Method for amplifying circular DNA

Abstract

Provided is a method with which it is possible to simply and exponentially amplify circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for amplifying circular DNA, which comprises an isothermal reaction of a reaction mixture produced by mixing circular DNA having a replication initiation sequence (oriC) with a reaction solution containing the following enzyme groups: (1) a first enzyme group that catalyzes replication of the circular DNA, (2) a second enzyme group that catalyzes replication of the circular DNA, (3) a third enzyme group that catalyzes replication of the circular DNA, (4) a fourth enzyme group that catalyzes replication of the circular DNA, and (5) a third enzyme group that catalyzes replication of the circular DNA. (2) a second enzyme group which catalyzes a linking reaction of an okazaki fragment to synthesize two sister circular DNAs forming a catenary body; and (3) a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs.

Description

[0001] This application is a divisional application of the invention patent application of the same name 201580063334.6 filed on November 18, 2015. [Technical field]

[0002] The present invention relates to a method for amplifying circular DNA, and more particularly to a method for exponentially amplifying circular DNA in a cell-free system. [Background technology]

[0003] DNA cloning technology, which has become the foundation for the development of biotechnology, is a method of amplifying circular DNA modulated by cyclization of DNA fragments as a plasmid in cells such as Escherichia coli. When amplifying circular DNA using DNA cloning technology using cells, a cumbersome procedure such as cell culture and extraction and purification of amplified products becomes necessary. In addition, since it is necessary to produce genetically modified organisms in order to perform DNA cloning using cells, there are limitations on the experimental environment.

[0004] As a method for amplifying DNA in a test tube, polymerase chain reaction (PCR) is generally used. However, in the test tube DNA amplification method by PCR, circular DNA cannot be amplified directly. As a test tube amplification method for circular DNA, there are rolling circle amplification (RCA) and the like (non-patent document 1, patent document 1, patent document 2, patent document 3). However, in order to amplify circular DNA by the rolling circle amplification method, it is necessary to design primers that are specific to the target DNA each time. In addition, the direct amplification product by the rolling circle amplification method is a linear DNA. In order to circularize the obtained amplification product, further circularization steps such as incubation with a recombinase become necessary. Although there is also a report of replicating the minichromosome (oriC circular DNA) of Escherichia coli, separating it, and obtaining a method of a monomeric replication product (non-patent document 2), it is only a circular DNA of 6kbp that can be amplified. In addition, although there is a report on the use of a purified enzyme group derived from E. coli to reconstruct the replication of the E. coli minichromosome (Non-Patent Document 3), there is no known method for repeating the replication cycle in a single test tube to exponentially amplify long circular DNA in a test tube.

[0005] Thus, in order to amplify circular DNA using conventional in vitro DNA amplification methods, it is necessary to bind primers to template DNA, and the amplification product is linear DNA. In addition, there is a problem that the size of DNA that can be amplified is only a few kb.

[0006] As a technique for modulating long DNA, the SLIC method or the Gibson Assembly method are proposed, but when any of these techniques are used, the use of cells is also required in order to finally modulate long-chain circular DNA (Non-patent Document 4). As a method for making long-chain circular DNA using cells, for example, a method using Bacillus subtilis is reported (Non-patent Document 5).

[0007] [Prior art literature]

[0008] [Patent Literature]

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-229950

[0010] Patent Document 2: Japanese Patent Application Laid-Open No. 2008-161182

[0011] Patent Document 3: Japanese Patent Application No. 2012-501173

[0012]

Non-patent literature

[0013] Non-patent literature 1: Fakruddin M et al., J Pharm Bioallied Sci. 2013, 5: 245-252

[0014] Non-patent document 2: Peng H & Marians KJ. PNAS. 1993, 90: 8571-8575

[0015] Non-patent document 3: Kaguni JM & Kornberg A. Cell. 1984, 38: 183-90

[0016] Non-patent literature 4: Chao R et al., FEMS YEAST Res. 2014

[0017] Non-patent document 5: Tsuge et al., Nucleic Acids Res. 2003, 31: e133 [Summary of the invention]

[0018] [Technical issues to be solved by the invention]

[0019] The present invention aims to provide a method for simply and exponentially amplifying circular DNA, especially long-chain circular DNA, in a cell-free system.

[0020] [Technical solution to the problem]

[0021] As a result of repeated and intensive research to solve the above-mentioned problems, the present inventors found that a reaction mixture generated by mixing a circular DNA having a replication initiation sequence (origin of chromosome (oriC)) with a reaction solution containing the following enzyme group was subjected to an isothermal reaction:

[0022] (1) The first enzyme group catalyzing the replication of circular DNA;

[0023] (2) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0024] (3) a third enzyme group that catalyzes the separation reaction of two sister circular DNAs,

[0025] The circular DNA can be amplified exponentially by repeating the cycle of "initiation of replication (breakage of DNA double bonds)·elongation (progression of replication forks)·separation of replicated sister DNAs (Decatenation)".

[0026] That is, the present invention includes the inventions of the following embodiments, although not limited thereto. [1]

[0028] A method for amplifying circular DNA, comprising the following steps:

[0029] (1) A step of forming a reaction mixture containing a reaction solution of the following enzyme group and a circular DNA serving as a template:

[0030] The first enzyme group to catalyze the replication of circular DNA;

[0031] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0032] The third enzyme group catalyzes the separation reaction of the two sister circular DNAs.

[0033] The circular DNA contains a replication initiation sequence (origin of chromosome (oriC)) capable of binding to an enzyme having DnaA activity; and

[0034] (2) A step of maintaining the reaction mixture formed in the step (1) under isothermal conditions. [2]

[0036] [1] The method, wherein

[0037] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0038] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0039] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity. [3]

[0041] [2] The method according to claim 1, wherein the replication initiation sequence capable of binding to an enzyme having DnaA activity is a replication initiation sequence of Escherichia coli. [4]

[0043] The method according to any one of [2] or [3], wherein the size of the circular DNA is greater than 50 kb. [5]

[0045] The method according to any one of [2] to [4], wherein the one or more nucleoid proteins are a combination of IhfA and IhfB. [6]

[0047] The method according to any one of [2] to [5], wherein the enzyme or enzyme group having DNA gyrase activity is a combination of GyrA and GyrB. [7]

[0049] The method according to any one of [2] to [6], wherein the enzyme having DNA helicase activity is DnaB helicase. [8]

[0051] The method according to any one of [2] to [7], wherein the enzyme having DNA helicase loading activity is a DnaC helicase loading activity. [9]

[0053] The method according to any one of [2] to [8], wherein the enzyme having DNA priming activity is DnaG priming enzyme.

[10]

[0055] The method according to any one of [2] to [9], wherein the enzyme having DNA clamp activity is DnaN clamp.

[11]

[0057] The method according to any one of [2] to

[10] , wherein the enzyme or enzyme group having DNA polymerase III activity is a combination of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE.

[12]

[0059] The method according to any one of [2] to

[11] , wherein the enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB)), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, an enzyme or enzyme group having DNA polymerase III activity, an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity are derived from Escherichia coli.

[13]

[0061] The method according to any one of [1] to

[12] , wherein the reaction solution in step (1) further contains a component that inhibits the formation of by-products.

[14]

[0063] The method according to any one of [1] to

[13] , wherein the isothermal condition in step (2) is a constant temperature in the range of 25°C to 50°C.

[15]

[0065] A composition for amplifying circular DNA, comprising

[0066] The first enzyme group to catalyze the replication of circular DNA;

[0067] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0068] The third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[16]

[0070]

[15] The composition, wherein

[0071] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0072] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0073] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[17]

[0075] A kit for amplifying circular DNA, comprising

[0076] The first enzyme group to catalyze the replication of circular DNA;

[0077] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0078] The third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[18]

[0080]

[17] The kit, wherein

[0081] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0082] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0083] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[0084] [Effects of the invention]

[0085] The present invention provides a method for exponentially amplifying circular DNA, especially long-chain circular DNA, without using E. coli cells or plasmid vectors. According to the present invention, temperature control cycles or primers are not required for amplifying circular DNA, and amplification of long-chain circular DNA exceeding 200 kb is also possible. In addition, the amplified product obtained by the present invention is a copy of the same circular structure as the original template. Furthermore, after connecting multiple DNA fragments, they are directly added to the reaction system, so that only the modulation by connecting circularized DNA can be specifically amplified.

Brief Description of the Drawings

[0086]

Figure 1

[0087]

Figure 2

[0088]

Figure 3

[0089]

Figure 4

[0090]

Figure 5

[0091]

Figure 6

[0092]

Figure 7

[0093]

Figure 8

[0094]

Fig. 9

[0096] <Circular DNA>

[0097] The circular DNA used as a template preferably has double bonds. The circular DNA used as a template is not particularly limited as long as it contains a replication initiation sequence (origin of chromosome (oriC)) that can bind to an enzyme having DnaA activity. Examples include natural circular DNA such as circular chromosomes of microorganisms, natural circular DNA cut by enzyme treatment, etc., connected to other DNA fragments to make it circular, and all artificially synthesized circular DNA. As the replication initiation sequence (origin of chromosome (oriC)) that can bind to an enzyme having DnaA activity (hereinafter, sometimes referred to as "replication initiation sequence"), a known replication initiation sequence present in bacteria such as Escherichia coli and Bacillus subtilis can be obtained from public databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / ). In addition, the replication initiation sequence can also be obtained by cloning a DNA fragment that can bind to an enzyme having DnaA activity and analyzing its base sequence.

[0098] The circular DNA used as a template in the present invention may be a circular DNA originally containing a replication initiator sequence, or may be a circular DNA originally not containing a replication initiator sequence into which a replication initiator sequence has been introduced.

[0099] The circular DNA used as a template in the present invention may contain a gene sequence of a drug resistance marker such as kanamycin, ampicillin, or tetracycline, depending on the purpose.

[0100] The circular DNA used as a template in the present invention may be purified or in the form of a suspension of a bacterial extract containing the circular DNA. In addition, a single circular DNA may be used as a template, or a mixture of multiple circular DNAs such as a DNA library may be used as a template in one test tube.

[0101] The amount of template DNA used per reaction is not particularly limited, and one molecule of circular DNA may be used as a template per reaction.

[0102] The length of the circular DNA used as a template in the present invention is not limited, and can be set to, for example, 1 kb (1000 bases long) or more, 5 kb (5000 bases long) or more, 8 kb (8,000 bases long) or more, 10 kb (10,000 bases long) or more, 50 kb (50,000 bases long) or more, 100 kb (100,000 bases long) or more, 200 kb (200,000 bases long) or more, 500 kb (500,000 bases long) or more, 1000 kb (1,000,000 bases long) or more, or 2000 kb (2,000,000 bases long) or more.

[0103] In the present invention, the circular DNA described above is used as a template and can be amplified at least 10 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, or 10000 times.

[0104] <First, Second and Third Enzyme Groups>

[0105] The present invention relates to a method of isothermally reacting a reaction mixture generated by mixing a circular DNA having a replication initiation sequence (origin of chromosome (oriC)) with a reaction solution containing the following enzyme group:

[0106] (1) The first enzyme group catalyzing the replication of circular DNA;

[0107] (2) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0108] (3) A third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[0109] Without being limited by theory, the present invention repeats Figure 1 Replication cycles are shown, exponentially amplifying circular DNA.

[0110] <(1) The first enzyme group catalyzing the replication of circular DNA>

[0111] As the first enzyme group that catalyzes the replication of circular DNA, for example, the enzyme group described in Kaguni JM & Kornberg A. Cell. 1984, 38: 183-90 can be used. Specifically, as the first enzyme group, one of the following enzymes or enzyme groups, or a combination of the enzymes or enzyme groups can be exemplified: an enzyme with DnaA activity, one or more nucleoproteins, an enzyme or enzyme group with DNA gyrase activity, a single-stranded DNA binding protein (SSB), an enzyme with DNA helicase activity, an enzyme with DNA helicase loading activity, an enzyme with DNA priming enzyme activity, an enzyme with DNA clamp activity, and an enzyme or enzyme with DNA polymerase III activity.

[0112] The first enzyme group may be contained in the reaction solution in a range of 1 nM to 10 μM. Preferably, the first enzyme group is contained in the reaction solution in a range of 1 nM to 5 μM, 1 nM to 3 μM, or 1 nM to 1.5 μM, but is not limited thereto.

[0113] The enzyme having DnaA activity is not particularly limited in biological origin as long as it has the same initiator activity as DnaA, which is an initiator protein of Escherichia coli, and DnaA derived from Escherichia coli can be suitably used. DnaA may be contained in the reaction solution in a range of 1 nM to 10 μM, and may preferably be contained in a range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 200 nM, or 50 nM to 150 nM, but is not limited thereto.

[0114] Nucleoid protein refers to a protein contained in the nucleoid. As long as the one or more nucleoid proteins used in the present invention are enzymes with the same activity as the nucleoid proteins of Escherichia coli, their biological sources are not particularly limited. For example, IHF from Escherichia coli, i.e., a combination of IhfA and IhfB, or HU, i.e., a complex of hupA and hupB, can be suitably used. IHF or HU can be contained in the reaction solution in the range of 1nM to 10μM, and can also be preferably contained in the range of 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 200nM, 1nM to 100nM, but is not limited thereto.

[0115] As the enzyme or enzyme group having DNA gyrase activity, there is no particular limitation on its biological origin as long as it is an enzyme having the same activity as the DNA gyrase of Escherichia coli, and for example, a combination of GyrA and GyrB derived from Escherichia coli can be suitably used. The combination of GyrA and GyrB can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 200 nM, and 1 nM to 100 nM, but is not limited thereto.

[0116] As the single-stranded DNA binding protein (SSB), there is no particular limitation on its biological origin as long as it is an enzyme having the same activity as the single-stranded DNA binding protein of Escherichia coli, and for example, SSB derived from Escherichia coli can be suitably used. SSB can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 500 nM, and 100 nM to 500 nM, but is not limited thereto.

[0117] As the enzyme having DNA helicase activity, there is no particular limitation on its biological origin as long as it has the same activity as DnaB of Escherichia coli, and for example, DnaB derived from Escherichia coli can be suitably used. DnaB can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 200 nM, and 1 nM to 100 nM, but is not limited thereto.

[0118] As the enzyme having DNA helicase loading activity, there is no particular limitation on its biological origin as long as it has the same activity as DnaC of Escherichia coli, and for example, DnaC derived from Escherichia coli can be suitably used. DnaC can be contained in the reaction solution in the range of 1nM to 10μM, and can also be preferably contained in the range of 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 200nM, and 1nM to 100nM, but is not limited thereto.

[0119] As the enzyme having DNA priming activity, there is no particular limitation on its biological origin as long as it has the same activity as DnaG of Escherichia coli, and for example, DnaG derived from Escherichia coli can be suitably used. DnaG can be contained in the reaction solution in the range of 1 nM to 10 μM, preferably in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 500 nM, 100 nM to 500 nM, but is not limited thereto.

[0120] As the enzyme having DNA clamping activity, there is no particular limitation on its biological origin as long as it has the same activity as DnaN of Escherichia coli, and for example, DnaN derived from Escherichia coli can be suitably used. DnaN can be contained in the reaction solution in the range of 1nM to 10μM, and can also be preferably contained in the range of 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 200nM, and 1nM to 100nM, but is not limited thereto.

[0121] As the enzyme or enzyme group having DNA polymerase III* activity, there is no particular limitation on its biological origin as long as it has the same activity as the DNA polymerase III* complex of Escherichia coli, and for example, a combination of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli can be suitably used. The DNA polymerase III* complex may be contained in the reaction solution in the range of 1 nM to 10 μM, and may be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 100 nM, or 1 nM to 50 nM, but is not limited thereto.

[0122] <(2) A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer>

[0123] In the present invention, two sister circular DNAs forming a concatemer refer to two circular DNAs synthesized by a DNA replication reaction in a linked state.

[0124] Examples of the second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs forming a concatemer include one enzyme or a combination of enzymes selected from the group consisting of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0125] The second enzyme group may be contained in the reaction solution in the range of 1 nM to 10 μM. Preferably, the second enzyme group is contained in the reaction solution in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 200 nM, but is not limited thereto.

[0126] The enzyme having RNase H activity is not particularly limited in biological origin as long as it has activity to decompose the RNA chain of RNA:DNA hybrids, and for example, RNase H derived from Escherichia coli can be suitably used. RNase H can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 200 nM, and 1 nM to 100 nM, but is not limited thereto.

[0127] As an enzyme having DNA polymerase I activity, there is no particular limitation on its biological origin as long as it has the same activity as DNA polymerase I of Escherichia coli, and for example, DNA polymerase I derived from Escherichia coli can be suitably used. DNA polymerase I can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 200 nM, and 1 nM to 100 nM, but is not limited thereto.

[0128] As an enzyme having DNA ligase activity, there is no particular limitation on its biological origin as long as it has the same activity as DNA ligase of Escherichia coli, and for example, DNA ligase derived from Escherichia coli can be suitably used. DNA ligase can be contained in the reaction solution in the range of 1nM to 10μM, preferably 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 200nM, and 1nM to 100nM, but is not limited thereto.

[0129] <(3) The third enzyme group catalyzing the separation reaction of two sister circular DNAs>

[0130] As the third enzyme group for catalyzing the separation reaction of two sister circular DNAs, the enzyme group described in Peng & Marians 1993 PNAS can be used. Specifically, as the third enzyme group, one enzyme or a combination of the enzymes selected from the following can be exemplified: an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[0131] The third enzyme group may be contained in the reaction solution in the range of 1 nM to 10 μM. Preferably, the third enzyme group is contained in the reaction solution in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 200 nM, but is not limited thereto.

[0132] As an enzyme having topoisomerase IV activity, there is no particular limitation on its biological origin as long as it has the same activity as topoisomerase IV of Escherichia coli, and for example, topoisomerase IV derived from Escherichia coli as a complex of ParC and ParE can be suitably used. Topoisomerase IV can be contained in the reaction solution in the range of 1nM to 10μM, and can also be preferably contained in the range of 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 100nM, 1nM to 50nM, but is not limited thereto.

[0133] As an enzyme having topoisomerase III activity, there is no particular limitation on its biological origin as long as it has the same activity as topoisomerase III of Escherichia coli, and for example, topoisomerase III derived from Escherichia coli can be suitably used. Topoisomerase III can be contained in the reaction solution in the range of 1 nM to 10 μM, and can also be preferably contained in the range of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 1 nM to 200 nM, and 1 nM to 100 nM, but is not limited thereto.

[0134] As an enzyme having RecQ activity, there is no particular limitation on its biological origin as long as it has the same activity as RecQ of Escherichia coli, and for example, RecQ derived from Escherichia coli can be suitably used. RecQ can be contained in the reaction solution in the range of 1nM to 10μM, preferably 1nM to 5μM, 1nM to 3μM, 1nM to 1.5μM, 1nM to 1.0μM, 1nM to 500nM, 1nM to 200nM, and 1nM to 100nM, but is not limited thereto.

[0135] Particularly preferably, as the first enzyme group, a combination of DnaA, IHF, GyrA and GyrB, SSB, DnaB and DnaC, DnaG, DnaN, and polIII* derived from E. coli can be used; as the second enzyme group, a combination of RNase H, DNA polymerase I, and DNA ligase derived from E. coli can be used; and, as the third enzyme group, a combination of topoisomerase IV, topoisomerase III, and RecQ derived from E. coli can be used. Furthermore, the concentration of each enzyme when using the combination of the above enzyme groups is exemplified below, but is not limited thereto.

[0136]

Table 1

[0137]

[0138] The first, second and third enzyme groups may be commercially available or extracted from microorganisms and purified as needed. The extraction and purification of enzymes derived from microorganisms may be appropriately carried out using methods available to those skilled in the art.

[0139] Alternatively, a reaction mixture for amplification of circular DNA may be prepared by directly mixing a reaction solution of a cell-free protein expression system containing the above enzyme with a circular DNA serving as a template.

[0140] The cell-free protein expression system may be a cell-free translation system containing total RNA, mRNA, or in vitro transcription products of RNA consisting of a sequence complementary to the base sequence of the gene encoding the above-mentioned enzyme as a template RNA, or may be a cell-free transcription-translation system containing genes encoding each enzyme or expression vectors containing genes encoding each enzyme as template DNA.

[0141] <Circular DNA Amplification Method>

[0142] The method of the present invention includes the step of forming a reaction mixture containing a reaction solution of the first to third enzyme groups and a circular DNA serving as a template.

[0143] The composition of the reaction solution containing the first to third enzyme groups is not particularly limited as long as it can carry out DNA replication reaction. For example, the first to third enzyme groups can be added to a solution in which rNTP, dNTP, a magnesium ion source, an ATP source, etc. are added to a buffer such as Tris hydrochloric acid buffer. In addition, the above-mentioned reaction solution may also contain additional components such as components that inhibit the generation of by-products. As specific reaction solutions, those described in the embodiments described below can be exemplified.

[0144] The method of the present invention further comprises a step of keeping the reaction mixture warm under isothermal conditions. As isothermal conditions, there are no particular restrictions as long as the DNA replication reaction can be carried out, and for example, the isothermal conditions can be set to a certain temperature in the range of 20 to 80° C., which is the optimum temperature of DNA polymerase, or a certain temperature in the range of 25° C. to 50° C., or about 30° C. The warming time can be appropriately set according to the amount of the amplified product of the target circular DNA, and for example, can be set to 1 to 24 hours.

[0145] The method of the present invention may further include a step of purifying the amplified circular DNA product after the step of isothermally incubating the reaction mixture, depending on the purpose. The purification of circular DNA can be appropriately carried out using a method available to those skilled in the art.

[0146] The circular DNA amplified by the method of the present invention can be used for subsequent purposes such as transformation directly or by appropriately purifying the reaction mixture after the reaction.

[0147] <Composition and kit for amplifying circular DNA>

[0148] The present invention also relates to a composition for amplifying circular DNA, comprising a first enzyme group that catalyzes the replication of circular DNA; a second enzyme group that catalyzes the ligation reaction of Okazaki fragments to synthesize two sister circular DNAs that form a concatemer; and a third enzyme group that catalyzes the separation reaction of the two sister circular DNAs. The composition of the present invention may contain additional components such as stabilizers, reducing agents, reaction buffers, etc., as long as they do not hinder the effects of the present invention.

[0149] In addition, the present invention also relates to a kit for amplifying circular DNA, which comprises a first enzyme group that catalyzes the replication of circular DNA; a second enzyme group that catalyzes the ligation reaction of Okazaki fragments to synthesize two sister circular DNAs that form a concatemer; and a third enzyme group that catalyzes the separation reaction of the two sister circular DNAs. The kit of the present invention may contain a mixture of the above enzymes packaged as one, or may contain the above enzymes individually, or a combination of several of them packaged individually.

[0150] In addition, the present invention also relates to a kit for amplifying circular DNA, which comprises a first enzyme group that catalyzes the replication of circular DNA; a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and a cell-free protein expression system for each enzyme of the third enzyme group that catalyzes the separation reaction of the two sister circular DNAs.

[0151] The kit of the present invention may be a concentrate containing a reaction buffer or an additional component containing a reaction buffer or the like, depending on the purpose. [Example]

[0152] Hereinafter, the present invention will be specifically described based on examples. Note that the present invention is not limited to the scope described in the following examples.

[0153] [Example 1: Amplification of circular DNA]

[0154] <Materials and Methods>

[0155] Template DNA was added to the reaction solution of the composition shown in Table 2, mixed on ice, and then incubated in an incubator at 30°C for 1 hour, 2 hours, or 3 hours. The total volume per reaction was set to 10 μl. After the reaction at 30°C, the reaction product was subjected to agarose gel electrophoresis (0.5% 1×TAE, 150V, 100 minutes, 14°C), and DNA was detected using SyberGreen (Takara Bio Co., Ltd.).

[0156]

Table 2

[0157]

[0158]

[0159] In the table, SSB represents SSB derived from Escherichia coli, IHF represents a combination of IhfA and IhfB derived from Escherichia coli, DnaG represents DnaG derived from Escherichia coli, DnaN represents DnaN derived from Escherichia coli, PolIII* represents a DNA polymerase III* complex composed of a combination of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli, DnaB represents DnaB derived from Escherichia coli, DnaC represents DnaC derived from Escherichia coli, DnaA represents RNase H derived from Escherichia coli, Ligase represents DNA ligase derived from Escherichia coli, PolI represents DNA polymerase I derived from Escherichia coli, GyrA represents GyrA derived from Escherichia coli, GyrB represents GyrB derived from Escherichia coli, Topo IV represents a combination of ParC and ParE derived from Escherichia coli, TopoIII represents topoisomerase III derived from Escherichia coli, and RecQ represents RecQ derived from Escherichia coli.

[0160] SSB was prepared by purifying an Escherichia coli strain expressing SSB through a process of ammonium sulfate precipitation and ion exchange column chromatography.

[0161] IHF was prepared by purifying from an Escherichia coli strain co-expressing IhfA and IhfB using a process of ammonium sulfate precipitation and affinity column chromatography.

[0162] DnaG was prepared by purifying a DnaG-expressing Escherichia coli strain through the steps of ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography.

[0163] DnaN was prepared by purifying a DnaN-expressing Escherichia coli strain through a process of ammonium sulfate precipitation and anion exchange column chromatography.

[0164] PolIII* was prepared by purifying from a strain of Escherichia coli that co-expresses DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE using a process of ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

[0165] DnaB and DnaC were prepared by purifying from a strain of Escherichia coli that co-expresses DnaB and DnaC using a process of ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

[0166] DnaA was prepared by purifying a DnaA-expressing Escherichia coli strain through the steps of ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography.

[0167] GyrA and GyrB were prepared by purifying a mixture of a GyrA-expressing Escherichia coli strain and a GyrB-expressing Escherichia coli strain through the steps of ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

[0168] Topo IV was prepared by purifying a mixture of a ParC-expressing Escherichia coli strain and a ParE-expressing Escherichia coli strain through a process of ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

[0169] Topo III was prepared by purifying a Topo III-expressing Escherichia coli strain through a process of ammonium sulfate precipitation and affinity column chromatography.

[0170] RecQ was prepared by purifying a RecQ-expressing Escherichia coli strain through the steps of ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

[0171] As RNase H, Ligase, and Poll, commercially available enzymes derived from Escherichia coli (Takara Bio Co., Ltd.) were used.

[0172] As template DNA, 9.6 kb circular DNA (circular DNA with replication initiation sequence oriC, kanamycin resistance (Km)), 80 kb long-chain circular DNA (circular DNA with replication initiation sequence oriC, kanamycin resistance (Km)), 200 kb long-chain circular DNA (circular DNA with replication initiation sequence oriC, kanamycin resistance (Km)), or DNA circularized by in vitro ligation were used.

[0173] 9.6 kb circular DNA and 80 kb and 200 kb long circular DNA were prepared by intracellular recombination reaction in E. coli cells. Specifically, using E. coli expressing the recombinant proteome of λ phage, circular DNA of desired length containing a kanamycin resistance cassette and a region containing oriC in the E. coli chromosome was prepared by intracellular recombination reaction.

[0174] The method for preparing circularized DNA by in vitro ligation is shown in Figure 2. Specifically, a PCR fragment (2.3 kb) having the replication initiation sequence oriC and kanamycin resistance (Km) was connected to a PCR fragment (hereinafter referred to as dnaA-dnaN fragment) (2.6 kb) having the dnaA gene and dnaN gene sequences by the Gibson assembly method. The two PCR fragments were connected to form a circular structure via the same 20-base sequence (referred to as H1, H2) attached to both ends. The reaction was carried out by mixing the above two PCR fragments with Gibson Assembly Master Mix (NEB) and reacting at 50°C for 15 minutes. After the reaction, 0.1 μl of the reaction solution (10-fold diluted solution per 1 μl of the reaction solution) was used as a template DNA and directly added to 10 μl of the amplification reaction system (composition of Table 2), and reacted at 30°C for 1 hour.

[0175] <Result 1> Using 9.6 kb circular DNA (0.08 ng, about 10 7 When using a cyclic molecule) as a template

[0176] The detection results of the amplified products by SyberGreen are shown in Figure 3 .

[0177] Although byproducts (reaction intermediates) were also observed, a circular DNA amplification product having a supercoiled structure (indicated by a black frame) was confirmed.

[0178] The amount of circular DNA amplification was determined by transforming Escherichia coli DH5α strain using the reaction solution as it is, culturing in agar medium containing kanamycin, and counting the number of colonies. The results are shown in Table 3. As a control, a reaction solution with an incubation time of 0 hour was used.

[0179]

Table 3

[0180] Insulation time 0 hours 3 hours Transformants (colony number) 9 <![CDATA[5.4×10 4 ]]>

[0181] According to the method of the present invention, a 9.6 kb circular DNA can be amplified approximately 6,000 times as a circular DNA.

[0182] <Result 2> When 80kb and 200kb long circular DNA were used as templates

[0183] The detection results of the amplified products by SyberGreen are shown in Figure 4 .

[0184] A circular DNA amplification product with a supercoiled structure can be confirmed (indicated by a black frame or arrow).

[0185] It was found that even when a circular DNA as long as 80 kb or 200 kb was used as a template, a good amplification product was obtained by the method of the present invention.

[0186] <Result 3> When DNA circularized by in vitro ligation was used as a template

[0187] The detection results of the amplified products by SyberGreen are shown in Figure 5 .

[0188] It was found that the method of the present invention can also yield a good amplification product when a DNA circularized by in vitro ligation is used as a template.

[0189] The result of transforming Escherichia coli DH5α strain using the solution after the reaction as it is, culturing in agar medium containing kanamycin, and counting the number of colonies to determine the amount of circular DNA amplification is shown in Table 4. As a control, a sample not subjected to amplification reaction was used.

[0190]

Table 4

[0191] Amplification reaction - + Transformants (colony number) <![CDATA[3.2×10 2 ]]> <![CDATA[6.6×10 5 ]]>

[0192] It was found that the DNA circularized by the Gibson Assembly method was amplified approximately 2,000 times as a circular molecule by the method of the present invention.

[0193] [Example 2: Amplification of circular DNA from a small number of template molecules]

[0194] Using the 9.6 kb circular DNA described in Example 1, an amplification reaction was carried out in the same manner as in Example 1.

[0195] (2-1) Discussion on amplification efficiency

[0196] The amplification reaction was performed by adding 9.6 kb circular DNA to 10 μl of the amplification reaction solution (Example 1, Table 2) until 1 to 1000 molecules were contained as circular molecules, and incubating at 30°C for 3 hours. The reaction product was subjected to 0.5% agarose gel electrophoresis and stained with SybrGreen (Takara Bio Co., Ltd.) to detect the amplified DNA ( Figure 6 In addition, the total DNA amount of the amplified byproducts was quantified using a PicoGreen detection kit (ThermoFisher). Figure 6 b: PicoGreen method). The amplified product was directly transformed into E. coli, and the number of kanamycin-resistant colonies was determined to quantify the amplified amount of circular DNA molecules ( Figure 6 b: transformation method) The degree of amplification relative to the initial DNA amount was determined from the quantitative results and shown in a graph.

[0197] From the above results, it is known that one molecule of circular DNA can be amplified to about 10 11 molecules (approximately 100 billion-fold amplification).

[0198] (2-2) Discussion on doubling time

[0199] The above circular DNA was added to 80 μl of the amplification reaction solution (Example 1, Table 2) and incubated at 30°C to perform an amplification reaction. The circular DNA was added to 10 μl of the reaction solution. 5 By sampling over time, the samples were directly transformed into E. coli, and the number of kanamycin-resistant colonies was determined to quantify the number of amplified circular DNA molecules ( Figure 7 ).

[0200] From the above results, it was confirmed that the doubling time of the 9.6 kb circular DNA molecule was about 5 minutes.

[0201] [Example 3: Amplification of a single circular DNA clone from a mixture]

[0202] A single circular DNA clone was amplified from a mixture of the 9.6 kb circular DNA and the 12.0 kb circular DNA described in Example 1 (circular DNA having the replication initiator sequence oriC, kanamycin resistance (Km)).

[0203] The 12.0 kb circular DNA was prepared by intracellular recombination reaction in E. coli cells. Specifically, using E. coli cells expressing the recombinant proteome of λ phage, a circular DNA of a desired length containing a cassette consisting of oriC and a kanamycin resistance gene and a region of a part of the E. coli chromosome was prepared by intracellular recombination reaction.

[0204] The mixture of the two circular DNAs was diluted to 15 molecules or 1.5 molecules each in the reaction solution and added to 10 μl of the amplification reaction solution (Example 1, Table 2), and incubated at 30°C for 6 hours to perform an amplification reaction. The reaction product was subjected to 0.5% agarose gel electrophoresis and stained with SybrGreen (Takara Bio Co., Ltd.) to detect the amplified DNA ( Figure 8 ).

[0205] As a result, in the reaction solution where the circular DNA was diluted to 1.5 molecules, only one clone was amplified for each reaction sample. This shows that even if the template DNA is a mixture, a single circular DNA clone can be amplified by diluting it to the 1-molecule level in the reaction solution.

[0206] [Example 4: Subculture and Amplification]

[0207] Using lacZ circular DNA, experiments were conducted on the passage and amplification of circular DNA.

[0208] lacZ circular DNA (9.0 kb) was prepared by ligating a double-stranded DNA fragment (1.0 kb) containing oriC, a double-stranded DNA fragment (4.6 kb) containing a kanamycin resistance gene (Km), and a double-stranded DNA fragment (3.4 kb) containing a lacZ (β-galactosidase) gene. The lacZ circular DNA was added to 10 μl of the amplification reaction solution (Example 1, Table 2) to contain 1,000 molecules, and the amplification reaction was carried out by incubating at 30°C for 3 hours, which was used as the first passage. The amplification reaction product at the above passage number was diluted 10 5 The amplification product of each passage was subjected to 0.5% agarose gel electrophoresis and stained with SybrGreen (Takara Bio Co., Ltd.) to detect ( Fig. 9 ). In addition, a portion of the amplified product was transformed into Escherichia coli, and the degree of DNA amplification was quantified by the number of kanamycin-resistant colonies. From this value, it was calculated whether the exponential amplification was repeated for several generations and displayed as the total number of generations.

[0209] The results show that the circular DNA can still be effectively amplified after 10 passages. Figure 7 ), if the circular DNA is amplified to a certain extent, the amplification rate reaches a peak as the substrate or enzyme runs out. On the one hand, the results of this example show that the exponential amplification of the circular DNA can be repeated semi-permanently by subculturing a part of the amplification reaction with a new reaction solution. That is, the method of the present invention is a method that can amplify the circular DNA like the subculturing and proliferation of cells.

[0210] [Example 5: Incidence of replication errors in amplification reactions]

[0211] Since the circular DNA template in Example 4 contains the lacZ gene, if E. coli transformed with this circular gene is cultured on an X-gal plate, colonies expressing the lacZ gene normally (lacZ + ) The colony is blue because it can degrade X-gal. The lacZ gene cannot function normally due to the introduction of replication errors (lacZ - ) Since X-gal cannot be decomposed, it appears white. That is, the replication error in the amplified circular DNA can be judged by the color of the E. coli transformed with this circular gene on the X-gal plate.

[0212] The amplified reaction products of each passage sample were transformed into E. coli directly and cultured on X-gal plates to determine the lacZ -Since then, lacZ - The error rate per generation of the replication cycle was calculated based on the appearance rate and the total number of generations determined in Example 4 according to the method of Barnes (Barnes WM Gene. 1992, 112, 29-35). The results are shown in Table 5 below.

[0213]

Table 5

[0214] Generation Number Total generations <![CDATA[lacZ - Colony appearance rate (%)]]> Error rate (per base) 1 25 <0.045 <![CDATA[<3.7×10 -8 ]]> 2 44 <0.074 <![CDATA[<3.4×10 -8 ]]> 5 106 0.1 <![CDATA[1.9×10 -8 ]]> 10 201 0.093 <![CDATA[0.93×10 -8 ]]>

[0215] The above results show that the replication error is about 1 position per 100 million bases (an average of 1.4x 10 -8 This is about the same level as the mutation rate in cells (strains without mismatch repair systems) and is about 10,000 times more accurate than Taq polymerase.

[0216] This manual also includes the following contents:

[0217] 1. A method for amplifying circular DNA, comprising the following steps:

[0218] (1) A step of forming a reaction mixture containing a reaction solution of the following enzyme group and a circular DNA serving as a template:

[0219] The first enzyme group to catalyze the replication of circular DNA;

[0220] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0221] The third enzyme group catalyzes the separation reaction of the two sister circular DNAs.

[0222] The circular DNA contains a replication initiation sequence (origin of chromosome (oriC)) capable of binding to an enzyme having DnaA activity; and

[0223] (2) A step of maintaining the reaction mixture formed in the step (1) under isothermal conditions.

[0224] 2. The method of embodiment 1, wherein

[0225] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0226] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0227] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[0228] 3. The method described in embodiment 2, wherein the replication initiation sequence capable of binding to an enzyme having DnaA activity is the replication initiation sequence of Escherichia coli.

[0229] 4. The method according to any one of embodiments 2 or 3, wherein the size of the circular DNA is greater than 50 kb.

[0230] 5. The method according to any one of embodiments 2 to 4, wherein the one or more nucleoid proteins are a combination of IhfA and IhfB.

[0231] 6. The method according to any one of embodiments 2 to 5, wherein the enzyme or enzyme group having DNA gyrase activity is a combination of GyrA and GyrB.

[0232] 7. The method according to any one of embodiments 2 to 6, wherein the enzyme having DNA helicase activity is DnaB helicase.

[0233] 8. The method according to any one of embodiments 2 to 7, wherein the enzyme having DNA helicase loading activity is a DnaC helicase loading activity.

[0234] 9. The method according to any one of embodiments 2 to 8, wherein the enzyme having DNA priming activity is DnaG priming enzyme.

[0235] 10. The method according to any one of embodiments 2 to 9, wherein the enzyme having DNA clamp activity is DnaN clamp.

[0236] 11. The method according to any one of embodiments 2 to 10, wherein the enzyme or enzyme group having DNA polymerase III activity is a combination of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE.

[0237] 12. The method according to any one of embodiments 2 to 11, wherein the enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, an enzyme or enzyme group having DNA polymerase III activity, an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity are derived from Escherichia coli.

[0238] 13. The method according to any one of embodiments 1 to 12, wherein the reaction solution in step (1) further includes a component that inhibits the formation of by-products.

[0239] 14. The method according to any one of embodiments 1 to 13, wherein the isothermal condition in step (2) is a constant temperature in the range of 25°C to 50°C.

[0240] 15. A composition for amplifying circular DNA, comprising:

[0241] The first enzyme group to catalyze the replication of circular DNA;

[0242] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0243] The third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[0244] 16. The composition of embodiment 15, wherein

[0245] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0246] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0247] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[0248] 17. A kit for amplifying circular DNA, comprising:

[0249] The first enzyme group to catalyze the replication of circular DNA;

[0250] A second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0251] The third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[0252] 18. The kit of embodiment 17, wherein

[0253] The first enzyme group catalyzing the replication of circular DNA is an enzyme having DnaA activity, one or more nucleoplastins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III activity.

[0254] The second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer is a combination of an enzyme having RNase H activity, an enzyme having DNA polymerase I activity, and an enzyme having DNA ligase activity.

[0255] The third enzyme group that catalyzes the separation reaction of two sister circular DNAs is a combination of an enzyme having topoisomerase IV activity, an enzyme having topoisomerase III activity, and an enzyme having RecQ activity.

[0256] This manual also includes the following contents:

[0257] 1. A method for exponentially amplifying circular DNA by repeating replication cycles, comprising the step of forming a reaction mixture containing a reaction solution of the following enzyme group and a circular DNA serving as a template:

[0258] (a) The first enzyme group catalyzing the replication of circular DNA;

[0259] (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0260] (c) a third enzyme group that catalyzes the separation reaction of two sister circular DNAs,

[0261] The circular DNA contains a replication initiation sequence (origin of chromosome (oriC)) that can bind to an enzyme with DnaA activity.

[0262] 2. The method according to item 1, wherein the third enzyme group comprises at least two enzymes selected from the group consisting of an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity, and an enzyme having RecQ activity.

[0263] 3. The method of item 1 or 2, wherein

[0264] The first enzyme group includes an enzyme having DnaA activity, one or more nucleoproteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamping activity, and an enzyme or enzyme group having DNA polymerase III* activity.

[0265] The second enzyme group includes a combination of an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity.

[0266] 4. The method according to any one of items 1 to 3, wherein the size of the circular DNA is 8 kb or more.

[0267] 5. The method according to any one of items 1 to 4, wherein the reaction solution further contains tRNA.

[0268] 6. The method according to any one of items 1 to 5, wherein the reaction solution further contains potassium glutamate.

[0269] 7. The method according to any one of items 1 to 6, comprising the step of keeping the reaction mixture warm under isothermal conditions.

[0270] 8. The method described in item 7, wherein the heat preservation step is performed for more than 1.5 hours.

[0271] 9. The method according to any one of items 1 to 8, wherein in the step of forming the reaction mixture, the circular DNA serving as a template is present in a range of 1 molecule to 15 pM.

[0272] 10. A kit for amplifying circular DNA, comprising:

[0273] (a) The first enzyme group catalyzing the replication of circular DNA;

[0274] (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0275] (c) A third enzyme group catalyzing the separation reaction of two sister circular DNAs, which contains at least two enzymes selected from an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity, and an enzyme having RecQ activity.

[0276] 11. The kit according to item 10, wherein

[0277] The first enzyme group includes an enzyme having DnaA activity, one or more nucleoproteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamping activity, and an enzyme or enzyme group having DNA polymerase III* activity.

[0278] The second enzyme group includes a combination of an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity.

[0279] 12. The kit according to item 10 or 11, further comprising tRNA.

[0280] 13. The kit according to any one of items 10 to 12, further comprising potassium glutamate.

[0281] 14. A kit for exponentially amplifying circular DNA by repeated replication cycles, comprising:

[0282] (a) The first enzyme group catalyzing the replication of circular DNA;

[0283] (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0284] (c) The third enzyme group catalyzes the separation reaction of two sister circular DNAs.

[0285] 15. A kit for amplifying circular DNA, comprising:

[0286] (a) The first enzyme group catalyzing the replication of circular DNA;

[0287] (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer; and

[0288] (c) a third enzyme group that catalyzes the separation reaction of the two sister circular DNAs; and

[0289] An additional construct, wherein the additional construct describes a method for exponentially amplifying circular DNA by repeating replication cycles in a reaction mixture containing the reaction solutions of the first, second and third enzyme groups and circular DNA serving as a template.

Claims

1. A method for exponentially amplifying circular DNA by repeated replication cycles, comprising: A step of forming a single reaction mixture comprising: (a) a first enzyme group that catalyzes the replication of circular DNA, which includes an enzyme having DnaA activity, one or more nucleoproteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III* activity; (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer, which includes a combination of an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity; (c) a third enzyme group for catalyzing the separation reaction of two sister circular DNAs, which comprises an enzyme selected from the group consisting of an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity, and an enzyme having RecQ activity; (d) circular DNA serving as a template; and (e) tRNA, and a step of amplifying the circular DNA by heating the reaction mixture under isothermal conditions of 20°C to 80°C, The circular DNA contains a replication initiation sequence (origin of chromosome (oriC)) that can bind to an enzyme having DnaA activity.

2. The method according to claim 1, wherein the third enzyme group comprises at least two enzymes selected from the group consisting of an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity, and an enzyme having RecQ activity.

3. The method according to any one of claims 1 to 2, wherein the size of the circular DNA is greater than 8 kb.

4. The method according to any one of claims 1 to 3, wherein the reaction solution further contains potassium glutamate.

5. The method according to claim 1, wherein the heat preservation step is performed for more than 1.5 hours.

6. The method according to any one of claims 1 to 5, wherein in the step of forming the reaction mixture, the circular DNA serving as a template is present in a range of 1 molecule to 15 pM.

7. The method according to any one of claims 1 to 6, wherein the circular DNA is amplified by incubating the reaction mixture under isothermal conditions of 20°C to 50°C.

8. The method according to any one of claims 1 to 7, wherein the circular DNA is amplified by incubating the reaction mixture under isothermal conditions of 25°C to 50°C.

9. A kit for use in the method according to any one of claims 1 to 8, comprising: (a) a first enzyme group that catalyzes the replication of circular DNA, which includes an enzyme having DnaA activity, one or more nucleoproteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III* activity; (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer, which includes a combination of an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity; (c) a third enzyme group that catalyzes the separation reaction of two sister circular DNAs, which contains at least two enzymes selected from the following: an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity, and an enzyme having RecQ activity; and (d) tRNA.

10. The kit according to claim 9, further comprising potassium glutamate.

11. A kit for use in the method according to any one of claims 1 to 8, comprising: (a) a first enzyme group that catalyzes the replication of circular DNA, which includes an enzyme having DnaA activity, one or more nucleoproteins, an enzyme or enzyme group having DNA gyrase activity, a single-strand DNA binding protein (SSB), an enzyme having DNA helicase activity, an enzyme having DNA helicase loading activity, an enzyme having DNA priming activity, an enzyme having DNA clamp activity, and an enzyme or enzyme group having DNA polymerase III* activity; (b) a second enzyme group that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a concatemer, which includes a combination of an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity; (c) a third enzyme group for catalyzing the separation reaction of two sister circular DNAs, which comprises an enzyme selected from the following: an enzyme having topoisomerase III activity, an enzyme having topoisomerase IV activity and an enzyme having RecQ activity; and (d) tRNA.

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