Method for amplifying target nucleic acid and corresponding kit

Abstract

Provided are a method for amplifying a target nucleic acid, a product obtained by means of the method and a corresponding kit. In particular, the present invention relates to a method for amplifying a target nucleic acid. The method comprises the following steps: step (1): providing a first double-stranded nucleic acid, wherein the first double-stranded nucleic acid contains a first strand and a second strand, the first strand contains the same nucleic acid sequence as that of the target nucleic acid, the second strand comprises a nucleic acid sequence that is inversely complementary to the target nucleic acid, and the first strand and the second strand together contain a first nickase recognition site; and step (2): contacting the double-stranded nucleic acid with an amplification starting mixture and performing a reaction under appropriate conditions, wherein the amplification starting mixture comprises a first nickase, a first exonuclease and a first nucleic acid polymerase, and the first nickase can recognize the first nickase recognition site and form a nick in the first strand.

Description

Method for amplifying a target nucleic acid and corresponding kit

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410419077.6, filed on April 8, 2024. The entire contents of the prior application are considered part of the disclosure of this application and are hereby incorporated in its entirety.

[0003] Reference to a Sequence Listing

[0004] The Sequence Listing created on April 7, 2024, is 35,701 bytes, and is named D-CF240285-SequenceListing.xml, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0005] The present disclosure relates to the field of molecular cloning, in particular to a method for amplifying a target nucleic acid, a product obtained by the method and a corresponding kit. BACKGROUND

[0006] To date, almost all known DNA replication systems require a free hydroxyl group as the acceptor for the addition of nucleotides by DNA polymerases to initiate replication. In response to this requirement, several different solutions have been reported in nature: (1) in all cellular forms of life as well as in many viruses and plasmids, a specialized ribonucleotide RNA polymerase named “primase” generates a short RNA primer that is subsequently elongated by DNA polymerases; (2) in retroelements and retroviruses, reverse transcriptases use tRNA to initiate replication; (3) in protein primer systems of viruses like bacteriophage Phi29, a hydroxyl group is exploited from the side chain of a specific serine, threonine or tyrosine as the initial site for DNA replication; (4) in viruses that follow the rolling circle replication model, an endonuclease generates a nick in the DNA strand, creating a free 3’ hydroxyl group that is elongated by DNA polymerases.

[0007] As mentioned above, in specific cases, a nick in a DNA strand and its association with certain DNA polymerases (such as T7 DNA polymerase) can lead to DNA replication in the absence of primers or primases. However, such a nick and its association with major nucleic acid polymerases (such as Phi29 DNA polymerase) failed to initiate DNA replication (Jonejia and Huang, p. 11, Anal Biochem, 2011).

[0008] In the current nickase-mediated DNA isothermal amplification, the reaction usually contains the following five steps. i. At a suitable temperature, primers hybridize to the template nucleic acid strand, wherein the tail of the primer carries a specific recognition region of the nickase. ii. DNA polymerase extends from the 3' end of the primer to synthesize double-stranded DNA. iii. The nickase recognizes the specific region of the primer and only cuts one of the nucleic acid strands, introducing a nick site and exposing the 3' end. iv. DNA polymerase starts from this nick and synthesizes new double-stranded nucleic acid using the other uncut strand as a template, and displaces the old strand as a template for the next round of nucleic acid synthesis. At the same time, the recognition region of the nickase is restored to the newly synthesized double-stranded nucleic acid. v. By repeating the above cutting, extending and displacing steps, the target nucleic acid is continuously amplified. However, the disadvantages of this method are also obvious, first, non-specific amplification is easy to occur, second, the length of the amplified fragment cannot exceed 200 bp (Qian and Wu, Anal Chim Acta. 2019 Mar 7: 1050: 1-15.).

[0009] Therefore, there is a need for a new method to facilitate such a reaction. SUMMARY

[0010] To solve the above technical problems, the inventors of the present application have provided a method for amplifying a target nucleic acid, which enables the amplification of the target nucleic acid without primers or primase by using the method.

[0011] In a first aspect, the present application provides a method for amplifying a target nucleic acid, the method comprising the following steps:

[0012] Step (1): providing a first double-stranded nucleic acid, the first double-stranded nucleic acid comprising a first strand and a second strand, the first strand comprising a nucleic acid sequence identical to the target nucleic acid, the second strand comprising a nucleic acid sequence complementary to the target nucleic acid, the first strand and the second strand together comprising a first nickase recognition site;

[0013] Step (2): contacting the double-stranded nucleic acid with an amplification initiation mixture and reacting under suitable conditions, the amplification initiation mixture comprising a first nickase, a first exonuclease and a first nucleic acid polymerase, wherein the first nickase is capable of recognizing the first nickase recognition site and forming a nick on the first strand.

[0014] In a second aspect, the present application provides a product obtained by the method of the first aspect of the present application.

[0015] In a second aspect, the present application provides a kit comprising a first nickase, a first exonuclease and a first polymerase. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 shows a schematic diagram of a method for amplifying a target nucleic acid according to one embodiment of the present application.

[0017] Figure 2 shows a schematic diagram of a method for amplifying a target nucleic acid according to another embodiment of the present application.

[0018] Figure 3 shows expression identification of Phi29 E20 clone strains. M: protein Marker; lanes 1 / 3 / 5 / 7: Phi29 E20 strains ① / ② / ③ / ④ - supernatant; lanes 2 / 4 / 6 / 8: Phi29 E20 strains ① / ② / ③ / ④ - precipitate.

[0019] Figure 4 shows expression identification of Phi29 E20 purified protein. M: protein Marker; lanes 1 / 2 / 3 / 4 / 5: loading 3 / 6 / 9 / 12 / 15 μL.

[0020] Figure 5 shows expression identification of Ccdc111 clone strains. M: protein Marker; lanes 1 / 3 / 5 / 7: Ccdc111 strains ① / ② / ③ / ④ - supernatant; lanes 2 / 4 / 6 / 8: Ccdc111 strains ① / ② / ③ / ④ - precipitate.

[0021] Figure 6 shows expression identification of Ccdc111 purified protein. M: protein Marker; lanes 1 / 2 / 3 / 4: loading 0.2 / 0.5 / 1 / 2 μL.

[0022] Figure 7 shows amplification of single-stranded DNA. M. DNA marker; 1. addition of 0.5 U Exo III; 2. addition of 0.05 U Exo III; 3. addition of 0.005 U Exo III; 4. addition of 0.0005 U Exo III; 5. no addition of Exo III.

[0023] Figure 8 shows amplification of bidirectional or unidirectional companion DNA. M. DNA marker; 1. bidirectional companion DNA 2.0; 2. M13 F companion DNA; 3. M13 R companion DNA.

[0024] Figure 9 shows amplification of double-stranded DNA, wherein exonuclease III is used. (A) double-stranded DNA product after amplification, (B) DNA product after TelN treatment. M. DNA marker; 1. addition of primase Ccdc111 to the system; 2. addition of primase Tth to the system; 3. addition of duplex 3 (corresponding to M13 R companion DNA) to the system; 4. addition of duplex 1 (corresponding to bidirectional companion DNA 2.0) to the system. Arrow indicates the amplified product of > 30 kb.

[0025] Figure 10 shows the cascade amplification reaction of double stranded DNA amplification. (A) Double stranded DNA product after amplification, (B) DNA product after TelN treatment. M. DNA marker; 1. supplemented dNTP, Phi29 E20; 2. supplemented H20; 3. supplemented dNTP. Arrow indicates the amplified product of >30 kb.

[0026] Figure 11 shows the amplification of double stranded DNA, where T5 exonuclease is used. (A) Double stranded DNA product after amplification, (B) DNA product after TelN treatment. M. DNA marker; 1. 5U T5 exonuclease added to the system; 2. 0.5U T5 exonuclease added to the system; 3. 0.05U T5 exonuclease added to the system; 4. 0.5U exonuclease III added to the system.

[0027] Figure 12 shows the linearization enzyme digestion efficiency of double stranded DNA product. M. DNA marker; 1. Double stranded DNA product; 2. Exonuclease treated double stranded DNA product; 3. BspQ I linearized double stranded DNA product; 4. BspQ I linearization enzyme digestion and exonuclease treated double stranded DNA product. DETAILED DESCRIPTION

[0028] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, particle size, percentages, ratios, time, temperature, and so forth, in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. At the very least, each numerical parameter should at least be construed in light of the number of significant digits and ordinary rounding approaches at the time of the disclosure or the skilled person's understanding.

[0029] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Every numerical range given throughout this specification will include every narrower numerical range falling within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0030] As described in the background section, Phi29 DNA polymerase or similar nucleic acid polymerase cannot initiate strand extension of a target nucleic acid with a nick in the absence of a primer or primase, and thus cannot perform amplification of the target nucleic acid. To address this problem, the inventors of the present application have creatively proposed a method for amplifying a target nucleic acid, by which amplification of the target nucleic acid can be performed in the absence of a primer or primase.

[0031] In a first aspect, the present application provides a method for amplifying a target nucleic acid, the method comprising the following steps:

[0032] Step (1): providing a first double-stranded nucleic acid, the first double-stranded nucleic acid comprising a first strand and a second strand, the first strand comprising a nucleic acid sequence identical to the target nucleic acid, the second strand comprising a nucleic acid sequence reverse complementary to the target nucleic acid, the first strand and the second strand together comprising a first nickase recognition site;

[0033] Step (2): contacting the double-stranded nucleic acid with an amplification initiation mixture and reacting under suitable conditions, the amplification initiation mixture comprising a first nickase, a first exonuclease, and a first nucleic acid polymerase, wherein the first nickase is capable of recognizing the first nickase recognition site and forming a nick on the first strand.

[0034] In the case of only performing step (1) and step (2), the resulting amplification product comprises a single-stranded product comprising the target nucleic acid.

[0035] In some embodiments, the first double-stranded nucleic acid is circular.

[0036] As used in the present application, "circular double-stranded nucleic acid" is not particularly limited and can be any circular form of nucleic acid double-strand, which can be DNA or RNA, or a hybrid of DNA and RNA, as long as the nick site sequence of the first nickase can be successfully introduced. In some embodiments, the circular double-stranded nucleic acid can be a plasmid with an expression element.

[0037] In some embodiments, in the context of "contacting the double-stranded nucleic acid with an amplification initiation mixture and reacting under suitable conditions", "reacting" includes the first nickase forming a nick on the first strand; the first exonuclease cutting from the nick in the direction of 3'→5' or 5'→3' to form an enlarged gap; and the first nucleic acid polymerase binding the sequence on the 3' side of the nick, initiating extension and performing amplification.

[0038] In some embodiments, in the context of "contacting the double-stranded nucleic acid with an amplification initiation mixture and reacting under suitable conditions", the "suitable conditions" can vary depending on the specific type of enzyme selected, the length of the target nucleic acid, and the like, and can be selected by a person skilled in the art as appropriate. In some embodiments, the "suitable conditions" can include a reaction temperature of 4-75°C, 4-70°C, 10-70°C, 15-70°C, 20-70°C, 25-70°C, 25-68°C, 60-70°C, 62-68°C, 25-60°C, 25-50°C, 25-40°C, 30-40°C, 36-38°C, or about 37°C; and / or a reaction time of 10 minutes to overnight, 0.5-10 hours, 1-8 hours, 2-6 hours, 3-5 hours, or about 4 hours. In some embodiments, the "suitable conditions" can include a reaction temperature of 36-38°C; and / or a reaction time of 3-5 hours. In some embodiments, the "suitable conditions" can include a reaction temperature of about 37°C; and / or a reaction time of about 4 hours.

[0039] In some embodiments, the amplification products are cleaved into uniform single-stranded nucleic acid fragments.

[0040] In some embodiments, the amplification products are cleaved into uniform single-stranded nucleic acid fragments by one or more of the following:

[0041] (A) adding a single-strand specific endonuclease to the reaction;

[0042] (B) adding an oligonucleotide complementary to at least one of the amplification products (newly synthesized strand) to the reaction, and adding a double-strand specific endonuclease;

[0043] (C) designing the first strand to contain a palindromic nucleic acid sequence, and adding a double-strand specific endonuclease that recognizes the palindromic nucleic acid sequence to the reaction;

[0044] (D) designing the first strand to contain a DNAzyme nucleic acid sequence, and self-cleaving under suitable conditions.

[0045] Figure 1 shows a schematic diagram of a method of amplifying a target nucleic acid according to an embodiment of the present application, in which a circular double-stranded DNA is used, a nicking enzyme is used to form a nick on one of the DNA strands (containing the target nucleic acid), a first exonuclease (not shown in the figure) cleaves from the nick in the 3'→5' or 5'→3' direction to form an enlarged gap, enabling a Phi29 DNA polymerase or similar nucleic acid polymerase to bind to the sequence on the 3' side of the nick, initiating synthesis of a new DNA strand using the intact strand without the nick (containing the reverse complement of the target nucleic acid) as a template, thereby amplifying the target nucleic acid.

[0046] In some embodiments, the amplification initiation mixture does not include primers or primase.

[0047] In some embodiments, the amplification initiation mixture further includes dNTPs, a buffer, pyrophosphatase, DTT, water, or one or more of the components known in the art to be useful in nucleic acid amplification.

[0048] Compared to the technical solutions using primers or primase, the products obtained by the technical solutions of the present application show higher cleavage efficiency when subjected to subsequent enzyme cleavage. In addition, compared to the technical solutions using primers, the use of the technical solutions of the present application can reduce the cost when amplifying. In addition, compared to the technical solutions using primase, the use of the technical solutions of the present application can improve the uniformity of the products when amplifying.

[0049] As used in the present application, enzymes (including nucleic acid polymerases, nicking enzymes, exonucleases, primases, etc.) include wild types and mutants thereof, which retain at least part of the function / activity of the wild types.

[0050] In some embodiments, "nucleic acid polymerase" refers to a general term for a class of enzymes that catalyze the synthesis of DNA, RNA, including DNA polymerase and RNA polymerase.

[0051] In some embodiments, the first nucleic acid polymerase is a DNA polymerase. In some embodiments, the first nucleic acid polymerase includes a nucleic acid polymerase that cannot utilize the nicked initiation nucleic acid for extension in the absence of primers or primase. In some embodiments, the first nucleic acid polymerase includes Phi29 DNA nucleic acid polymerase, Bst DNA nucleic acid polymerase, Bsu DNA nucleic acid polymerase, Klenow DNA nucleic acid polymerase. In some embodiments, the first nucleic acid polymerase includes wild type Phi29 DNA nucleic acid polymerase or a mutant thereof.

[0052] In some embodiments, the first nucleic acid polymerase includes an amino acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 11.

[0053] The His tag sequence for purification is included in SEQ ID NO. 1, and both SEQ ID NO. 1 itself and the amino acid sequence obtained by deleting the His tag sequence from SEQ ID NO. 1 (SEQ ID NO. 11) belong to the first nucleic acid polymerase described in the present application, and the embodiments related thereto fall within the scope of protection of the present application.

[0054] As used herein, "nickase" encompasses both an enzyme or functional analog thereof having only a nicking function (forming a nick on one strand of a double-stranded nucleic acid) and an enzyme or functional analog thereof having a nicking function in addition to other functions.

[0055] In some embodiments, the first nickase comprises a nicking endonuclease or a functional analog thereof, an enzyme or functional analog thereof having a nicking function. In some embodiments, the nicking endonuclease comprises, but is not limited to, Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, Nt.BsmAI. In some embodiments, the enzyme having a nicking function comprises, but is not limited to, a CRISPR nickase, a ZFN nickase, a TALE nickase. In some embodiments, the first nickase comprises a Nt.BspQI nickase, a Nt.CviPII nickase, a Nt.BstNBI nickase, a Nb.BsrDI nickase, a Nb.BtsI nickase, a Nt.AlwI nickase, a Nb.BbvCI nickase, a Nt.BbvCI nickase, a Nb.BsmI nickase, a Nb.BssSI nickase, a Nt.BsmA nickase, a CRISPR nickase, a ZFN nickase, a TALE nickase, or a functional analog thereof.

[0056] As used herein, "exonuclease" encompasses all enzymes or analogs thereof capable of enlarging a nick on a nucleic acid strand, forming a gap.

[0057] In some embodiments, the first exonuclease comprises a 3'→5' exonuclease, a 5'→3' exonuclease.

[0058] In some embodiments, the first exonuclease comprises exonuclease I, II, III, IV, V, VI, VII, VIII, T7 exonuclease, T5 exonuclease, RecJf exonuclease, lambda exonuclease, exonuclease T, BAL-31 nuclease, mung bean nuclease, mycobacterial nuclease, DNase I, snake venom phosphodiesterase, spleen phosphodiesterase, Lactobacillus acidophilus nuclease.

[0059] In some embodiments, the method further comprises:

[0060] Step (3): adding to the product of step (2) an amplification continuation mixture and reacting under suitable conditions, the amplification continuation mixture comprising: (i) a first oligonucleotide comprising a sequence reverse complementary to the target nucleic acid; (ii) a primase; or (iii) a second double-stranded nucleic acid comprising a second nickase recognition site, a second nickase capable of recognizing the second nickase recognition site and forming a nick on each of the two strands of the second double-stranded nucleic acid, wherein the 3' side of the nick on one strand comprises a sequence identical to the target nucleic acid and the 3' side of the nick on the other strand comprises a sequence reverse complementary to the target nucleic acid.

[0061] In some embodiments, the method further comprises:

[0062] Step (3): adding to the product of step (2) an amplification continuation mixture and reacting under suitable conditions, the amplification continuation mixture comprising: (i) a first oligonucleotide comprising a sequence reverse complementary to the target nucleic acid and a second oligonucleotide comprising a sequence identical to the target nucleic acid; (ii) a primase; or (iii) a second double-stranded nucleic acid comprising a second nickase recognition site, a second nickase capable of recognizing the second nickase recognition site and forming a nick on each of the two strands of the second double-stranded nucleic acid, wherein the 3' side of the nick on one strand comprises a sequence identical or reverse complementary to the target nucleic acid and the 3' side of the nick on the other strand comprises a sequence reverse complementary to the target nucleic acid.

[0063] In the case where step (1), step (2) and step (3) are performed, the resulting amplification product comprises a double-stranded product, wherein one strand comprises the target nucleic acid and the other strand comprises the reverse complementary sequence of the target nucleic acid.

[0064] In some embodiments, in the context of "adding to the product of step (2) an amplification continuation mixture and reacting under suitable conditions", "reacting" comprises the second nickase forming a nick on one strand; and the second nucleic acid polymerase binding to the sequence on the 3' side of the nick, initiating extension, performing amplification; or the second nickase forming a nick on each of the two strands; and the second nucleic acid polymerase binding to the sequence on the 3' side of the nick on each of the two strands, initiating extension, performing amplification.

[0065] In some embodiments, in the context of "adding an amplification continuation mixture to the product of step (2) and reacting under suitable conditions", the "suitable conditions" can vary depending on the specific type of enzyme selected, the length of the target nucleic acid, and the like, and can be selected by a person skilled in the art as appropriate. In some embodiments, the "suitable conditions" can include a reaction temperature of 4-75°C, 4-70°C, 10-70°C, 15-70°C, 20-70°C, 25-70°C, 25-68°C, 60-70°C, 62-68°C, 25-60°C, 25-50°C, 25-40°C, 30-40°C, 36-38°C, or about 37°C; and / or a reaction time of 10 minutes to overnight, 0.5-10 hours, 2-8 hours, 3-7 hours, 4-6 hours, or about 5 hours. In some embodiments, the "suitable conditions" can include a reaction temperature of 36-38°C; and / or a reaction time of 4-6 hours. In some embodiments, the "suitable conditions" can include a reaction temperature of about 37°C; and / or a reaction time of about 5 hours.

[0066] In some embodiments, the amplification continuation mixture further comprises one or more of dNTPs, a buffer, pyrophosphatase, DTT, water, or components known in the art to be useful in nucleic acid amplification.

[0067] In some embodiments, the second nucleic acid polymerase and / or dNTPs can be supplemented during the reaction of step (3). By doing so, a cascade amplification of the double-stranded product can be achieved.

[0068] By employing the technical solutions of the present application, the amplification order / timing of the two strands in the double-stranded product can be artificially controlled, one strand can be amplified first, and then the amplification of the other strand can be initiated under controlled conditions; or both strands can be simultaneously initiated for amplification.

[0069] In some embodiments, the second double-stranded nucleic acid is linear.

[0070] FIG. 2 shows a schematic diagram of a method of amplifying a target nucleic acid according to another embodiment of the present application, in which a circular double-stranded DNA (shown at the bottom of FIG. 2) is used, a nick is formed on one DNA strand (containing the target nucleic acid) by a first nicking enzyme, a first exonuclease (not shown in the figure) cuts along the 3'→5' or 5'→3' direction from the nick to form an enlarged gap, a Phi29 DNA polymerase or a similar nucleic acid polymerase is able to bind the sequence on the 3' side of the nick to initiate synthesis of a new DNA strand using the intact strand (containing the reverse complement of the target nucleic acid) without the nick as a template, thereby amplifying the target nucleic acid. On this basis, a linear double-stranded DNA (shown at the top of FIG. 2) is used, a nick is formed on one DNA strand by a second nicking enzyme (the 3' side of the nick includes the reverse complement of the target nucleic acid), a T7 DNA polymerase or a similar nucleic acid polymerase is able to bind the sequence on the 3' side of the nick to initiate synthesis of a new DNA strand using the intact strand (the 5' side of the position corresponding to the nick includes the same sequence as the target nucleic acid) without the nick as a template, thereby amplifying the partner DNA, which is able to bind to the 3' end of the target nucleic acid in reverse complement, thereby synthesizing a DNA strand in reverse complement to the target nucleic acid, thereby amplifying double-stranded DNA.

[0071] In some embodiments, the length of the partner DNA can be 5-500 nt. In some embodiments, the length of the partner DNA can be 5-200 nt. In some embodiments, the length of the partner DNA can be 5-100 nt. In some embodiments, the length of the partner DNA can be 5-50 nt. In some embodiments, the length of the partner DNA can be 10-40 nt. In some embodiments, the length of the partner DNA can be 10-30 nt. In some embodiments, the length of the partner DNA can be 10-20 nt.

[0072] In some embodiments, the second nucleic acid polymerase includes any nucleic acid polymerase that is able to initiate nucleic acid extension using a nick.

[0073] In some embodiments, the second nucleic acid polymerase includes a T7 DNA nucleic acid polymerase, a T4 DNA nucleic acid polymerase, a Phi29 DNA nucleic acid polymerase, a Bst DNA nucleic acid polymerase, a Bsu DNA nucleic acid polymerase, a Klenow DNA nucleic acid polymerase.

[0074] In some embodiments, the first nucleic acid polymerase and the second nucleic acid polymerase can be the same or different.

[0075] In some embodiments, the second nicking enzyme comprises a nicking endonuclease or a functional analog thereof, an enzyme with nicking function or a functional analog thereof. In some embodiments, the nicking endonuclease comprises, but is not limited to, Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, Nt.BsmAI. In some embodiments, the enzyme with nicking function comprises, but is not limited to, a CRISPR nicking enzyme, a ZFN nicking enzyme, a TALE nicking enzyme. In some embodiments, the second nicking enzyme comprises a Nt.BspQI nicking enzyme, a Nt.CviPII nicking enzyme, a Nt.BstNBI nicking enzyme, a Nb.BsrDI nicking enzyme, a Nb.BtsI nicking enzyme, a Nt.AlwI nicking enzyme, a Nb.BbvCI nicking enzyme, a Nt.BbvCI nicking enzyme, a Nb.BsmI nicking enzyme, a Nb.BssSI nicking enzyme, a Nt.BsmA nicking enzyme, a CRISPR nicking enzyme, a ZFN nicking enzyme, a TALE nicking enzyme, or a functional analog thereof.

[0076] In some embodiments, the first nicking enzyme and the second nicking enzyme can be the same or different.

[0077] In some embodiments, the method further comprises:

[0078] Step (4): adding an enzyme with double-strand cleavage activity to the product of step (3).

[0079] In some embodiments, the enzyme with double-strand cleavage activity further has an activity of generating covalently closed ends at the cleavage site. In some embodiments, the enzyme with double-strand cleavage activity comprises a telomerase. In some embodiments, the telomerase comprises, but is not limited to, a TelN telomerase.

[0080] In some embodiments, the first double-stranded nucleic acid comprises a specific recognition sequence of the enzyme with double-strand cleavage activity. In some embodiments, the target nucleic acid does not comprise a specific recognition sequence of the enzyme with double-strand cleavage activity.

[0081] In some embodiments, the product of step (4) comprises a linear nucleic acid product with both ends closed.

[0082] In some embodiments, the method further comprises:

[0083] Step (5): adding a second exonuclease to the product of step (4).

[0084] In some embodiments, the product of step (5) does not comprise a non-closed nucleic acid.

[0085] In some embodiments, the second exonuclease comprises a 3'→5' exonuclease, a 5'→3' exonuclease.

[0086] In some embodiments, the second exonuclease comprises exonuclease I, II, III, IV, V, VI, VII, VIII, T7 exonuclease, T5 exonuclease, RecJf exonuclease, lambda exonuclease, exonuclease T, BAL-31 nuclease, mung bean nuclease, mycobacterial nuclease, DNase I, snake venom phosphodiesterase, spleen phosphodiesterase, Lactobacillus acidophilus nuclease.

[0087] In a second aspect, the present application provides a product obtained by the method of the first aspect of the present application.

[0088] In a second aspect, the present application provides a kit comprising the first nicking enzyme, the first exonuclease and the first polymerase.

[0089] In some embodiments, the kit further comprises a first double-stranded nucleic acid comprising a first nicking enzyme recognition site, the first nicking enzyme being capable of recognizing the first nicking enzyme recognition site and forming a nick on one strand of the first double-stranded nucleic acid.

[0090] In some embodiments, the kit further comprises a second double-stranded nucleic acid comprising a second nicking enzyme recognition site, the second nicking enzyme being capable of recognizing the second nicking enzyme recognition site and forming a nick on one strand of the second double-stranded nucleic acid or forming a nick on each of the two strands of the second double-stranded nucleic acid.

[0091] In some embodiments, the kit further comprises a second nicking enzyme.

[0092] In some embodiments, the kit further comprises a second nucleic acid polymerase.

[0093] The various embodiments and preferences provided above in relation to the present application can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by the combination are all considered as part of the disclosure of the present application.

[0094] The technical solutions of the present application will be described more clearly and explicitly below with reference to the examples in an exemplary manner. It should be understood that these examples are only for illustrative purposes and are by no means intended to limit the protection scope of the present application. The protection scope of the present application is only defined by the claims.

[0095] Examples

[0096] Unless otherwise specified, the techniques or conditions in the examples were performed according to the techniques or conditions described in the literature or according to the product manual. Unless otherwise specified, the reagents or instruments used were conventional products that can be obtained commercially.

[0097] Example 1: Preparation of Phi29 E20 DNA polymerase

[0098] 1. Construction of Phi29 E20 expression plasmid:

[0099] The amino acid sequence of Phi29 E20 DNA polymerase is shown in SEQ ID NO. 1 (including the His tag sequence for purification, i.e., His tag sequence + SEQ ID NO. 11). The Phi29 E20 gene sequence was synthesized (Jinsuibiological) and ligated to the pET-30a(+) expression vector digested with Nde I and BamH I, and transformed into E. coli DH5α to obtain the correct cloned plasmid by sequencing.

[0100] 2. Expression of Phi29 E20 recombinant protein:

[0101] The obtained correct cloned plasmid was transformed into BL21(DE3) host bacteria, and the transformed monoclonal strain was inoculated in a test tube.

[0102] 1) The monoclonal bacteria were cultured at 37°C on a shaker for 2 h, and 0.5 mM IPTG was added to induce the expression of Phi29 E20 protein, and the culture was continued at 16°C for 16 h. The bacteria were collected and numbered appropriately.

[0103] 2) The centrifugally collected bacteria were frozen at -80°C for 30 min; 2.7 mL PBS was added to resuspend the bacteria; then 300 μL 10% Triton solution and 10 μL lysozyme (100 mg / mL) were added, respectively, and incubated at room temperature for 30 min; and the bacteria were frozen and thawed 3 times at -80°C / 30°C;

[0104] 3) 1.5 mL of the sample was taken and centrifuged at 12000 rpm for 10 min, and the supernatant was taken to obtain the supernatant sample; the precipitate was resuspended with an equal volume (1.5 mL) of PBS, mixed well, and obtained as the precipitate sample;

[0105] 4) 15 μL of the sample was taken and loaded onto a 10% precast PAGE Gel (Jinsuibiological, M01010C) and run electrophoresis at 180 V for 40 min;

[0106] 5) The sample was stained with ExBlue protein super fast staining solution (Beijing Zhuangmeng Biological, ZD305A) for 30 min, and then decolorized with tap water for 1 h;

[0107] According to the electrophoresis result of Figure 3, the clone of No. 1 strain has the best expression, and therefore No. 1 clone is selected as the strain for large-scale expression culture.

[0108] 3. Purification of Phi29 E20 recombinant protein:

[0109] 1) Centrifugation at 8000 rpm for 5 min, discard the supernatant, collect the bacterial body and freeze at -80℃ for 30 min; resuspend the bacterial liquid with 180 ml PBS; add 20 ml 10% Triton solution, 800 μl lysozyme (100 mg / ml), 200 μl PMSF (100 mM), mix well, and place on ice for 30 min; after freezing and thawing 3 times at -80℃ / 30℃, perform ultrasonic crushing; centrifuge at 10000 rpm for 5 min, collect the supernatant

[0110] 2) Washing: mix the Ni-binding magnetic beads (Genscribio, L00295), take 1.4 ml magnetic beads to a centrifuge tube, place on the magnetic stand, and remove the supernatant; add 15 mL solution (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4), mix well, and place on the magnetic stand, remove the supernatant, and repeat 2 times;

[0111] 3) Binding: mix the magnetic beads with the sample, and rotate incubate at room temperature for 4 h; separate the supernatant and magnetic beads with the magnetic stand, and transfer the supernatant to another bottle;

[0112] 4) Washing: add 15 mL solution (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4) to the magnetic beads, mix well, and place on the magnetic stand, remove the supernatant, and repeat 3 times, and then add 15 mL solution (10 mM Tris-HCl, 75 mM NaCl, 20 mM imidazole, pH 7.4) to the magnetic beads, mix well, and place on the magnetic stand, remove the supernatant;

[0113] 5) Elution: add 6 mL (10 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 7.4) to the magnetic beads, mix well, and incubate at room temperature for 5 min; place on the magnetic stand, and transfer the elution collection liquid to a centrifuge tube;

[0114] 6) Take sample, load 10% precast PAGE Gel (Genscribio, M01010C) at 180 V voltage for 40 min;

[0115] 7) Stain with ExBlue protein super fast staining solution for 30 min, and then decolorize with tap water for 1 h;

[0116] The purified sample was subjected to SDS-PAGE electrophoresis detection, and the results are shown in Figure 4. The expression of the target protein was clear, and the band size was consistent. The activity was 60 KU / mL*10 mL.

[0117] Example 2: Preparation of Ccdc111 primase

[0118] 1. Construction of Ccdc111 expression plasmid:

[0119] The amino acid sequence of Ccdc111 is shown in SEQ ID NO. 9. The Ccdc111 gene sequence was synthesized (Jinsuibiological) and ligated to the pET-30a(+) expression vector digested by Nde I and BamH I. The transformed E. coli DH5α was sequenced to obtain the correct cloned plasmid.

[0120] 2. Expression of Ccdc111 recombinant protein:

[0121] The obtained correct cloned plasmid was transformed into BL21(DE3) host bacteria, and the transformed monoclonal strain was inoculated in a test tube.

[0122] 1) The monoclonal bacteria were cultured at 37℃ for 2h, and 0.5mM IPTG was added to induce the expression of Ccdc111 protein. The culture was continued at 16℃ for 16h, and the bacteria were collected and numbered reasonably.

[0123] 2) The centrifugally collected bacteria were frozen at -80℃ for 30min; 2.7mL PBS was added to resuspend the bacteria; then 300μL 10%Triton solution and 10μL lysozyme (100mg / mL) were added respectively, and incubated at room temperature for 30min; freeze-thawed 3 times at -80℃ / 30℃;

[0124] 3) 1.5mL sample was taken and centrifuged at 12000rpm for 10min. The supernatant was taken to obtain the supernatant sample; the precipitate was resuspended with equal volume (1.5mL) of PBS, mixed well to obtain the precipitate sample;

[0125] 4) 15μL sample was taken and loaded onto 10%precast PAGE Gel (Jinsuibiological, M01010C) and run electrophoresis at 180V for 40min;

[0126] 5) ExBlue protein super fast staining solution (Beijing Zhuangmeng Biological, ZD305A) was used for staining for 30min, and then tap water was used for decolorization for 1h;

[0127] According to the electrophoresis results in Figure 5, the expression of No. 4 strain was the best, so No. 4 clone was selected as the strain for large-scale expression culture.

[0128] 3. Purification of Ccdc111 recombinant protein:

[0129] 1) 8000 rpm centrifugation for 5 min, discard the supernatant, collect the bacteria and freeze at -80℃ for 30 min; resuspend the bacteria in 180 ml PBS; add 20 ml 10% Triton solution, 800 μl lysozyme (100 mg / ml), 200 μl PMSF (100 mM), mix well, and place on ice for 30 min; freeze-thaw 3 times at -80℃ / 30℃, then ultrasonic breakage; centrifuge at 10000 rpm for 5 min, collect the supernatant

[0130] 2) Wash: mix the Ni-binding magnetic beads (Genscript, L00295), take 1.4 ml of magnetic beads into a centrifuge tube, place on the magnetic stand, and remove the supernatant; add 15 mL solution (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4), mix well, and place on the magnetic stand, remove the supernatant, repeat 2 times;

[0131] 3) Binding: mix the magnetic beads with the sample, rotate incubation at room temperature for 4 h; separate the supernatant and magnetic beads with the magnetic stand, and transfer the supernatant to another bottle;

[0132] 4) Wash: add 15 mL solution (10 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.4) to the magnetic beads, mix well, and place on the magnetic stand, remove the supernatant, repeat 3 times, and then add 15 mL solution (10 mM Tris-HCl, 75 mM NaCl, 20 mM imidazole, pH 7.4) to the magnetic beads, mix well, and place on the magnetic stand, remove the supernatant;

[0133] 5) Elution: add 6 mL (10 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 7.4) to the magnetic beads, mix well, and incubate at room temperature for 5 min; place on the magnetic stand, and transfer the elution collection liquid to a centrifuge tube;

[0134] 6) Take sample, load 10% precast PAGE Gel (Genscript, M01010C) at 180 V for 40 min;

[0135] 7) Stain with ExBlue protein super fast staining solution for 30 min, then decolorize with tap water for 1 h; take the purified sample for SDS-PAGE electrophoresis detection, the results are shown in Figure 6, the expression of the target protein is clear, and the band size is consistent, and the activity is 100 KU / mL*2 mL.

[0136] Example 3: Amplification of single-stranded DNA

[0137] According to Table 1, in a 200 μL Eppendorf tube, add each component, mix well, add H2O, 10*buffer, plasmid containing nickase recognition site (N0096, SEQ ID NO. 8), dNTP, Nt.BbvCI nickase (NEB, R0632L), Phi29 E20, 0.0005 U, 0.005 U, 0.05 U or 0.5 U exonuclease III (Exo III (SEQ ID NO. 6), Suzhou Coastal Protein, M007), pyrophosphatase (Suzhou Coastal Protein, M036), DTT, prepare 100 μL reaction system, mix well.

[0138] Table 1 Single-stranded DNA production system

[0139] Incubate at 37°C for 4 h, take 0.5 μL of each reaction for 1% agarose gel identification, 125 V voltage, electrophoresis for 45 min. As can be seen from Figure 7, when 0.05 U or 0.5 U Exo III is added to the system, the brightness of the DNA product is significantly improved, indicating that the reaction effectively amplifies to produce single-stranded DNA.

[0140] Example 4: Production of partner DNA

[0141] Double-stranded body 1 (corresponding to bidirectional partner DNA 2.0) formed by SEQ ID NO. 3 and its reverse complementary sequence, double-stranded body 2 (corresponding to M13F partner DNA) formed by SEQ ID NO. 4 and its reverse complementary sequence, and double-stranded body 3 (corresponding to M13R partner DNA) formed by SEQ ID NO. 5 and its reverse complementary sequence were used as the second double-stranded nucleic acid for forming partner DNA.

[0142] According to Table 2, in a 200 μL Eppendorf tube, add H2O, 10*buffer, double-stranded body 1 / 2 / 3, dNTP, Sequenase (SEQ ID NO. 7, Thermo Fisher, 70775Z1000UN), Nt.BbvCI nickase (NEB, R0632L), pyrophosphatase (Suzhou Coastal Protein, M036), DTT, prepare 100 μL reaction system, mix well.

[0143] Table 2 Partner DNA production system

[0144] 37°C incubation for 4h, 80°C inactivation for 20min. Each group of reaction was sampled 4μL for gel electrophoresis analysis by 4%-20% TBE PAGE precast gel (Bi Yun Tian, D0185S), 150V voltage, electrophoresis for 35min. The results were shown in Figure 8, it could be obviously seen that duplex 1 / 2 / 3 containing nickase recognition site produced corresponding partner DNA under the action of nickase and Sequenase.

[0145] Example 5: Amplification of double-stranded DNA, in which exonuclease III was used

[0146] According to Table 3, in 200μL Eppendorf tube, each component was added, H2O, 10*buffer, plasmid containing nickase recognition site (N0096), dNTP, Nt.BbvCI nickase (NEB, R0632L), Phi29 E20, exonuclease III (Suzhou Jinan Protein, M007), pyrophosphatase (Suzhou Jinan Protein, M036), DTT were added in turn, mixed well, 37°C incubation for 4h to produce single-stranded DNA; then primerase Tth (Kaiyou Bio, TPP-TE101-C) or primerase Ccdc111 or duplex 1 / 3 (partner DNA template) and Sequenase mixture were added in the reaction, 37°C incubation for 5h to synthesize complementary strand using amplified single-stranded DNA as template. Each group of reaction was sampled 0.15μL for 1% agarose gel identification, 125V voltage, electrophoresis for 45min; according to Table 4, 40μL amplified sample was simultaneously sampled for TelN telomerase (Kaiyou Bio, TLN-BE001) treatment, 0.5μL of each group of reaction was sampled after TelN treatment for 1% agarose gel identification, 125V voltage, electrophoresis for 45min.

[0147] As shown in Figure 9, after adding primerase Tth or duplex 1 / 3 (partner DNA template) and Sequenase mixture in the system, >30kb DNA fragments (A) were amplified, and after the larger DNA fragments were treated with TelN enzyme (double-stranded DNA specific recognition enzyme), DNA fragments of corresponding target size (B) were produced, indicating that double-stranded DNA was effectively amplified in the reaction.

[0148] Table 3: Double-stranded DNA production system

[0149] Table 4: TelN enzyme digestion system

[0150] Example 6: Cascade amplification reaction of double-stranded DNA amplification

[0151] In the amplification product of Example 4, Group 3 (bi-directional partner DNA 2.0), the double-stranded DNA amplification product was used as a template according to Table 5; different components were added and mixed thoroughly, and incubated at 37°C for 13 h; 0.5 μL of each reaction was sampled for 1% agarose gel identification at 125 V for 45 min; 40 μL of each reaction was sampled for TelN enzyme treatment according to Table 4, and 0.5 μL of each reaction was sampled for 1% agarose gel identification after TelN treatment at 125 V for 45 min. As shown in FIG. 10, compared with the addition of ddH2O or dNTPs only, the further addition of dNTPs and Phi29 E20 resulted in further amplification of the target double-stranded DNA product.

[0152] Table 5: Cascade amplification system for double-stranded DNA

[0153] Example 7: Amplification of double-stranded DNA using T5 exonuclease

[0154] According to Table 6, in a 200 μL Eppendorf tube, each component was added in the following order: H2O, 10*buffer, plasmid containing nickase recognition site (N00313, SEQ ID NO. 10), dNTP, Nt.BbvCI nickase (NEB, R0632L), Phi29 E20, 0.05 U, 0.5 U or 5 U T5 exonuclease (Suzhou Jinan Protein, M035) or 0.5 U exonuclease III (Suzhou Jinan Protein, M007), pyrophosphatase (Suzhou Jinan Protein, M036), DTT, mixed thoroughly, and incubated at 37°C for 4 h to generate single-stranded DNA; then double-stranded body 1 (partner DNA template, corresponding to bi-directional partner DNA 2.0) and Sequenase mixture were added to the reaction, and incubated at 37°C for 5 h to synthesize the complementary strand using the amplified single-stranded DNA as a template. 0.15 μL of each reaction was sampled for 1% agarose gel identification at 125 V for 45 min; 40 μL of each reaction was sampled for TelN telomerase (Kaiyou Bio, TLN-BE001) treatment according to Table 4, and 0.5 μL of each reaction was sampled for 1% agarose gel identification after TelN treatment at 125 V for 45 min.

[0155] As shown in Figure 11, in the single-stranded DNA amplification system, after adding 5U T5 exonuclease or adding 0.5U exonuclease III, duplex 1 / 3 (companion DNA template) and Sequenase mixture, the amplification produced >30kb DNA fragments (A), and showed a dose-dependent relationship to T5 exonuclease, and after the larger DNA fragments were treated with TelN enzyme (double-stranded DNA specific recognition enzyme), DNA fragments of the corresponding size were produced (B), indicating that the reaction effectively amplified to produce double-stranded DNA. With the increase of T5 exonuclease in the single-stranded DNA amplification system, the yield of double-stranded DNA increased, and the yield of 5U T5 exonuclease in the system was basically the same as that of 0.5U exonuclease III.

[0156] Table 6 T5 exonuclease in the production of double-stranded DNA system

[0157] Example 8: Linearization enzyme digestion efficiency reaction of double-stranded DNA amplification product

[0158] The template sequence commonly used for mRNA expression of green fluorescent protein is shown as SEQ ID NO. 12. Using the mRNA commonly used plasmid, the amplification was carried out according to Example 6; then according to Table 7, in a 200 μL Eppendorf tube, H2O, 10*buffer, double-stranded DNA amplification product, BspQ I (Kaiyou Bio, BSP-BE101) were added in turn and mixed well, and incubated at 37°C for 2h for linearization enzyme digestion; 1 μL of each group of reaction was sampled for 1% agarose gel identification, 125V voltage, electrophoresis for 45min; at the same time, 20 μL of each group of reaction was sampled and added with H2O, 10*buffer, T5 exonuclease (Suzhou Jinan Protein, M035) and exonuclease 3 (Suzhou Jinan Protein, M007) according to Table 7, and incubated at 37°C for 4h. After exonuclease treatment, 2 μL of each group of reaction was sampled for 1% agarose gel identification, 125V voltage, electrophoresis for 45min. 10 uL of sample was quantified by dsDNA quantification kit (Thermo Fisher, Q33232);

[0159] ​As shown in FIG. 12, the BspQ I linearization enzyme digestion of the double-stranded DNA amplification product produced a DNA fragment of about 150 bp, and the exonuclease treatment could digest the BspQ I linearization product completely, while the product without BspQ I linearization enzyme digestion had no change after the exonuclease treatment, indicating that the linearization enzyme digestion efficiency of the double-stranded DNA amplification product was high. Meanwhile, the dsDNA quantification kit quantitative results showed that the double-stranded DNA concentration of the group treated by BspQ I linearization enzyme digestion and exonuclease treatment was 0.17 ng / μL, and the double-stranded DNA concentration of the group treated by exonuclease treatment only was 10.50 ng / μL, indicating that the linearization enzyme digestion efficiency of the double-stranded DNA amplification product could reach 98.4%.

[0160] Table 7 Linearization enzyme digestion system of double-stranded DNA amplification product

[0161] The above only describes the embodiments of the present disclosure, and does not limit the protection scope of the present disclosure; any equivalent structure or equivalent flow transformation made by using the content of the present disclosure specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the protection scope of the present disclosure.

Claims

1. A method for amplifying a target nucleic acid, comprising the following steps: Step (1): providing a first double-stranded nucleic acid, wherein the first double-stranded nucleic acid comprises a first strand and a second strand, wherein the first strand comprises a nucleic acid sequence identical to the target nucleic acid, the second strand comprises a nucleic acid sequence reversely complementary to the target nucleic acid, and the first strand and the second strand together comprise a first nickase recognition site; Step (2): contacting the double-stranded nucleic acid with an amplification starting mixture and reacting under appropriate conditions, wherein the amplification starting mixture includes a first nicking enzyme, a first nuclease and a first nucleic acid polymerase, wherein the first nicking enzyme is capable of recognizing the first nicking enzyme recognition site and forming a nick on the first chain.

2. The method according to claim 1, further comprising: Step (3): Add an amplification continuation mixture to the product of step (2) and react under appropriate conditions, wherein the amplification continuation mixture includes: (i) a first oligonucleotide, wherein the first oligonucleotide includes a sequence that is reverse complementary to the target nucleic acid; (ii) a primer enzyme; or (iii) a second double-stranded nucleic acid, a second nicking enzyme, and a second nucleic acid polymerase, wherein the second double-stranded nucleic acid includes a second nicking enzyme recognition site, and the second nicking enzyme is capable of recognizing the second nicking enzyme recognition site and forming a nick on one chain of the second double-stranded nucleic acid, and the 3' side of the nick includes a sequence that is reverse complementary to the target nucleic acid.

3. The method according to claim 1, further comprising: Step (3): Add an amplification continuation mixture to the product of step (2) and react under appropriate conditions, wherein the amplification continuation mixture includes: (i) a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide includes a sequence that is reverse complementary to the target nucleic acid, and the second oligonucleotide includes a sequence that is identical to the target nucleic acid; (ii) a primer; or (iii) a second double-stranded nucleic acid, a second nickase, and a second nucleic acid polymerase, wherein the second double-stranded nucleic acid contains a second nickase recognition site, and the second nickase is capable of recognizing the second nickase recognition site and forming nicks on the two chains of the second double-stranded nucleic acid, respectively, wherein the 3' side of the nick on one chain includes a sequence that is identical to or reverse complementary to the target nucleic acid, and the 3' side of the nick on the other chain includes a sequence that is reverse complementary to the target nucleic acid.

4. The method according to claim 2 or 3, further comprising: Step (4): adding an enzyme having double-strand cleavage activity to the product of step (3). 5 . The method according to claim 4 , wherein the enzyme having double-stranded cleavage activity further has an activity of generating a covalently closed end at the cleavage site, and optionally, the enzyme having double-stranded cleavage activity comprises telomerase.

6. The method according to claim 5, further comprising: Step (5): Add a second exonuclease to the product of step (4).

7. The method according to any one of the preceding claims, wherein the first double-stranded nucleic acid is circular.

8. The method according to any one of the preceding claims, wherein the second double-stranded nucleic acid is linear.

9. The method of any one of the preceding claims, wherein the amplification initiation mixture does not include primers or primases.

10. The method according to any one of the preceding claims, wherein the first nucleic acid polymerase comprises a nucleic acid polymerase that cannot utilize a nick to initiate nucleic acid extension in the absence of a primer or a primer enzyme, optionally comprising Phi29 DNA nucleic acid polymerase, Bst DNA nucleic acid polymerase, Bsu DNA nucleic acid polymerase, or Klenow DNA nucleic acid polymerase.

11. The method of any one of the preceding claims, wherein the first nucleic acid polymerase comprises the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

11.

12. A method according to any one of the preceding claims, wherein the first nickase and the second nickase independently include Nt.BspQI nickase, Nt.CviPII nickase, Nt.BstNBI nickase, Nb.BsrDI nickase, Nb.BtsI nickase, Nt.AlwI nickase, Nb.BbvCI nickase, Nt.BbvCI nickase, Nb.BsmI nickase, Nb.BssSI nickase, Nt.BsmA nickase, CRISPR nickase, ZFN nickase, TALE nickase or their functional analogs.

13. The method of any one of the preceding claims, wherein the first exonuclease comprises exonuclease I, II, III, IV, V, VI, VII, VIII, T7 exonuclease, T5 exonuclease, RecJf exonuclease, lambda exonuclease, exonuclease T, BAL-31 nuclease, mung bean nuclease, micrococcal nuclease, DNase I, snake venom phosphodiesterase, spleen phosphodiesterase, Lactobacillus acidophilus nuclease.

14. The method according to any one of the preceding claims, wherein the second nucleic acid polymerase comprises T7 DNA polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Bst DNA polymerase, Bsu DNA polymerase, or Klenow DNA polymerase.

15. The method of any one of the preceding claims, wherein the second exonuclease comprises exonuclease I, II, III, IV, V, VI, VII, VIII, T7 exonuclease, T5 exonuclease, RecJf exonuclease, lambda exonuclease, exonuclease T, BAL-31 nuclease, mung bean nuclease, micrococcal nuclease, DNase I, snake venom phosphodiesterase, spleen phosphodiesterase, Lactobacillus acidophilus nuclease.

16. A product obtained by the method of any one of claims 1 to 15.

17. A kit comprising a first nickase, a first exonuclease and a first nucleic acid polymerase.

18. The kit according to claim 17 further comprises a first double-stranded nucleic acid, wherein the first double-stranded nucleic acid comprises a first nicking enzyme recognition site, and the first nicking enzyme is capable of recognizing the first nicking enzyme recognition site and forming a nick on one chain of the first double-stranded nucleic acid.

19. The kit according to claim 17 or 18, further comprising a second double-stranded nucleic acid, wherein the second double-stranded nucleic acid comprises a second nickase recognition site, and the second nickase is capable of recognizing the second nickase recognition site and forming a nick on one chain of the second double-stranded nucleic acid or forming nicks on both chains of the second double-stranded nucleic acid.

20. A kit according to any one of claims 17-19, further comprising a second nickase.

21. The kit according to any one of claims 17 to 20, further comprising a second nucleic acid polymerase.

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