second temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The second preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0184] In some embodiments, the first heating rate and the second heating rate are 0.1 to 5.0°C / s. Exemplarily, the first heating rate and the second heating rate can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5 ℃ / s, 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0185] In some embodiments, S1600 is performed under variable temperature conditions, including:

[0186] In S1600, the reaction system of S1600 is pulse heated at least once, so that the temperature of the reaction system of S1600 is raised to a third temperature; wherein the duration of one pulse is the third preset duration.

[0187] By adopting pulse heating, the hydrogen bonds between GC in the secondary structure formed in the template molecule can be more effectively destroyed, making the secondary structure easier to melt, which is beneficial to the binding of the template molecule to the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0188] In some embodiments, the third temperature is 60°C to 85°C, and the third preset time is 1 to 10s. The third temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The third preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0189] In some embodiments, the heating rate of pulse heating is 0.1-5.0°C / s. The heating rate of pulse heating can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5°C / s , 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0190] In some embodiments, after one pulse ends and before the next pulse is applied to the reaction system of S1600, the temperature of the reaction system of S1600 is restored to the temperature before the pulse heating. In this way, the hydrogen bonds between GCs in the secondary structure formed in the template molecule can be repeatedly destroyed, so that the secondary structure in the template molecule is completely opened, which is beneficial to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0191] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain betaine, Mg 2+ NH 4 + , formamide, wherein the concentration of betaine is greater than 0 mol / L and less than or equal to 4 mol / L, Mg 2+ The concentration of NH 4 + The concentration of is greater than 0 mmol / L and less than or equal to 100 mmol / L, and the volume percentage concentration of formamide is less than or equal to 15%. The amplification method provided by the present application comprises configuring betaine with a concentration greater than 0 mol / L and less than or equal to 4 mol / L, Mg with a concentration of 0.5 to 4 mmol / L in the first solution, the second solution, and the fourth solution. 2+ , NH with a concentration greater than 0mmol / L and less than or equal to 100mmol / L 4 +, at least one of formamide with a volume percentage concentration less than or equal to 15%, so that the formation of secondary structure in the template molecule can be reduced, the amplification efficiency can be improved, and it is beneficial to the combination of the template molecule and the amplification primer and the extension of the amplification primer, thereby reducing the GC preference.

[0192] Specifically, the inventors of the present application have found through research that betaine can reduce the formation of secondary structures caused by GC enrichment in template molecules, improve the amplification efficiency of GC-rich sequences, and thus reduce GC preference. The inventors believe that this may be due to the fact that betaine can increase the hydration of guanine- and cytosine-rich regions in template molecules, affect the structure of template molecules, change their flexibility, and help DNA polymerases extend along template molecules, thereby reducing the formation of secondary structures within template molecules, improving amplification efficiency, and reducing GC preference. In addition, betaine can also reduce the melting temperature of sequences rich in GC content, which is conducive to more uniform denaturation of double-stranded molecules, reducing the melting stability differences caused by different GC contents, thereby reducing GC preference. Furthermore, betaine can stabilize the template molecule-protein complex, help maintain the stability of the template molecule during amplification, and reduce the polymerase dissociation caused by GC-rich regions. The inventors of the present application have also found through research that betaine plays the above-mentioned role or has the above-mentioned effect within a certain concentration range. If the concentration of betaine exceeds this range, amplification will be inhibited.

[0193] In some embodiments, the concentration of betaine in the first solution is greater than 0 mol / l and less than or equal to 3 mol / l. For example, the concentration of betaine in the first solution can be 0.5 mol / l, 1 mol / l, 1.5 mol / l, 2 mol / l, 2.5 mol / l, 3 mol / l, or a concentration between any two values.

[0194] In some embodiments, the concentration of betaine in the second solution may be 1.5 to 2.5 mol / l. For example, the concentration of betaine in the second solution may be 1.5 mol / l, 1.6 mol / l, 1.7 mol / l, 1.8 mol / l, 1.9 mol / l, 2.0 mol / l, 2.1 mol / l, 2.2 mol / l, 2.3 mol / l, 2.4 mol / l, 2.5 mol / l, or a concentration between any two values.

[0195] In some embodiments, the concentration of betaine in the fourth solution is 1.5-2.5 mol / l. For example, the concentration of betaine in the fourth solution can be 1.5 mol / l, 1.6 mol / l, 1.7 mol / l, 1.8 mol / l, 1.9 mol / l, 2.0 mol / l, 2.1 mol / l, 2.2 mol / l, 2.3 mol / l, 2.4 mol / l, 2.5 mol / l, or a concentration between any two values.

[0196] In some embodiments, Mg 2+ The concentration can be 0.5mmol / L, 1mmol / L, 1.5mmol / L, 2mmol / L, 2.5mmol / L, 3mmol / L, 3.5mmol / L, 4mmol / L or a concentration between the above two values.

[0197] The inventor of the present application has also found through research that Mg 2+ It can be used as an active auxiliary agent of polymerase, which helps to activate the activity of polymerase, so that the polymerase catalyzes the formation of a phosphodiester bond between the 3'-OH of the amplification primer bound to the template molecule and the phosphate group of the nucleotide or nucleotide analog, thereby combining the nucleotide or nucleotide analog with the amplification primer, and making the nucleotide or nucleotide analog bind to the template molecule in a complementary base pairing manner, that is, incorporating the nucleotide or nucleotide analog into the template molecule, thereby improving the amplification efficiency and reducing the GC preference, thereby helping to reduce the intensity difference of the signal generated by the high AT and high GC target polynucleotide molecular clusters during the sequencing process, and improving the accuracy of sequencing. In addition, Mg 2+ The concentration of Mg is also critical to the amplification process. 2+ If the concentration is too low, amplification will not proceed, and if it is too high, base mismatches will increase during amplification. 2+ concentration.

[0198] In some embodiments, in S400, the first amplification primer is extended using the first solution, Mg 2+ The concentration of Mg in the first solution is 2-4 mmol / l, and the concentration of betaine in the first solution is 0.1-2 mol / l. 2+ Cooperating with betaine at a lower concentration can improve amplification efficiency, reduce GC preference, and facilitate extension of the first amplification primer to generate an extended chain of the first amplification primer.

[0199] In S1000 or in S1600, the fourth solution is replaced by the second solution, Mg 2+The concentration of Mg in the second solution is 2-4 mmol / l, and the concentration of betaine in the second solution is 0.1-2 mol / l; or, in S1000 or S1600, the fourth solution is replaced by the second solution, Mg 2+ The concentration of betaine in the second solution is 1-1.5 mmol / l, and the concentration of betaine in the second solution is 2-3 mol / l.

[0200] The inventors of the present application have found that, during the extension process of the first amplification primer and / or the second amplification primer of S1000 or S1600, the use of Mg containing 2 to 4 mmol / l 2+ and 0.1-2 mol / l of betaine as the second solution to replace the fourth solution, or using 1-1.5 mmol / l of Mg 2+ The invention speculates that this may be due to the fact that a higher concentration of Mg in S400 is used to replace the fourth solution with a second solution of 2-3 mol / l betaine, which can effectively extend the first amplification primer and / or the second amplification primer to generate an extended chain of the first amplification primer and / or an extended chain of the second amplification primer. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0201] In some embodiments, in S400, the first amplification primer is extended using the second solution, Mg 2+ The concentration of Mg in the second solution is 2-4 mmol / l, and the concentration of betaine in the second solution is 0.1-2 mol / l. 2+ Cooperating with betaine at a lower concentration can improve amplification efficiency, reduce GC preference, and facilitate extension of the first amplification primer to generate an extended chain of the first amplification primer.

[0202] In S1000 or S1600, the fourth solution is replaced by the first solution, Mg 2+ The concentration of Mg in the first solution is 2-4 mmol / l, and the concentration of betaine in the first solution is 0.1-2 mol / l; or, in S1000 or S1600, the first solution is used to replace the fourth solution, Mg 2+ The concentration of betaine in the first solution is 1-1.5 mmol / l, and the concentration of betaine in the first solution is 2-3 mol / l.

[0203] The inventors of the present application have found that, during the extension process of the first amplification primer and / or the second amplification primer of S1000 or S1600, the use of Mg containing 2 to 4 mmol / l 2+and 0.1-2 mol / l of betaine in the first solution to replace the fourth solution, or use 1-1.5 mmol / l of Mg 2+ The inventors speculate that this may be due to the fact that a higher concentration of Mg in S40 is used to replace the fourth solution with the first solution of 2-3 mol / l betaine. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0204] In some embodiments, in S400, the first amplification primer is extended using the first solution, Mg 2+ The concentration of betaine in the first solution is 2-4 mmol / l, the concentration of betaine in the first solution is 0.1-2 mol / l, and in S1000 or S1600, the fourth solution is replaced by the second solution, Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; or,

[0205] In S400, the first amplification primer is extended using the second solution, Mg 2+ The concentration of betaine in the second solution is 2-4 mmol / l, the concentration of betaine in the second solution is 0.1-2 mol / l, and in S1000 or S1600, the fourth solution is replaced by the first solution, Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l;

[0206] The first solution or the second solution also contains dNTPs, and dNTPs includes dATP, dTTP, dCTP, and ddTP, and the sum of the contents of dATP and dTTP is equal to the sum of the contents of dGTP and dCTP. The amplification primer is extended using the first solution or the second solution, and in particular, in S40, a base solution having a balanced ratio, i.e., the sum of the contents of dATP and dTTP is equal to the sum of the contents of dGTP and dCTP, is configured in the first solution or the second solution, which is conducive to amplifying all target polynucleotide molecules as much as possible. Exemplarily, dATP, dTTP, dCTP, and ddTP can exist in equal concentrations, for example, the concentrations of dATP, dTTP, dCTP, and ddTP can all be 200 μmol / l, 100 μmol / l, 50 μmol / l, 10 μmol / l, 5 μmol / l, 1 μmol / l, etc.

[0207] In some embodiments, in S400, the first amplification primer is extended using the first solution, Mg 2+ The concentration of betaine in the first solution is 2-4 mmol / l, the concentration of betaine in the first solution is 0.1-2 mol / l, and in S1000 or S1600, the fourth solution is replaced by the second solution, Mg 2+ The concentration of the betaine in the second solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the second solution is 2 to 3 mol / l; or,

[0208] In S400, the first amplification primer is extended using the second solution, Mg 2+ The concentration of betaine in the second solution is 2-4 mmol / l, the concentration of betaine in the second solution is 0.1-2 mol / l, and in S1000 or S1600, the fourth solution is replaced by the first solution, Mg 2+ The concentration of the betaine in the first solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the first solution is 2 to 3 mol / l;

[0209] The second solution or the first solution also contains dNTPs, and the dNTPs include dATP, dTTP, dCTP, and ddTP, and the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP. The first solution or the second solution is used to extend the amplification primer, especially in S1000 or in S1600, the first solution or the second solution is configured with a base solution with an unbalanced ratio, that is, the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP, which is conducive to increasing the probability of complementary pairing of dGTP and dCTP with GC bases on the template molecule, preferentially amplifying sequences with high GC content, thereby enhancing the amplification of GC bases in the template molecule, improving the amplification efficiency, and reducing GC preference. Exemplarily, the sum of the contents of dATP and dTTP is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, etc. of the sum of the contents of dGTP and dCTP.

[0210] The inventor of the present application has also found through research that NH 4 + It can improve the reaction activity of polymerase, thereby improving amplification efficiency and reducing GC preference. The inventors speculate that NH 4 + Improving the polymerase activity may be related to NH 4 +It is related to the ability to change the charge distribution on the surface of the polymerase and / or change the spatial morphology of the polymerase, making it easier for the polymerase to bind to the reaction site of the template molecule and / or the amplification primer, catalyzing the formation of a phosphodiester bond between the 3'-OH of the amplification primer and the phosphate group of the nucleotide or nucleotide analogue, thereby combining the nucleotide or nucleotide analogue with the amplification primer, and allowing the nucleotide or nucleotide analogue to bind to the nucleic acid template in a base complementary pairing manner, that is, incorporating the nucleotide or nucleotide analogue into the template molecule.

[0211] In some embodiments, NH 4 + The concentration can be 0.1mmol / L, 1mmol / L, 5mmol / L, 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or a concentration between any two values.

[0212] In some embodiments, NH 4 + NH provided by at least one of ammonium sulfate, ammonium chloride and ammonium acetate 4 + That is to say, the first solution, the second solution, and the fourth solution of the present application all contain at least one of ammonium sulfate, ammonium chloride, and ammonium acetate, or at least one of ammonium sulfate, ammonium chloride, and ammonium acetate is added when the first solution, the second solution, and the fourth solution are prepared.

[0213] In addition, the inventors of the present application have also found that formamide can not only be used as a denaturing agent, but also can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. This difference in effect is mainly related to the concentration of formamide in the solution. For example, when the volume percentage concentration of formamide reaches or exceeds 60%, such as 60-100%, it is used as a denaturing agent, and when the volume percentage concentration of formamide is greater than or equal to 3% and less than or equal to 15%, then formamide can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. Therefore, by controlling the concentration of formamide, formamide can be controlled to play different roles and achieve different purposes.

[0214] In some embodiments, the volume percentage concentration of formamide in the first solution, the second solution, and the fourth solution can be 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15% or a volume percentage concentration between any two values.

[0215] In the embodiments of the present application, the first solution, the second solution and the fourth solution all contain DMSO (dimethyl sulfoxide), and the volume percentage concentration of DMSO in the first solution, the second solution and the fourth solution is 1-5%.

[0216] Since sequences with high GC content in template molecules are often difficult to amplify due to their easy formation of secondary structures, DMSO can improve the spatial morphology of template molecules with high GC content, reduce the formation of secondary structures, and enable the polymerase to extend along the template molecules, thereby improving the amplification efficiency and reducing GC preference.

[0217] In some embodiments, the volume percentage concentration of DMSO in the first solution, the second solution, and the fourth solution can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a concentration between any two values.

[0218] In the embodiment of the present application, the first solution, the second solution and the fourth solution further contain Trisbase (tris(hydroxymethyl)aminomethane), and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10-500 mmol / l. Among them, Trisbase can provide a suitable buffer environment and ionic strength for amplification.

[0219] In some embodiments, the concentration of Trisbase in the first solution, the second solution, and the fourth solution can be 10 mmol / l, 15 mmol / l, 20 mmol / l, 25 mmol / l, 30 mmol / l, 35 mmol / l, 40 mmol / l, 45 mmol / l, 50 mmol / l, 55 mmol / l, 60 mmol / l, 65 mmol / l, 70 mmol / l, 75 mmol / l, 80 mmol / l, 85 mmol / l, 90 mmol / l, 95 mmol / l, 100 mmol / l, 110 mmol / l, 120 mmol / l, 130 mmol / l, 140 mmol / l, 150 mmol / l, 160 mmol / l, 170 mmol / l, 180 mmol / l, 190 mmol / l, 200 mmol / l, 210 mmol / l, 190mmol / l, 200mmol / l, 210mmol / l, 220mmol / l, 230mmol / l, 240mmol / l, 250mmol / l, 260mmol / l, 270mmol / l, 280mmol / l, 290mmol / l, 300mmol / l, 310mmol / l, 320mmol / l, 330mmol / l, 340mmol / l, 350mmol / l, 360mmol / l, 370mmol / l, 380mmol / l, 390mmol / l, 400mmol / l, 410mmol / l, 420mmol / l, 430mmol / l, 440mmol / l, 450mmol / l, 460mmol / l, 470mmol / l, 480mmol / l, 490mmol / l, 500mmol / l or a concentration between any two of the values.

[0220] In an embodiment of the present application, the first solution, the second solution, and the fourth solution further contain single-stranded binding protein and / or bovine serum albumin. Single-stranded binding protein and / or bovine serum albumin can protect DNA polymerase and effectively promote the amplification of template molecules and reduce GC preference. The concentration of single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; the volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution is 0.05 to 2%. Illustratively, the concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution can be 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.75 mg / ml, 0.8 mg / ml, 0.85 mg / ml, 0.9 mg / ml, 0.95 mg / ml, 1 mg / ml or a concentration between any two values. The volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0221] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain 10 to 50 mmol / l of tetramethylammonium chloride or tetraethylammonium chloride. Although the inventors do not know the reason, they have found through research that tetramethylammonium chloride or tetraethylammonium chloride can promote the amplification of template molecules, thereby reducing GC preference. Exemplarily, the concentration of tetramethylammonium chloride or tetraethylammonium chloride can be 10 mmol / l, 20 mmol / l, 30 mmol / l, 40 mmol / l, 50 mmol / l, 60 mmol / l, or a concentration between any two values.

[0222] In the embodiment of the present application, the first solution, the second solution, and the fourth solution further contain a surfactant with a volume percentage concentration of 0.01 to 2%. Exemplarily, the surfactant is a nonionic surfactant, and the nonionic surfactant is selected from one or more of Tween 20, DD (dodecyl dimethyl amine oxide), DDM (dodecyl-β-D-maltoside) and TritonX-100 (polyethylene glycol octylphenyl ether).

[0223] In some embodiments, the concentration of the surfactant can be 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0224] In addition to the above two amplification methods, Figure 1 and Figure 2 As shown, the embodiment of the present application also provides a third amplification method, which comprises:

[0225] S2000, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer.

[0226] Wherein, the surface of the solid phase carrier can be, for example, the surface of a sequencing chip, and illustratively, the surface of the solid phase carrier includes a number of nanopores arranged in an array. The size of the nanopore and the inter-hole spacing can be determined according to actual conditions. The first amplification primer and the second amplification primer are fixed on the inner surface of the nanopore by surface chemical modification, and the first amplification primer and the second amplification primer are, for example, single-stranded oligonucleotides, and the first amplification primer and the second amplification primer can be fixed on the surface of the solid phase carrier by means known in the art, such as covalent bonding or physical adsorption. The target polynucleotide can be, for example, a single-stranded nucleic acid molecule or a nucleic acid analog, and at least a portion of one end of the target polynucleotide is complementary hybridized with the first amplification primer in a base complementary pairing manner to form a hybrid complex, thereby fixing the target polynucleotide on the surface of the solid phase carrier. The first amplification primer complementary hybridized with the target polynucleotide can be used as the starting point for synthesizing the complementary chain of the target polynucleotide, and the synthesis of the complementary chain of the target polynucleotide is initiated under the action of DNA polymerase and under conditions suitable for polymerase chain reaction.

[0227] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0228] S4000, using the target polynucleotide as a template, using the first solution or the second solution to extend the first amplification primer to obtain an extended chain of the first amplification primer.

[0229] Wherein, the first solution or the second solution contains the components required for extension, such as DNA polymerase, nucleotides (such as A, T, C, G) or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, with the target polynucleotide as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer, thereby extending the first amplification primer, and the extended chain obtained by the first amplification primer is the complementary chain of the target polynucleotide. Exemplarily, the DNA polymerase is selected from one or more of rTaq, Canace, Pfu, KOD, Phusion, Primerstar, Tth, BST and BSU. Preferably, the concentration of the DNA polymerase is 4U / ml.

[0230] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0231] S6000, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent.

[0232] By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the target polynucleotide and its complementary chain, for example, to break the hydrogen bond between the target polynucleotide and its complementary chain, so that the double-stranded molecule is unzipped to form a single-stranded molecule to remove the target polynucleotide. Exemplarily, the denaturing agent can be, for example, a strong acid (such as HCl), a strong base (such as NaOH), urea, methanol, ethanol, an amide compound, guanidine hydrochloride, guanidine isothiocyanate, etc. Preferably, the denaturing agent is an amide compound, such as formamide.

[0233] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0234] S8000, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer.

[0235] Wherein, the free end of the extended chain of the newly synthesized first amplification primer contains a part that can be complementary hybridized with the second amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize with the second amplification primer on the surface of the solid phase carrier, so as to extend the second amplification primer using the extended chain of the first amplification primer as a template to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer.

[0236] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0237] S10000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer to obtain the extended chain of the second amplification primer.

[0238] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, the nucleotides or nucleotide analogs are bound to the 3' end of the second amplification primer using the extended chain of the first amplification primer as a template, thereby extending the second amplification primer to obtain the extended chain of the second amplification primer, which is the complementary chain of the extended chain of the first amplification primer.

[0239] like Figure 1 and Figure 2 As shown, the amplification method provided in the embodiment of the present application further includes:

[0240] S12000, replacing the first solution or the second solution with the third solution.

[0241] The third solution contains a denaturing agent. By replacing the first solution or the second solution after the reaction with the third solution, the denaturing agent in the third solution can be used to weaken or destroy the molecular force between the extended chain of the first amplification primer and the extended chain of the second amplification primer, for example, to break the hydrogen bond between the extended chain of the first amplification primer and the extended chain of the second amplification primer, so that the double-stranded molecule is unzipped to form a single-stranded molecule.

[0242] The amplification method provided in the embodiment of the present application further includes:

[0243] S14000, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively.

[0244] Wherein, the free end of the extended chain of the first amplification primer contains a part that can be complementary hybridized with the second amplification primer, and the free end of the extended chain of the second amplification primer contains a part that can be complementary hybridized with the first amplification primer. The fourth solution contains components required for hybridization. Exemplarily, the fourth solution can be the first solution or the second solution that does not contain DNA polymerase and nucleotides or nucleotide analogs. Replacing the third solution with the fourth solution can make the free end of the extended chain of the first amplification primer hybridize and bind with the second amplification primer on the surface of the solid phase carrier, and the free end of the extended chain of the second amplification primer hybridize and bind with the first amplification primer on the surface of the solid phase carrier.

[0245] The amplification method provided in the embodiment of the present application further includes:

[0246] S16000, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates.

[0247] The first solution or the second solution contains components required for extension, such as DNA polymerase, nucleotides or nucleotide analogs, etc. Under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, respectively:

[0248] Using the extended chain of the first amplification primer as a template, a nucleotide or a nucleotide analog is bound to the 3' end of the second amplification primer, and the second amplification primer is extended to obtain the extended chain of the second amplification primer, i.e., the complementary chain of the extended chain of the first amplification primer, i.e., the target polynucleotide; and,

[0249] Using the extended chain of the second amplification primer as a template, nucleotides or nucleotide analogs are bound to the 3' end of the first amplification primer to extend the first amplification primer, thereby obtaining the extended chain of the first amplification primer, i.e., the complementary chain of the extended chain of the second amplification primer, i.e., the complementary chain of the target polynucleotide.

[0250] The amplification method provided in the embodiment of the present application further includes:

[0251] S18000, repeat S12000-S16000 at least once.

[0252] In this way, multiple target polynucleotides can be obtained, that is, a target polynucleotide molecular cluster is formed.

[0253] The amplification method provided in the present application performs a first round of amplification on the target polynucleotide through steps S2000 to S4000 to generate an extended chain of the first amplification primer on the surface of the solid phase carrier; then amplifies the extended chain of the first amplification primer through steps S6000 to S10000 to generate an extended chain of the second amplification primer on the surface of the solid phase carrier; then uses the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates to perform cyclic amplification according to steps S12000 to 16000 to obtain a molecular cluster of the target polynucleotide, so as to amplify the signal generated by the incorporation of nucleotides or nucleotide analogs into the target polynucleotide during the sequencing of the target polynucleotide, thereby making it easier to detect the signal and improving the efficiency and accuracy of signal detection.

[0254] In the embodiment of the present application, in S10000 or S16000, the first solution or the second solution contains dATP, dTTP, dCTP and ddTP, and the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP.

[0255] The amplification primer is extended by using the first solution or the second solution, especially in S10000 or S16000, a base solution with an unbalanced ratio, i.e., the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP, is configured in the first solution or the second solution, which is conducive to increasing the probability of complementary pairing between dGTP and dCTP and the GC base on the template molecule, preferentially amplifying sequences with high GC content, thereby enhancing the amplification of GC bases in the template molecule, improving the amplification efficiency, and reducing GC preference. Exemplarily, the sum of the contents of dATP and dTTP is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, etc. of the sum of the contents of dGTP and dCTP.

[0256] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain betaine, Mg 2+ NH 4 + , formamide, wherein the concentration of betaine is greater than 0 mol / L and less than or equal to 4 mol / L, Mg 2+ The concentration of NH 4 + The concentration of is greater than 0 mmol / L and less than or equal to 100 mmol / L, and the volume percentage concentration of formamide is less than or equal to 15%. The amplification method provided by the present application comprises configuring betaine with a concentration greater than 0 mol / L and less than or equal to 4 mol / L, Mg with a concentration of 0.5 to 4 mmol / L in the first solution, the second solution, and the fourth solution. 2+, NH with a concentration greater than 0mmol / L and less than or equal to 100mmol / L 4 + , at least one of formamide with a volume percentage concentration less than or equal to 15%, so that the formation of secondary structure in the template molecule can be reduced, the amplification efficiency can be improved, and it is beneficial to the combination of the template molecule and the amplification primer and the extension of the amplification primer, thereby reducing the GC preference.

[0257] The inventors of the present application have found through research that betaine can reduce the formation of secondary structures caused by GC enrichment in template molecules, improve the amplification efficiency of GC-rich sequences, and thus reduce GC preference. The inventors believe that this may be due to the fact that betaine can increase the hydration of guanine- and cytosine-rich regions in template molecules, affect the structure of template molecules, change their flexibility, and help DNA polymerase extend along template molecules, thereby reducing the formation of secondary structures in template molecules, improving amplification efficiency, and reducing GC preference. In addition, betaine can also reduce the melting temperature of sequences rich in GC content, which is conducive to more uniform denaturation of double-stranded molecules, reducing the melting stability differences caused by different GC contents, thereby reducing GC preference. Furthermore, betaine can stabilize the template molecule-protein complex, help maintain the stability of the template molecule during amplification, and reduce the polymerase dissociation caused by GC-rich regions. The inventors of the present application have also found through research that betaine plays the above-mentioned role or has the above-mentioned effect within a certain concentration range. If the concentration of betaine exceeds this range, the amplification will be inhibited.

[0258] Mg 2+ It can be used as an active auxiliary agent of polymerase, which helps to activate the activity of polymerase, so that the polymerase catalyzes the formation of a phosphodiester bond between the 3'-OH of the amplification primer bound to the template molecule and the phosphate group of the nucleotide or nucleotide analog, thereby combining the nucleotide or nucleotide analog with the amplification primer, and making the nucleotide or nucleotide analog bind to the template molecule in a complementary base pairing manner, that is, incorporating the nucleotide or nucleotide analog into the template molecule, thereby improving the amplification efficiency and reducing the GC preference, thereby helping to reduce the intensity difference of the signal generated by the high AT and high GC target polynucleotide molecular clusters during the sequencing process, and improving the accuracy of sequencing. In addition, Mg 2+ The concentration of Mg is also critical to the amplification process. 2+ If the concentration is too low, amplification will not proceed, and if it is too high, base mismatches will increase during amplification. 2+ concentration.

[0259] In some embodiments, in S4000, the first amplification primer is extended using the first solution, and in S10000 and S16000, the fourth solution is replaced by the first solution or the second solution, wherein the Mg in the first solution is 2+ The concentration of Mg in the second solution is 2-4 mmol / l, the concentration of betaine is 0.1-2 mol / l, and the concentration of Mg in the second solution is 0.1-2 mol / l. 2+ The concentration of Mg is 2-4 mmol / l, and the concentration of betaine is 0.1-2 mol / l. Alternatively, in S4000, the first amplification primer is extended using the first solution, and in S10000 and S16000, the fourth solution is replaced by the first solution or the second solution, and the Mg content in the first solution is 0.1-2 mol / l. 2+ The concentration of Mg in the second solution is 2-4 mmol / l, the concentration of betaine is 0.1-2 mol / l, and the concentration of Mg in the second solution is 0.1-2 mol / l. 2+ The concentration of is 1-1.5 mmol / l, and the concentration of betaine is 2-3 mol / l.

[0260] In S4000, the first amplification primer is extended using the first solution, Mg 2+ The concentration of Mg in the first solution is 2-4 mmol / l, and the concentration of betaine in the first solution is 0.1-2 mol / l. 2+ The combination of betaine with a lower concentration can improve the amplification efficiency, reduce GC preference, and facilitate the extension of the first amplification primer to generate an extended chain of the first amplification primer. In the extension process of the first amplification primer and / or the second amplification primer of S10000 or S16000, Mg-containing 2-4 mmol / l 2+ and 0.1-2 mol / l of betaine in the first solution or the second solution to replace the fourth solution, or using 1-1.5 mmol / l of Mg 2+ The first solution or the second solution containing 2 to 3 mol / l betaine can effectively extend the first amplification primer and / or the second amplification primer to generate an extended chain of the first amplification primer and / or the extended chain of the second amplification primer. The inventors speculate that this may be due to the use of a higher concentration of Mg in the first round of amplification. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0261] In some embodiments, in S4000, the first amplification primer is extended using the second solution, Mg 2+ The concentration of Mg in the second solution is 2-4 mmol / l, and the concentration of betaine in the second solution is 0.1-2 mol / l. 2+Cooperating with betaine at a lower concentration can improve amplification efficiency, reduce GC preference, and facilitate extension of the first amplification primer to generate an extended chain of the first amplification primer.

[0262] In S10000 or S16000, the fourth solution is replaced by the first solution or the second solution, Mg 2+ The concentration of betaine in the first solution or the second solution is 2-4 mmol / l, and the concentration of betaine in the first solution or the second solution is 0.1-2 mol / l; or, in S10000 or S16000, the first solution or the second solution is used to replace the fourth solution, Mg 2+ The concentration of betaine in the first solution or the second solution is 1-1.5 mmol / l, and the concentration of betaine in the first solution or the second solution is 2-3 mol / l.

[0263] The inventors of the present application have found that, during the extension process of the first amplification primer and / or the second amplification primer of S10000 or S16000, the use of Mg containing 2 to 4 mmol / l 2+ and 0.1-2 mol / l of betaine in the first solution or the second solution to replace the fourth solution, or using 1-1.5 mmol / l of Mg 2+ The first solution or the second solution containing 2 to 3 mol / l betaine can effectively extend the first amplification primer and / or the second amplification primer to generate an extended chain of the first amplification primer and / or the extended chain of the second amplification primer. The inventors speculate that this may be due to the use of a higher concentration of Mg in the first round of amplification. 2+ This is related to the fact that the lower concentration of betaine cooperates to improve the amplification efficiency and reduce the GC preference, thereby providing more feasible solutions for the subsequent amplification steps.

[0264] In some embodiments, in S4000 , the first solution or the second solution contains dATP, dTTP, dCTP, and ddTP, and the sum of the contents of dATP and dTTP is equal to the sum of the contents of dGTP and dCTP.

[0265] The amplification primer is extended by using the first solution or the second solution, and in particular, in S4000, a base solution having a balanced ratio, i.e., a base solution having a content of dATP and dTTP equal to a content of dGTP and dCTP, is configured in the first solution or the second solution, which is conducive to amplifying all target polynucleotide molecules as much as possible. Exemplarily, dATP, dTTP, dCTP, and ddTP can be present in equal concentrations, for example, the concentrations of dATP, dTTP, dCTP, and ddTP can all be 200 μmol / l, 100 μmol / l, 50 μmol / l, 10 μmol / l, 5 μmol / l, 1 μmol / l, etc.

[0266] In some embodiments, the concentration of betaine in the fourth solution is 1.5-2.5 mol / l. For example, the concentration of betaine in the fourth solution can be 1.5 mol / l, 1.6 mol / l, 1.7 mol / l, 1.8 mol / l, 1.9 mol / l, 2.0 mol / l, 2.1 mol / l, 2.2 mol / l, 2.3 mol / l, 2.4 mol / l, 2.5 mol / l, or a concentration between any two values.

[0267] The inventor of the present application has also found through research that NH 4 + It can improve the reaction activity of polymerase, thereby improving amplification efficiency and reducing GC preference. The inventors speculate that NH 4 + Improving the polymerase activity may be related to NH 4 + It is related to the ability to change the charge distribution on the surface of the polymerase and / or change the spatial morphology of the polymerase, making it easier for the polymerase to bind to the reaction site of the template molecule and / or the amplification primer, catalyzing the formation of a phosphodiester bond between the 3'-OH of the amplification primer and the phosphate group of the nucleotide or nucleotide analogue, thereby combining the nucleotide or nucleotide analogue with the amplification primer, and allowing the nucleotide or nucleotide analogue to bind to the nucleic acid template in a base complementary pairing manner, that is, incorporating the nucleotide or nucleotide analogue into the template molecule.

[0268] In some embodiments, NH 4 + The concentration can be 0.1mmol / L, 1mmol / L, 5mmol / L, 10mmol / L, 20mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or a concentration between any two values.

[0269] In some embodiments, NH 4 + NH provided by at least one of ammonium sulfate, ammonium chloride and ammonium acetate 4 + That is to say, the first solution, the second solution, and the fourth solution of the present application all contain at least one of ammonium sulfate, ammonium chloride, and ammonium acetate, or at least one of ammonium sulfate, ammonium chloride, and ammonium acetate is added when the first solution, the second solution, and the fourth solution are prepared.

[0270] In addition, the inventors of the present application have also found that formamide can not only be used as a denaturing agent, but also can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. This difference in effect is mainly related to the concentration of formamide in the solution. For example, when the volume percentage concentration of formamide reaches or exceeds 60%, such as 60-100%, it is used as a denaturing agent, and when the volume percentage concentration of formamide is greater than or equal to 3% and less than or equal to 15%, then formamide can reduce the stability of double-stranded molecules in the secondary structure of the template molecule, destroy the hydrogen bonds between the double-stranded molecules, melt the double-stranded molecules at a lower temperature and help the template molecule to bind to the amplification primer, which is beneficial to the amplification, thereby improving the amplification efficiency and reducing the GC preference. Therefore, by controlling the concentration of formamide, formamide can be controlled to play different roles and achieve different purposes.

[0271] In some embodiments, the volume percentage concentration of formamide in the first solution, the second solution, and the fourth solution can be 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15% or a volume percentage concentration between any two values.

[0272] In the embodiments of the present application, the first solution, the second solution and the fourth solution all contain DMSO (dimethyl sulfoxide), and the volume percentage concentration of DMSO in the first solution, the second solution and the fourth solution is 1-5%.

[0273] Since sequences with high GC content in template molecules are often difficult to amplify due to their easy formation of secondary structures, DMSO can improve the spatial morphology of template molecules with high GC content, reduce the formation of secondary structures, and enable the polymerase to extend along the template molecules, thereby improving the amplification efficiency and reducing GC preference.

[0274] In some embodiments, the volume percentage concentration of DMSO in the first solution, the second solution, and the fourth solution can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a concentration between any two values.

[0275] In the embodiment of the present application, the first solution, the second solution and the fourth solution further contain Trisbase (tris(hydroxymethyl)aminomethane), and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10-500 mmol / l. Among them, Trisbase can provide a suitable buffer environment and ionic strength for amplification.

[0276] In some embodiments, the concentration of Trisbase in the first solution, the second solution, and the fourth solution can be 10 mmol / l, 15 mmol / l, 20 mmol / l, 25 mmol / l, 30 mmol / l, 35 mmol / l, 40 mmol / l, 45 mmol / l, 50 mmol / l, 55 mmol / l, 60 mmol / l, 65 mmol / l, 70 mmol / l, 75 mmol / l, 80 mmol / l, 85 mmol / l, 90 mmol / l, 95 mmol / l, 100 mmol / l, 110 mmol / l, 120 mmol / l, 130 mmol / l, 140 mmol / l, 150 mmol / l, 160 mmol / l, 170 mmol / l, 180 mmol / l, 190 mmol / l, 200 mmol / l, 210 mmol / l, 190mmol / l, 200mmol / l, 210mmol / l, 220mmol / l, 230mmol / l, 240mmol / l, 250mmol / l, 260mmol / l, 270mmol / l, 280mmol / l, 290mmol / l, 300mmol / l, 310mmol / l, 320mmol / l, 330mmol / l, 340mmol / l, 350mmol / l, 360mmol / l, 370mmol / l, 380mmol / l, 390mmol / l, 400mmol / l, 410mmol / l, 420mmol / l, 430mmol / l, 440mmol / l, 450mmol / l, 460mmol / l, 470mmol / l, 480mmol / l, 490mmol / l, 500mmol / l or a concentration between any two of the values.

[0277] In an embodiment of the present application, the first solution, the second solution, and the fourth solution further contain single-stranded binding protein and / or bovine serum albumin. Single-stranded binding protein and / or bovine serum albumin can protect DNA polymerase and effectively promote the amplification of template molecules and reduce GC preference. The concentration of single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; the volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution is 0.05 to 2%. Illustratively, the concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution can be 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.75 mg / ml, 0.8 mg / ml, 0.85 mg / ml, 0.9 mg / ml, 0.95 mg / ml, 1 mg / ml or a concentration between any two values. The volume percentage concentration of bovine serum albumin in the first solution, the second solution, and the fourth solution can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0278] In the embodiment of the present application, the first solution, the second solution, and the fourth solution also contain 10 to 50 mmol / l of tetramethylammonium chloride or tetraethylammonium chloride. Although the inventors do not know the reason, they have found through research that tetramethylammonium chloride or tetraethylammonium chloride can promote the amplification of template molecules, thereby reducing GC preference. Exemplarily, the concentration of tetramethylammonium chloride or tetraethylammonium chloride can be 10 mmol / l, 20 mmol / l, 30 mmol / l, 40 mmol / l, 50 mmol / l, 60 mmol / l, or a concentration between any two values.

[0279] In the embodiment of the present application, the first solution, the second solution, and the fourth solution further contain a surfactant with a volume percentage concentration of 0.01 to 2%. Exemplarily, the surfactant is a nonionic surfactant, and the nonionic surfactant is selected from one or more of Tween 20, DD (dodecyl dimethyl amine oxide), DDM (dodecyl-β-D-maltoside) and TritonX-100 (polyethylene glycol octylphenyl ether).

[0280] In some embodiments, the concentration of the surfactant can be 0.01%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2%, or a concentration between any two values.

[0281] In the embodiments of the present application, S16000 is carried out under variable temperature conditions, S12000 and S14000 are carried out under constant temperature conditions, and the reaction temperature of S16000 is not lower than the reaction temperatures of S12000 and S14000.

[0282] The inventors of the present application have found through research that temperature can affect the stability of the secondary structure formed by high GC content in the template molecule, and the higher the temperature, the easier it is to cause the secondary structure in the template molecule to melt. Based on this discovery, the present application introduces variable temperature amplification during the isothermal amplification process to cause the secondary structure formed by high GC content in the template molecule to melt, thereby facilitating the binding of the template molecule to the amplification primer and the extension of the amplification primer, improving the amplification efficiency and reducing the GC preference.

[0283] Specifically, S16000 is performed under variable temperature conditions, including:

[0284] In S16000, the reaction system of S16000 is heated for the first time, so that the temperature of the reaction system of S16000 is raised to a first temperature, and the first temperature is maintained for a first preset time;

[0285] After the first preset time, the reaction system of S16000 is heated for the second time, so that the temperature of the reaction system of S16000 is increased from the first temperature to the second temperature, and the second temperature is maintained for a second preset time.

[0286] Since the temperature at which the polymerase exhibits the best activity may be different from the temperature at which the secondary structure melts, the amplification may not be able to take into account both the activity of the polymerase and the effect of the secondary structure melting. Therefore, the present application creatively proposes to adopt the above gradient heating to the reaction system of S16000, which can balance the activity of the polymerase in the reaction system and the effect of the melting of the secondary structure formed in the template molecule. For example, it is assumed that the polymerase exhibits the best activity at the first temperature, but the secondary structure cannot be melted well at this temperature. Therefore, it can be considered that after the first preset duration, the temperature of the reaction system is raised to the second temperature on the basis of the first temperature and the second preset duration of the second temperature is maintained. In this way, not only can the polymerase maintain a good activity to continue to play its function, but the secondary structure in the template molecule can be fully prompted to open its double-stranded structure well, which is conducive to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0287] In some embodiments, the first temperature is 60°C to 65°C, and the first preset time is 1 to 10s; and / or, the second temperature is 80°C to 85°C, and the second preset time is 1 to 10s. Exemplarily, the first temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or a temperature between any two values. The first preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values. The second temperature can be 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The second preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0288] In some embodiments, the first heating rate and the second heating rate are 0.1 to 5.0°C / s. Exemplarily, the first heating rate and the second heating rate can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5 ℃ / s, 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0289] In some embodiments, S16000 is performed under variable temperature conditions, including:

[0290] In S16000, the reaction system of S16000 is pulse-heated at least once, so that the temperature of the reaction system of S16000 is raised to a third temperature; wherein the duration of one pulse is the third preset duration.

[0291] By adopting pulse heating, the hydrogen bonds between GC in the secondary structure formed in the template molecule can be more effectively destroyed, making the secondary structure easier to melt, which is beneficial to the binding of the template molecule to the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0292] In some embodiments, the third temperature is 60°C to 85°C, and the third preset time is 1 to 10s. The third temperature can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, or a temperature between any two values. The third preset time can be 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, or a time between any two values.

[0293] In some embodiments, the heating rate of pulse heating is 0.1-5.0°C / s. The heating rate of pulse heating can be 0.1°C / s, 0.2°C / s, 0.3°C / s, 0.4°C / s, 0.5°C / s, 0.6°C / s, 0.7°C / s, 0.8°C / s, 0.9°C / s, 1°C / s, 1.1°C / s, 1.2°C / s, 1.3°C / s, 1.4°C / s, 1.5°C / s, 1.6°C / s, 1.7°C / s, 1.8°C / s, 1.9°C / s, 2°C / s, 2.1°C / s, 2.2°C / s, 2.3°C / s, 2.4°C / s, 2.5°C / s , 2.6℃ / s, 2.7℃ / s, 2.8℃ / s, 2.9℃ / s, 3℃ / s, 3.1℃ / s, 3.2℃ / s, 3.3℃ / s, 3.4℃ / s, 3.5℃ / s, 3.6℃ / s, 3.7℃ / s, 3.8℃ / s, 3.9℃ / s, 4℃ / s, 4.1℃ / s, 4.2℃ / s, 4.3℃ / s, 4.4℃ / s, 4.5℃ / s, 4.6℃ / s, 4.7℃ / s, 4.8℃ / s, 4.9℃ / s, 5℃ / s or a rate between any two values.

[0294] In some embodiments, after a pulse ends and before the next pulse is applied to the reaction system of S16000, the temperature of the reaction system of S16000 is restored to the temperature before the pulse heating. In this way, the hydrogen bonds between GCs in the secondary structure formed in the template molecule can be repeatedly destroyed, so that the secondary structure in the template molecule is completely opened, which is conducive to the combination of the template molecule with the amplification primer and the extension of the amplification primer, thereby improving the amplification efficiency and reducing the GC preference.

[0295] Finally, an embodiment of the present application further provides a kit, comprising the first solution, the second solution, the third solution and the fourth solution in any one of the above-mentioned amplification methods.

[0296] The present application will be described below in conjunction with specific embodiments. The experimental instruments and their manufacturer information involved in the embodiments of the present application are shown in Table 1:

[0297] Table 1

[0298] Experimental instruments model factory FASTASeq300 Sequencer FASTASeq300 Shenzhen Zhenmai Biotechnology Co., Ltd.

[0299] like Figure 3As shown, usually, the GC preference in the sequencing results can be intuitively displayed through the GC preference scatter plot. The horizontal axis of the figure is the GC percentage content of the target polynucleotide, and the vertical axis is the ratio of the sequencing abundance to the true abundance of the target polynucleotide. Specifically, assuming that the sequencing results have no preference, then the ratio of the sequencing abundance to the true abundance of target polynucleotides with different GC contents will be around 1, and the corresponding scatter plot should be nearly horizontally distributed, as shown by the green scatter points; assuming that the sequencing results are biased towards high GC, then the ratio of the sequencing abundance to the true abundance of target polynucleotides with high GC content will be greater than 1, and the corresponding scatter plot should be distributed upward, as shown by the red scatter points; assuming that the sequencing results are biased towards low GC, then the ratio of the sequencing abundance to the true abundance of target polynucleotides with low GC content will be less than 1, and the corresponding scatter plot should be distributed downward, as shown by the blue scatter points; the more horizontal the trend of the scatter plot, the lower the GC preference, and the more inclined, the higher the GC preference.

[0300] Example 1

[0301] In Example 1 of the present application, a FASTASEq 300 sequencer was used in a constant temperature amplification mode (amplification temperature was 55° C.), and amplification and sequencing were performed according to the following amplification method:

[0302] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0303] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0304] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0305] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0306] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the first solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0307] S120, replacing the first solution with a third solution;

[0308] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0309] S160, replacing the fourth solution with the first solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0310] S180, repeat S120-S160 30 times.

[0311] The first solution contains 5% DMSO, 2.5 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 200mmol / l Trisbase, 50mmol / l ammonium chloride, 1.5mg / ml BST polymerase, and 200mmol / l dATP, dTTP, dCTP, and dGTP respectively;

[0312] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0313] The fourth solution contains 5% DMSO, 2.5 mol / l betaine, 2.5 mmol / l Mg 2 + , 3% volume percent concentration of formamide, 200 mmol / l Trisbase, and 50 mmol / l ammonium chloride.

[0314] Comparative Example 1-1

[0315] Comparative Example 1-1 studied different Mg 2+ Effect of concentration on GC bias. Comparative Example 1-1 is based on Example 1 and only adjusts the concentration of Mg in the first solution and the fourth solution. 2+ In Comparative Example 1-1, the concentration of Mg in the first solution and the fourth solution is 2+ The concentrations were 1mmol / l, 2mmol / l or 3mmol / l. Figure 4 As shown: The red, green and blue scattered points represent the Mg in the first solution and the fourth solution respectively. 2+ GC preference scatter plot at concentrations of 1mmol / l, 2mmol / l, and 3mmol / l. Figure 4 As can be seen in the figure, as the GC content of the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. 2+ , with Mg 2+ As the concentration decreases, the GC preference scatter plot becomes more horizontal, and the GC preference decreases.

[0316] Comparative Example 1-2

[0317] Comparative Examples 1-2 studied the effect of different betaine concentrations on GC preference. Comparative Examples 1-2 only adjusted the concentrations of betaine in the first solution and the fourth solution based on Example 1. In Comparative Examples 1-2, the concentrations of betaine in the first solution and the fourth solution were all 1 mol / l, 2 mol / l or 3 mol / l. The results are shown in FIG. Figure 5 As shown: the red, green and blue scattered points represent the GC preference scatter plots when the betaine concentrations in the first solution and the fourth solution are 1 mol / l, 2 mol / l and 3 mol / l, respectively. Figure 5 It can be seen that as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. Betaine is added during the amplification process. As the concentration of betaine increases, the GC preference scatter plot tends to be more horizontal, and the GC preference decreases.

[0318] Comparative Examples 1-3

[0319] Comparative Examples 1-3 studied different NH 4 + The effect of the concentration of NH on GC preference. Comparative Examples 1-3 are based on Example 1 and only adjust the concentration of NH 4 + In Comparative Examples 1-3, NH 4 + The concentrations were 10mmol / l, 30mmol / l or 50mmol / l. Figure 6 As shown: The red, green and blue scattered points represent the NH 4 + GC preference scatter plots at concentrations of 10mmol / l, 30mmol / l, and 50mmol / l. Figure 6 As can be seen in the figure, as the GC content of the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. 4 + , along with NH 4 + As the concentration increases, the GC preference scatter plot becomes more horizontal and the GC preference decreases.

[0320] Example 2

[0321] In Example 2 of the present application, a FASTASEq 300 sequencer was used in a constant temperature amplification mode (amplification temperature was 55° C.), and amplification and sequencing were performed according to the following amplification method:

[0322] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0323] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0324] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0325] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0326] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the first solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0327] S120, replacing the first solution with a third solution;

[0328] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0329] S160, replacing the fourth solution with the first solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0330] S180, repeat S120-S160 30 times.

[0331] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 100 mmol / l Trisbase, 100 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0332] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0333] The fourth solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+, 3% volume percent concentration of formamide, 100 mmol / l Trisbase, and 100 mmol / l ammonium chloride.

[0334] Comparative Example 2-1

[0335] Comparative Example 2-1 studies the effect of different formamide concentrations on GC preference. Comparative Example 2-1 only adjusts the formamide concentrations in the first solution and the fourth solution based on Example 2. In Comparative Example 2-1, the formamide concentrations in the first solution and the fourth solution are 6%, 9%, 12%, and 15%. The results are as follows: Figure 7 As shown: the red, green and blue scattered points represent the GC preference scatter plots when the formamide concentrations in the first solution and the fourth solution are 6%, 9%, 12% and 15%, respectively. Figure 7 It can be seen that as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. Formamide is added during the amplification process. As the formamide concentration increases, the GC preference scatter plot tends to be more horizontal, and the GC preference decreases.

[0336] Example 3

[0337] In Example 3 of the present application, a FASTASEq 300 sequencer was used in a constant temperature amplification mode (amplification temperature was 50° C.), and amplification and sequencing were performed according to the following amplification method:

[0338] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0339] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0340] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0341] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0342] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the second solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0343] S120, replacing the second solution with the third solution;

[0344] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0345] S160, replacing the fourth solution with the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0346] S180, repeat S120-S160 30 times.

[0347] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 50 mmol / l Trisbase, 30 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0348] The second solution contains 5% DMSO, 2 mol / l betaine, 1 mmol / l Mg 2+ , 3% formamide by volume, 10 mmol / l Trisbase, 30 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0349] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0350] The fourth solution contains 5% DMSO, 2 mol / l betaine, 1 mmol / l Mg 2+ , 3% volume percentage concentration of formamide, 10mmol / l Trisbase, and 30mmol / l ammonium chloride.

[0351] Comparative Example 3-1

[0352] The comparative example studied the effect of different AT contents and GC contents on GC preference. Comparative Example 3-1 only adjusted the concentrations of dATP, dTTP, dCTP and dGTP in the first solution and the second solution on the basis of Example 3. In Comparative Example 3-1, the concentrations of dATP, dTTP, dCTP and dGTP in the first solution and the second solution are shown in Table 2 below (the content in the rightmost note in Table 2 can be simply understood as the ratio of the total concentration of dATP and dTTP to the total concentration of dCTP and dGTP, for example, 0.5AT:39.5CG means that the ratio of the total concentration of dATP and dTTP to the total concentration of dCTP and dGTP is 0.5:39.5, and so on). The results are as follows Figure 8 As shown. Figure 8 It can be seen that as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. However, when the total nucleotide concentration remains unchanged during the amplification process, as the dCTP and dGTP concentrations increase, the GC preference scatter plot gradually tends to be horizontal and eventually tilts upward. The GC preference first decreases and then increases. This may be related to the gradual shift from amplification biased towards low GC templates to amplification biased towards high GC templates during the amplification process.

[0353] Table 2

[0354]

[0355] Example 4

[0356] In Example 4 of the present application, a FASTASEq 300 sequencer was used, and a constant temperature amplification mode (amplification temperature was 50° C.) was adopted to perform amplification and sequencing according to the amplification methods in the following four schemes (Scheme A, Scheme B, Scheme C, and Scheme D):

[0357] Option A:

[0358] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0359] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0360] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0361] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0362] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the second solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0363] S120, replacing the second solution with the third solution;

[0364] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0365] S160, replacing the fourth solution with the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0366] S180, repeat S120-S16027 times.

[0367] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 50 mmol / l Trisbase, 30 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0368] The second solution contains 5% DMSO, 2 mol / l betaine, 1 mmol / l Mg 2+ , 3% formamide by volume, 10 mmol / l Trisbase, 30 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, 10 μmol / l dATP, 10 μmol / l dTTP, 390 μmol / l dCTP and 390 μmol / l dGTP;

[0369] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0370] The fourth solution contains 5% DMSO, 2 mol / l betaine, 1 mmol / l Mg 2+ , 3% volume percentage concentration of formamide, 10mmol / l Trisbase, and 30mmol / l ammonium chloride.

[0371] Option B:

[0372] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0373] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0374] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0375] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0376] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the first solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0377] S120, replacing the second solution with the third solution;

[0378] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0379] S160, replacing the fourth solution with the first solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0380] S180, repeat S120-S16027 times.

[0381] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 50 mmol / l Trisbase, 30 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0382] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0383] The fourth solution contains 5% DMSO, 2 mol / l betaine, 1 mmol / l Mg 2+ , 3% volume percentage concentration of formamide, 10mmol / l Trisbase, and 30mmol / l ammonium chloride.

[0384] Plan C

[0385] The difference between the amplification method of Scheme C and Scheme A and Scheme B is:

[0386] Scheme C firstly follows the amplification method of Scheme A and repeats S120-S160 17 times, then repeats S120-S160 in Scheme B 10 times.

[0387] Plan D

[0388] The difference between the amplification method of Scheme D and Scheme A and Scheme B is:

[0389] Scheme D was first performed according to the amplification method of Scheme A and repeated S120-S16022 times, and then repeated S120-S160 in Scheme B for 5 times.

[0390] The results are as follows Fig. 9 As shown, where:

[0391] Cyan represents the GC preference scatter plot of amplification using scheme A;

[0392] Purple represents the GC preference scatter plot of amplification using scheme B;

[0393] Red indicates the GC preference scatter plot for amplification using the C scheme;

[0394] Green represents the GC preference scatter plot of amplification using the D scheme;

[0395] As can be seen from the figure, as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. However, as the number of cycles of the A scheme increases during the amplification process, the scatter plot tends to be more horizontal, and the GC preference gradually decreases. This shows that introducing an unbalanced base ratio during the cyclic amplification process, that is, the sum of the contents of dATP and dTTP is less than the sum of the contents of dGTP and dCTP, can reduce the GC preference.

[0396] Example 5

[0397] In Example 5 of the present application, a FASTASEq300 sequencer was used in a constant temperature amplification mode (amplification temperature was 50° C.), and amplification and sequencing were performed according to the following amplification methods:

[0398] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0399] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0400] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0401] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0402] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the first solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0403] S120, replacing the second solution with the third solution;

[0404] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0405] S160, replacing the fourth solution with the first solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0406] S180, repeat S120-S160 30 times.

[0407] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 100 mmol / l Trisbase, 100 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, and 200 mmol / l each of dATP, dTTP, dCTP, and dGTP;

[0408] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0409] The fourth solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% volume percent concentration of formamide, 100 mmol / l Trisbase, and 100 mmol / l ammonium chloride.

[0410] Comparative Example 5-1

[0411] Comparative Example 5-1 studied the effect of the temperature of isothermal amplification on GC preference. Comparative Example 5-1 only adjusted the temperature of isothermal amplification based on Example 5. The results are as follows Fig.10 As shown, purple, cyan, green and red represent the GC preference scatter plots of amplification at 60°C, 55°C, 50°C and 45°C, respectively. As can be seen from the figure, as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that there is a GC preference in the sequencing results, but as the amplification temperature gradually decreases, the scatter plot tends to be more horizontal, and the GC preference gradually decreases.

[0412] Example 6

[0413] In Example 6 of the present application, a FASTASEq 300 sequencer was used in a constant temperature amplification mode (amplification temperature was 50° C.), and amplification was performed according to the following amplification methods, and pulse heating was introduced during the amplification process before sequencing:

[0414] S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer;

[0415] S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution to obtain an extended chain of the first amplification primer;

[0416] S60, replacing the first solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent;

[0417] S80, replacing the third solution with the fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer;

[0418] S100, using the extended chain of the first amplification primer as a template, replacing the fourth solution with the first solution to extend the second amplification primer to obtain the extended chain of the second amplification primer;

[0419] S120, replacing the second solution with the third solution;

[0420] S140, replacing the third solution with the fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively;

[0421] S160, replacing the fourth solution with the first solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates;

[0422] S180, repeat S120-S160 30 times.

[0423] The first solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% formamide by volume, 100 mmol / l Trisbase, 100 mmol / l ammonium chloride, 1.5 mg / ml BST polymerase, 4 U / ml rTaq enzyme, and 200 mmol / l dATP, dTTP, dCTP, and dGTP respectively;

[0424] The third solution contains 90% formamide and 0.05% Tween20 in a volume percentage concentration;

[0425] The fourth solution contains 5% DMSO, 1 mol / l betaine, 2.5 mmol / l Mg 2+ , 3% volume percent concentration of formamide, 100 mmol / l Trisbase, and 100 mmol / l ammonium chloride.

[0426] Comparative Example 6-1

[0427] Comparative Example 6-1 studied the effect of pulse heating on GC preference during constant temperature amplification. Fig.11 As shown in the figure, the constant temperature amplification was set to 50°C (red line), and the pulse heating cycle was set to 2 times, with the pulse temperatures being 65°C and 80°C respectively. Fig.12 As shown: red and cyan represent constant temperature amplification and pulse heating during constant temperature amplification, respectively. As can be seen from the figure, as the GC content on the target polynucleotide gradually increases, the sequencing abundance decreases, indicating that the sequencing results have a GC preference. However, after the pulse heating is introduced during the constant temperature amplification process, the scatter plot tends to be more horizontal, and the GC preference gradually decreases.

[0428] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "example", "certain examples", "specific examples" or "embodiments" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present application. In the present application, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0429] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An amplification method, characterized in that: include: S20, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer; S40, using the target polynucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer; S60, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent; S80, replacing the third solution with a fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer; S100, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer, thereby obtaining the extended chain of the second amplification primer; S120, replacing the first solution or the second solution with the third solution; S140, replacing the third solution with a fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively; S160, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates; S180, repeat S120-S160 at least once; Wherein, the first solution, the second solution and the fourth solution all contain betaine, Mg 2+ NH4 + , formamide, wherein the concentration of betaine is greater than 0 mol / l and less than or equal to 4 mol / l, and the Mg 2+ The concentration of NH4 + The concentration of the formamide is greater than 0 mmol / l and less than or equal to 100 mmol / l, and the volume percentage concentration of the formamide is less than or equal to 15%.

2. The amplification method according to claim 1, characterized in that The concentration of betaine in the first solution is greater than 0 mol / l and less than or equal to 3 mol / l; Optionally, the concentration of betaine in the second solution is 1.5 to 2.5 mol / l; Optionally, the concentration of betaine in the fourth solution is 1.5 to 2.5 mol / l; Optionally, the first solution, the second solution, and the fourth solution all contain DMSO, and the volume percentage concentration of the DMSO in the first solution, the second solution, and the fourth solution is 1-5%; Optionally, in the S40, the first amplification primer is extended using the first solution, and the Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l; Optionally, in the S100 or in the S160, the fourth solution is replaced by the second solution, the Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; Optionally, in the S100 or in the S160, the fourth solution is replaced by the second solution, the Mg 2+ The concentration of the betaine in the second solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the second solution is 2 to 3 mol / l; Optionally, in the S40, the first amplification primer is extended using a second solution, and the Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; Optionally, in S100 or in S160, the fourth solution is replaced by the first solution, and the Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l; Optionally, in S100, the fourth solution is replaced by the first solution, and the Mg 2+ The concentration of the betaine in the first solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the first solution is 2 to 3 mol / l; Optionally, both the first solution and the second solution contain dNTPs, and the concentration of the dNTPs in the first solution and the second solution is 20 to 1600 μmol / l. Optionally, the first solution or the second solution further comprises dNTPs, wherein the dNTPs comprise dATP, dTTP, dCTP, and ddTP, and the sum of the contents of the dATP and the dTTP is equal to the sum of the contents of the dGTP and the dCTP; Optionally, the second solution or the first solution further comprises dNTPs, wherein the dNTPs comprises dATP, dTTP, dCTP, and ddTP, and the sum of the contents of the dATP and the dTTP is less than the sum of the contents of the dGTP and the dCTP; optionally, the NH4 + NH4 provided for at least one of ammonium sulfate, ammonium chloride and ammonium acetate + . Optionally, the volume percentage concentration of the formamide is greater than or equal to 3% and less than or equal to 15%. Optionally, the first solution, the second solution and the fourth solution further comprise Trisbase, and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10 to 500 mmol / l; Optionally, the concentration of Trisbase in the first solution, the second solution, and the fourth solution is 10 to 200 mmol / l; Optionally, the first solution, the second solution, and the fourth solution further comprise single-stranded binding protein and / or bovine serum albumin; The concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; The volume percentage concentration of the bovine serum albumin in the first solution, the second solution and the fourth solution is 0.05-2%; Optionally, the first solution, the second solution and the fourth solution further comprise a surfactant, and the volume percentage concentration of the surfactant in the first solution, the second solution and the fourth solution is 0.01-2%; Optionally, the surfactant is a nonionic surfactant; Optionally, the nonionic surfactant is selected from one or more of Tween20, DD, DDM and Triton; Optionally, the first solution, the second solution, and the fourth solution further contain tetramethylammonium chloride or tetraethylammonium chloride, and the concentration of the tetramethylammonium chloride or tetraethylammonium chloride in the first solution, the second solution, and the fourth solution is 10 to 50 mmol / l; Optionally, the denaturing agent is selected from amides. Optionally, the denaturing agent is formamide, and the volume percentage concentration of the formamide in the third solution is 60 to 100%; Optionally, the first amplification primer and the second amplification primer are oligonucleotides.

3. The amplification method according to claim 1 or 2, characterized in that The S160 is carried out under variable temperature conditions, the S120 and the S140 are carried out under constant temperature conditions, and the reaction temperature of the S160 is not lower than the reaction temperatures of the S120 and the S140; Optionally, the S160 is performed under variable temperature conditions, including: In S160, the reaction system of S160 is heated for the first time, so that the temperature of the reaction system of S160 is raised to a first temperature, and the first temperature is maintained for a first preset time; After the first preset time, the reaction system of S160 is heated for a second time, so that the temperature of the reaction system of S160 is raised from the first temperature to a second temperature, and the second temperature is maintained for a second preset time; Optionally, the first temperature is 60° C. to 65° C., and the first preset time is 1 to 10 seconds; and / or, The second temperature is 80°C to 85°C, and the second preset time is 1 to 10 seconds; Optionally, the heating rates of the first heating and the second heating are 0.1 to 5.0°C / s; Optionally, the S160 is performed under variable temperature conditions, including: In S160, the reaction system of S160 is pulse-heated at least once to raise the temperature of the reaction system of S160 to a third temperature; The duration of one pulse is the third preset duration; Optionally, the third temperature is 60° C. to 85° C., and the third preset time is 1 to 10 seconds; Optionally, the heating rate of the pulse heating is 0.1 to 5.0°C / s; Optionally, after one pulse ends and before the next pulse is applied to the reaction system of S160, the temperature of the reaction system of S160 is restored to the temperature before the pulse heating.

4. An amplification method, characterized in that include: S200, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer; S400, using the target nucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer; S600, replacing the first solution or the second solution with a third solution to remove the target nucleotide, wherein the third solution contains a denaturing agent; S800, replacing the third solution with a fourth solution to allow the extended chain of the first amplification primer to combine with the second amplification primer; S1000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer, thereby obtaining the extended chain of the second amplification primer; S1200, replacing the first solution or the second solution with the third solution; S1400, replacing the third solution with a fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively; S1600, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates; S1800, repeat S1200-S1600 at least once; Wherein, the S1600 is carried out under variable temperature conditions, the S1200 and the S1400 are carried out under constant temperature conditions, and the reaction temperature of the S1600 is not lower than the reaction temperatures of the S1200 and the S1400.

5. The amplification method according to claim 4, characterized in that The S1600 is performed under variable temperature conditions, including: In S1600, the reaction system of S1600 is heated for the first time, so that the temperature of the reaction system of S1600 is raised to a first temperature, and the first temperature is maintained for a first preset time; After the first preset time, the reaction system of S1600 is heated for a second time, so that the temperature of the reaction system of S1600 is raised from the first temperature to a second temperature, and the second temperature is maintained for a second preset time; Optionally, the first temperature is 60°C to 65°C, and the first preset time is 1 to 10 seconds; The second temperature is 80°C to 85°C, and the second preset time is 1 to 10 seconds; Optionally, the heating rates of the first heating and the second heating are 0.1 to 5.0°C / s; Optionally, the S1600 is performed under variable temperature conditions, including: In S1600, the reaction system of S1600 is pulse-heated at least once to raise the temperature of the reaction system of S1600 to a third temperature; The duration of one pulse is the third preset duration; Optionally, the third temperature is 60° C. to 85° C., and the third preset time is 1 to 10 seconds; Optionally, the heating rate of the pulse heating is 0.1 to 5.0°C / s; Optionally, after one pulse ends and before the next pulse is applied to the reaction system of S1600, the temperature of the reaction system of S1600 is restored to the temperature before the pulse heating.

6. The amplification method according to claim 4 or 5, characterized in that The first solution, the second solution and the fourth solution all contain betaine, Mg 2+ NH 4+ , formamide, wherein the concentration of betaine is greater than 0 mol / l and less than or equal to 4 mol / l, and the Mg 2+ The concentration of NH4 + The concentration of the formamide is greater than 0 mmol / l and less than or equal to 100 mmol / l, and the volume percentage concentration of the formamide is less than or equal to 15%; Optionally, the concentration of betaine in the first solution is greater than 0 mol / l and less than or equal to 3 mol / l; Optionally, the concentration of betaine in the second solution is 1.5 to 2.5 mol / l; Optionally, the concentration of betaine in the fourth solution is 1.5 to 2.5 mol / l; Optionally, the first solution, the second solution, and the fourth solution all contain DMSO, and the volume percentage concentration of the DMSO in the first solution, the second solution, and the fourth solution is 1-5%; Optionally, in the S400, the first amplification primer is extended using the first solution, and the Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l; Optionally, in the S1000 or in the S1600, the fourth solution is replaced by the second solution, the Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; Optionally, in the S1000 or in the S1600, the fourth solution is replaced by the second solution, the Mg 2+ The concentration of the betaine in the second solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the second solution is 2 to 3 mol / l; Optionally, in the S400, the first amplification primer is extended using a second solution, and the Mg 2+ The concentration of the betaine in the second solution is 2-4 mmol / l, and the concentration of the betaine in the second solution is 0.1-2 mol / l; Optionally, in S1000 or in S1600, the fourth solution is replaced by the first solution to extend the second amplification primer, and the Mg 2+ The concentration of the betaine in the first solution is 2-4 mmol / l, and the concentration of the betaine in the first solution is 0.1-2 mol / l; Optionally, in S1000 or in S1600, the fourth solution is replaced by the first solution to extend the second amplification primer, and the Mg 2+ The concentration of the betaine in the first solution is 1 to 1.5 mmol / l, and the concentration of the betaine in the first solution is 2 to 3 mol / l; Optionally, the first solution and the second solution both contain dNTPs, and the concentration of the dNTPs in the first solution and the second solution is 20 to 1600 μmol / l; Optionally, the first solution or the second solution further comprises dNTPs, wherein the dNTPs comprise dATP, dTTP, dCTP, and ddTP, and the sum of the contents of the dATP and the dTTP is equal to the sum of the contents of the dGTP and the dCTP; Optionally, the second solution or the first solution further comprises dNTPs, wherein the dNTPs comprise dATP, dTTP, dCTP, and ddTP, and the sum of the contents of the dATP and the dTTP is less than the sum of the contents of the dGTP and the dCTP; Optionally, the NH4 + NH4 provided for at least one of ammonium sulfate, ammonium chloride and ammonium acetate + ; Optionally, the first solution, the second solution and the fourth solution further comprise Trisbase, and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10 to 500 mmol / l; Optionally, the concentration of Trisbase in the first solution, the second solution, and the fourth solution is 10 to 200 mmol / l; Optionally, the first solution, the second solution, and the fourth solution further comprise single-stranded binding protein and / or bovine serum albumin; The concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; The volume percentage concentration of the bovine serum albumin in the first solution, the second solution and the fourth solution is 0.05-2%; Optionally, the first solution, the second solution and the fourth solution further comprise a surfactant, and the volume percentage concentration of the surfactant in the first solution, the second solution and the fourth solution is 0.01-2%; Optionally, the surfactant is a nonionic surfactant; Optionally, the nonionic surfactant is selected from one or more of Tween20, DD, DDM and Triton; Optionally, the first solution, the second solution, and the fourth solution further contain tetramethylammonium chloride or tetraethylammonium chloride, and the concentration of the tetramethylammonium chloride or tetraethylammonium chloride in the first solution, the second solution, and the fourth solution is 10 to 50 mmol / l; Optionally, the denaturing agent is selected from amides; Optionally, the denaturing agent is formamide, and the volume percentage concentration of the formamide in the third solution is 60 to 100%; Optionally, the first amplification primer and the second amplification primer are oligonucleotides.

7. An amplification method, characterized in that: include: S2000, providing a solid phase carrier surface, wherein the solid phase carrier surface comprises a first amplification primer, a second amplification primer, and a target polynucleotide fixed thereon, and at least a portion of the target polynucleotide is complementarily hybridized with the first amplification primer; S4000, using the target polynucleotide as a template, extending the first amplification primer using the first solution or the second solution to obtain an extended chain of the first amplification primer; S6000, replacing the first solution or the second solution with a third solution to remove the target polynucleotide, wherein the third solution contains a denaturing agent; S8000, replacing the third solution with a fourth solution, so that the extended chain of the first amplification primer is combined with the second amplification primer; S10000, using the extended chain of the first amplification primer as a template, using the first solution or the second solution to replace the fourth solution to extend the second amplification primer, thereby obtaining the extended chain of the second amplification primer; S12000, replacing the first solution or the second solution with the third solution; S14000, replacing the third solution with a fourth solution, so that the extended chain of the first amplification primer and the extended chain of the second amplification primer are combined with the second amplification primer and the first amplification primer respectively; S16000, replacing the fourth solution with the first solution or the second solution, and extending the first amplification primer and the second amplification primer using the extended chain of the first amplification primer and the extended chain of the second amplification primer as templates; S18000, repeat S12000-S16000 at least once; Wherein, in the S10000 or the S16000, the first solution or the second solution contains dATP, dTTP, dCTP and ddTP, and the sum of the contents of the dATP and the dTTP is less than the sum of the contents of the dGTP and the dCTP.

8. The amplification method according to claim 7, characterized in that The first solution, the second solution and the fourth solution all contain betaine, Mg 2+ NH4 + , formamide, wherein the concentration of betaine is greater than 0 mol / l and less than or equal to 4 mol / l, and the Mg 2+ The concentration of NH 4+ The concentration of the formamide is greater than 0 mmol / l and less than or equal to 100 mmol / l, and the volume percentage concentration of the formamide is less than or equal to 15%; Optionally, in S4000, the first amplification primer is extended using the first solution, and in S10000 and S16000, the fourth solution is replaced by the first solution or the second solution, wherein the Mg content in the first solution is 2+ The concentration of Mg in the second solution is 2-4 mmol / l, the concentration of betaine is 0.1-2 mol / l, and the concentration of Mg in the second solution is 0.1-2 mol / l. 2+ The concentration of is 2-4 mmol / l, and the concentration of betaine is 0.1-2 mol / l; Optionally, in S4000, the first amplification primer is extended using the first solution, and in S10000 and S16000, the fourth solution is replaced by the first solution or the second solution, wherein the Mg content in the first solution is 2+ The concentration of Mg in the second solution is 2-4 mmol / l, the concentration of betaine is 0.1-2 mol / l, and the concentration of Mg in the second solution is 0.1-2 mol / l. 2+ The concentration of is 1-1.55mmol / l, and the concentration of betaine is 2-3mol / l; Optionally, in S4000, the first amplification primer is extended using the second solution; and in S10000 and S16000, the fourth solution is replaced by the first solution or the second solution, wherein the first solution contains Mg 2+ The concentration of Mg in the second solution is 2-4 mmol / l, the concentration of betaine is 0.1-2 mol / l, and the concentration of Mg in the second solution is 0.1-2 mol / l. 2+ The concentration of is 2-4 mmol / l, and the concentration of betaine is 0.1-2 mol / l; Optionally, in S4000, the first amplification primer is extended using the second solution; and in S10000 and S16000, the second amplification primer is extended using the first solution or the second solution instead of the fourth solution, wherein the first solution contains Mg 2+ The concentration of Mg in the second solution is 1-1.5 mmol / l, the concentration of betaine is 2-3 mol / l, and the concentration of Mg in the second solution is 1-1.5 mmol / l. 2+ The concentration of is 2-4 mmol / l, and the concentration of betaine is 0.1-2 mol / l; Optionally, in S4000, the first solution or the second solution contains dATP, dTTP, dCTP, and ddTP, and the sum of the contents of the dATP and the dTTP is equal to the sum of the contents of the dGTP and the dCTP; Optionally, the concentration of betaine in the fourth solution is 1.5 to 2.5 mol / l; Optionally, the NH4 + NH4 provided for at least one of ammonium sulfate, ammonium chloride and ammonium acetate + ; Optionally, the first solution, the second solution and the fourth solution further comprise Trisbase, and the concentration of Trisbase in the first solution, the second solution and the fourth solution is 10 to 500 mmol / l; Optionally, the concentration of Trisbase in the first solution, the second solution, and the fourth solution is 10 to 200 mmol / l; Optionally, the first solution, the second solution, and the fourth solution further comprise single-stranded binding protein and / or bovine serum albumin; The concentration of the single-stranded binding protein in the first solution, the second solution, and the fourth solution is 0.01 to 1 mg / ml; The volume percentage concentration of the bovine serum albumin in the first solution, the second solution and the fourth solution is 0.05-2%; Optionally, the first solution, the second solution and the fourth solution further comprise a surfactant, and the volume percentage concentration of the surfactant in the first solution, the second solution and the fourth solution is 0.01-2%; Optionally, the surfactant is a nonionic surfactant; Optionally, the nonionic surfactant is selected from one or more of Tween20, DD, DDM and Triton; Optionally, the first solution, the second solution, and the fourth solution further contain tetramethylammonium chloride or tetraethylammonium chloride, and the concentration of the tetramethylammonium chloride or tetraethylammonium chloride in the first solution, the second solution, and the fourth solution is 10 to 50 mmol / l; Optionally, the denaturing agent is selected from amides; Optionally, the denaturing agent is formamide, and the volume percentage concentration of the formamide in the third solution is 60 to 100%; Optionally, the first amplification primer and the second amplification primer are oligonucleotides.

9. The amplification method according to claim 7 or 8, characterized in that: The S16000 is carried out under variable temperature conditions, the S12000 and the S14000 are carried out under constant temperature conditions, and the reaction temperature of the S16000 is not lower than the reaction temperatures of the S12000 and the S14000; Optionally, the S16000 is performed under variable temperature conditions, including: In the S16000, the reaction system of the S16000 is heated for the first time, so that the temperature of the reaction system of the S16000 is raised to a first temperature, and the first temperature is maintained for a first preset time; After the first preset time, the reaction system of S16000 is heated for a second time, so that the temperature of the reaction system of S16000 is raised from the first temperature to a second temperature, and the second temperature is maintained for a second preset time; Optionally, the first temperature is 60°C to 65°C, and the first preset time is 1 to 10 seconds; The second temperature is 80°C to 85°C, and the second preset time is 1 to 10 seconds; Optionally, the heating rates of the first heating and the second heating are 0.1 to 5.0°C / s; Optionally, the S16000 is performed under variable temperature conditions, including: In S16000, the reaction system of S16000 is pulse-heated at least once to raise the temperature of the reaction system of S16000 to a third temperature; The duration of one pulse is the third preset duration; Optionally, the third temperature is 60° C. to 85° C., and the third preset time is 1 to 10 seconds; Optionally, the heating rate of the pulse heating is 0.1 to 5.0°C / s; Optionally, after one pulse ends and before the next pulse is applied to the reaction system of S16000, the temperature of the reaction system of S16000 is restored to the temperature before the pulse heating.

10. A kit, characterized in that: The first solution, the second solution, the third solution and the fourth solution comprising any one of the amplification methods of claims 1-9.

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