Method for producing circular DNA

Abstract

The present invention provides an efficient method for producing circular DNA. This method for producing circular DNA includes: (1) preparing circular DNA that will serve as a template, said circular DNA including (i) a replication initiation sequence capable of binding to an enzyme that has a DnaA activity, and (ii) a gyrase binding sequence, and said circular DNA not including a DnaA box cluster at a different position from the replication initiation sequence; (2) forming a reaction mixture of the circular DNA prepared in (1) and a reaction liquid including a first enzyme group that catalyzes replication of the circular DNA, a second enzyme group that synthesizes two sister circular DNAs forming a catenane, and a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) at a temperature of 80°C or lower, thereby obtaining a reaction product that includes the circular DNA, which has been amplified.

Description

Method for producing circular DNA

[0001] The present invention relates to a method for efficiently producing circular DNA in a cell-free system and to circular DNA used in said method. This application claims priority to Japanese Patent Application No. 2023-117904, filed on July 19, 2023, the contents of which are incorporated herein by reference.

[0002] With the advancement of biotechnology, technological developments for in vitro nucleic acid amplification have progressed. In particular, the Replication Cycle Reaction (RCR) method is a technology for amplifying circular DNA in a cell-free system (Patent Documents 1 to 3, Non-Patent Documents 1 and 2).

[0003] International Publication No. WO 2016 / 080424 International Publication No. WO 2017 / 199991 International Publication No. WO 2018 / 159669

[0004] Su'etsugu et al., Nucleic Acids Research, 2017, vol.45(20), p.11525-11534.Hasebe, et al., Life, 2018, vol.8(4), p.43.

[0005] Circular DNA, particularly circular double-stranded DNA, is used in a variety of applications, such as as a template plasmid for mRNA synthesis, and is therefore in high demand. Against this background, a primary object of the present invention is to provide an efficient method for producing circular DNA.

[0006] As a result of extensive research, the present inventors have found that in conventional PCR methods, the amplification efficiency can be further improved by providing a circular DNA to be amplified with a specific sequence, and have thus completed the present invention.

[0007] That is, the present invention relates to the following: [1] A method for producing a circular DNA, comprising: (1) preparing a template circular DNA, the circular DNA containing (i) a replication initiation sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence, and the circular DNA not containing a DnaA box cluster at a position different from that of the replication initiation sequence; (2) forming a reaction mixture with the circular DNA prepared in (1) above and a reaction solution containing: a first group of enzymes that catalyze the replication of the circular DNA; a second group of enzymes that synthesize two sister circular DNAs that form catenanes; and a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) above at a temperature of 80°C or less, thereby obtaining a reaction product containing the amplified circular DNA. [2] The method of [1], wherein the replication initiation sequence of (i) has a mutant double-strand cleavage region with a higher AT content than the double-strand cleavage region of a wild-type replication initiation sequence, and the double-strand cleavage region has L, M, and R elements. [3] The method of [1] or [2], wherein the gyrase binding sequence of (ii) is a sequence derived from bacteriophage Mu. [4] A method for producing circular DNA, comprising: (1) preparing a template circular DNA, the circular DNA (i) comprising a replication initiation sequence capable of binding to an enzyme having DnaA activity, and the replication initiation sequence having a mutant double-strand cleavage region with a higher AT content than the double-strand cleavage region of a wild-type replication initiation sequence, the double-strand cleavage region having L, M, and R elements; (2) forming a reaction mixture with the circular DNA prepared in (1) above and a reaction solution containing: a first group of enzymes that catalyze the replication of the circular DNA; a second group of enzymes that synthesize two sister circular DNAs that form catenanes; and a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) above at a temperature of 80°C or less, thereby obtaining a reaction product containing the amplified circular DNA.[5] The method of [4], further comprising, following (3), (4) diluting the reaction mixture two-fold or more with a solution not containing the first to third enzyme groups, and maintaining the resulting diluted solution under conditions in which the second and third enzyme groups act. [6] The method of any of [2] to [5], wherein the mutant double-strand cleavage region is a mutant double-strand cleavage region in which, among the L, M, and R elements constituting the double-strand cleavage region, the M and / or R elements have a higher AT content compared to wild-type M and / or R elements, the M element constituting the double-strand cleavage region is a mutant double-strand cleavage region having a repetitive AT sequence, and / or the R element constituting the double-strand cleavage region is a mutant double-strand cleavage region having four or more consecutive KAK (K is T or G) motif sequences. [7] The method of any of [1] to [6], wherein the circular DNA further comprises a ter sequence inserted outward relative to the replication initiation sequence, the ter sequence not adjacent to the replication initiation sequence, and the reaction solution of (2) further comprises a protein having activity of binding to the ter sequence and inhibiting replication. [8] The method of any of [1] to [7], wherein the circular DNA further comprises a ter sequence inserted outward relative to the replication initiation sequence capable of binding to an enzyme having DnaA activity, the ter sequence being a ter sequence into which a mutation has been introduced such that one or more bases of a wild-type ter sequence have been substituted, deleted, or inserted, and the reaction solution of (2) further comprises a protein having activity of binding to the ter sequence and inhibiting replication. [9] The method of any of [1] to [8], wherein the incubation of (3) comprises isothermal incubation.

[10] The method according to any one of [1] to [8], wherein the incubation in (3) above comprises incubation under temperature cycling, in which incubation at two temperatures not exceeding 65°C is repeated.

[11] The method of any of [1] to

[10] , wherein the first group of enzymes comprises an enzyme having DnaA activity, SSB, an enzyme having DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and an enzyme or group of enzymes having DNA polymerase III activity; the second group of enzymes comprises an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity; and the third group of enzymes comprises an enzyme having topoisomerase III activity or an enzyme having topoisomerase IV activity.

[12] The method of any of [1] to

[11] , wherein the reaction solution further comprises a linear DNA-specific exonuclease.

[13] The method of any of [1] to

[12] , wherein the reaction solution further comprises a single-stranded DNA-specific exonuclease.

[14] Circular DNA comprising (i) a replication origin sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence, wherein the circular DNA does not contain a DnaA box cluster at a position different from that of the replication origin sequence.

[15] Circular DNA comprising (i) a replication origin sequence capable of binding to an enzyme having DnaA activity, (ii) a gyrase binding sequence, and (iii) a plasmid replication origin, wherein the circular DNA does not contain a DnaA box cluster at a position different from that of the replication origin sequence.

[0008] According to the present invention, circular DNA can be efficiently produced, and circular DNA that can be efficiently produced by PCR can be provided.

[0009] 12A is a diagram of a gel electrophoresis in Example 5. FIG. 12B is a diagram of a gel electrophoresis in Example 6. FIG. 12C is a diagram of a gel electrophoresis in Example 7. FIG. 12D is a diagram of a gel electrophoresis in Example 8. FIG. 12E is a diagram of a gel electrophoresis in Example 9-1. FIG. 12F is a diagram of a gel electrophoresis in Example 9-2. FIG. 12G is a diagram of a gel electrophoresis in Example 10. FIG. 12G is a diagram of a gel electrophoresis in Example 11. FIG. 12G is a diagram of a gel electrophoresis in Example 12. FIG. 12H is a diagram of a gel electrophoresis in Example 13. FIG. 12A is a schematic diagram of the amplification cycle of circular DNA by the PCR method. FIG. 12B is a schematic diagram of the process of replication initiation from oriC. FIG. 12C is a diagram showing the results of Example 14-2. FIG. 13A shows the sequences of a wild-type and two oriC cassette mutants. FIG. 13B shows the results of amplifying DNA with different GC contents using three oriC cassettes. Figure 13C shows the results of real-time PCR performed using three types of oriC cassettes. Figure 14A shows the sequences of the wild-type and two types of oriC cassette mutants. Figure 14B is a schematic diagram showing the positions of mutations in four types of oriC cassette mutants. Figure 14C shows the results of real-time PCR performed using five types of oriC cassettes. Figure 14D shows the results of amplifying DNA with different GC contents using four types of oriC cassettes. Figure 15A shows the sequences of the wild-type and two types of oriC cassette mutants. Figure 15B is a schematic diagram showing the positions of mutations in three types of oriC cassette mutants. Figure 15C shows the results of real-time PCR performed using four types of oriC cassettes. Figure 15D shows the sequences of the wild-type and four types of oriC cassette mutants. Figure 145 shows the results of Example 14-5. Figure 16A shows the results of DNA amplification by PCR at different temperatures using seven types of oriC cassettes. Figure 16B shows the results of competitive PCR amplification using five types of oriC cassettes. Figure 16B is a graph showing changes in DNA concentration with reaction time in Example 15. Figure 16C is a gel electrophoresis diagram in Example 16. Figure 16D is a gel electrophoresis diagram in Example 17.18 is a graph showing the change in DNA concentration versus reaction time in Example 18. 19 is a graph showing the change in DNA concentration versus reaction time in Example 19.

[0010] Hereinafter, embodiments of the present invention (also referred to as the present embodiment) will be described, but the present invention is not limited thereto. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those skilled in the art.

[0011] <Circular DNA> In this embodiment, the circular DNA used as a template is preferably double-stranded. The circular DNA used as a template is not particularly limited as long as it contains a replication initiation sequence capable of binding to an enzyme having DnaA activity and has the characteristics described below. Examples of the circular DNA include a circular DNA obtained by cleaving natural circular DNA such as a circular chromosome of a microorganism by enzyme treatment or the like and then ligating another DNA fragment to the cleaved DNA, and an artificially synthesized circular DNA.

[0012] Examples of replication initiation sequences capable of binding to enzymes having DnaA activity include known replication initiation sequences present in bacteria such as Escherichia coli and Bacillus subtilis, which can be obtained from public databases such as NCBI. Alternatively, replication initiation sequences can be obtained by cloning a DNA fragment capable of binding to an enzyme having DnaA activity and analyzing its nucleotide sequence. The replication initiation sequence used in the present invention may be a sequence in which a mutation has been introduced into a known replication initiation sequence by substituting, deleting, or inserting one or more bases, and which is capable of binding to an enzyme having DnaA activity. The replication initiation sequence used in the present invention is preferably oriC and a modified sequence thereof, more preferably oriC derived from Escherichia coli and a modified sequence thereof. The circular DNA used as a template in this embodiment may be a circular DNA that originally contains a replication initiation sequence, or a circular DNA that does not originally contain a replication initiation sequence into which a replication initiation sequence has been introduced.

[0013] In one embodiment, the replication initiation sequence capable of binding to an enzyme having DnaA activity is oriC derived from Escherichia coli or a modified sequence thereof, and is a sequence having a length of 265 bp or more. In one embodiment, the replication initiation sequence capable of binding to an enzyme having DnaA activity is a sequence having SEQ ID NO: 4, or a modified sequence of SEQ ID NO: 4, which does not have mutations in the 12 or 13 bases at both ends of SEQ ID NO: 4. In one embodiment, the replication initiation sequence capable of binding to an enzyme having DnaA activity is a modified sequence of SEQ ID NO: 4, which is a sequence having SEQ ID NO: 29 (5' end) and / or SEQ ID NO: 30 (3' end) shown underlined below, or a sequence having a sequence in SEQ ID NO: 29 or 30 in which one or more bases have been deleted, substituted, or added.

[0014]

[0015] In one embodiment, the replication origin sequence capable of binding to an enzyme having DnaA activity is a modified oriC sequence in which one or more of the 11 DnaA boxes present in oriC have been deleted. The DnaA boxes in oriC are called R1, R5, τ2, I1, I2, R2, C3, C2, I3, C1, and R4, from the side closest to the DUE. In one embodiment, the modified oriC sequence is a sequence in which, of the 11 DnaA boxes, 1 to 6 selected from R2, C3, C2, I3, C1, and R4, for example, 1 to 5 selected from C3, C2, I3, C1, and R4, for example, C3, C2, I3, C1, and R4, have been deleted.

[0016] In one aspect, the circular DNA used in the method of this embodiment includes (i) a replication initiation sequence capable of binding to an enzyme having DnaA activity and (ii) a strong gyrase-binding sequence (SGS). The SGS is a binding sequence to which DNA gyrase binds, introducing negative supercoiling into DNA. Examples of SGS include the consensus sequence RNNNRNR[T / G]GRYC[G / T]YNYN[G / T]NY (R = A or G, Y = C or T, N = A, G, C, or T) (SEQ ID NO: 31) or a sequence containing its complementary sequence (Lockshon and Morris, Journal of Molecular Biology, 1985, vol. 181, pp. 63-74). The SGS may be derived from any biological species, as long as it contains the consensus sequence. For example, a phage-derived SGS or a plasmid-derived SGS can be used. Furthermore, the SGS may have a sequence preceding or following the consensus sequence, or may be a modified sequence of an SGS derived from any biological species. The modified sequence refers to a base sequence into which one or more bases of the base sequence before modification have been mutated by substitution, deletion, or insertion while retaining the function of increasing the amplification efficiency of the circular DNA containing it by the PCR method. In one aspect, from the viewpoint of fully obtaining the effect of increasing the amplification efficiency of the circular DNA in the replication initiation sequence, the SGS is preferably an SGS derived from a bacteriophage, more preferably an SGS derived from bacteriophage Mu (Mu-SGS) (Folarin et al., Bioengineering, 2019, vol.6(2):54.), and more preferably the sequence of SEQ ID NO:9 or a sequence including same.

[0017] The SGS may be located at any position on the circular DNA, and the distance from the replication initiation sequence is not particularly limited. In one embodiment, the SGS is adjacent to or close to the replication initiation sequence, and is located within 30 bases, 20 bases, 15 bases, 13 bases, or 10 bases from the end of the replication initiation sequence. In one embodiment, the SGS is adjacent to or close to the end of the double-strand cleavage region (DUE) of the replication initiation sequence, and is located within 30 bases, 20 bases, 15 bases, 13 bases, or 10 bases from the end of the replication initiation sequence on the DUE side.

[0018] In one aspect, the circular DNA used in the method of this embodiment comprises (i) a replication origin sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence, and does not comprise a DnaA box cluster at a position different from that of the replication origin sequence.In one aspect, the circular DNA used in the method of this embodiment comprises (i) a replication origin sequence capable of binding to an enzyme having DnaA activity, (ii) a gyrase binding sequence, and (iii) a plasmid replication origin, and does not comprise a DnaA box cluster at a position different from that of the replication origin sequence.

[0019] Circular DNA having such a structure can be efficiently amplified by the PCR method described below, and more specifically, is useful for one or more of amplification with a higher yield, amplification at a higher amplification rate, amplification with a smaller amount of enzyme, and accurate amplification from a lower concentration (e.g., one molecule) of circular DNA.

[0020] When the circular DNA of this embodiment has a plasmid replication origin, examples thereof include known plasmid replication origins such as p15A, F, ColE1, psC101, P1, R1, R6Kγ, λ, φB2, φB0, RK2, and P4, with p15A and F being particularly preferred. Examples of p15A replication origins include p15A, the replication origin of plasmids such as pACYC, or modified sequences thereof. Examples of F replication origins include fori, the replication origin of plasmids such as pBeloBAC, or modified sequences thereof. The modified sequence can be a base sequence into which a mutation has been introduced to substitute, delete, or insert one or more bases in the base sequence before modification while retaining its function as a plasmid replication origin. In one aspect, when the circular DNA of this embodiment has a plasmid replication origin, it is not ColE1 type. Examples of ColE1-type replication origins include pMB1, which is the replication origin of plasmids such as pUC, pGEM, pTZ, and pBR322, and ColE1, which is the replication origin of plasmids such as pBluescript.

[0021] In one aspect of this embodiment, the template circular DNA does not contain a DnaA box cluster at a position different from the replication initiation sequence. A DnaA box cluster is a region in which two or more DnaA boxes or sequences analogous thereto are present, in either forward or reverse orientation, and are adjacent to each other within 50 bases. The consensus sequence for the DnaA box is TT[A / T]TNCACA (DnaAB consensus sequence 1), and its reverse sequence is TGTGNA[A / T]AA (DnaAB consensus sequence 2). These analogous sequences are sequences in which one or more bases, for example, 1, 2, 3, 4, or 5 bases, preferably 1 to 3 bases, and more preferably 1 to 2 bases, are deleted, substituted, or added from DnaAB consensus sequence 1 or DnaAB consensus sequence 2. The p15A-type replication origin, p15A, and the F-type replication origin, fori, do not contain a DnaA box cluster, whereas the ColE1-type replication origin, pUC, contains a DnaA box cluster.

[0022] In one aspect, the circular DNA of this embodiment is a circular DNA that does not contain two or more, for example, three, of DnaAB consensus sequence 1, DnaAB consensus sequence 2, or sequences in which one to three, preferably one to two, bases are deleted, substituted, or added in these sequences, in a region that is different from the replication initiation sequence and is 100 bases, 90 bases, 80 bases, 70 bases, 60 bases, or preferably 50 bases (for example, 50 bases, 40 bases, or 30 bases in the case of two sequences). In one aspect, the circular DNA of this embodiment is not a circular DNA that contains, in this order, DnaAB consensus sequence 1 or a sequence analogous thereto, DnaAB consensus sequence 2 or a sequence analogous thereto, and DnaAB consensus sequence 2 or a sequence analogous thereto, within 50 bases. Here, the analogous sequences are sequences in which one to two bases are deleted, substituted, or added in the original sequence. In one aspect, the circular DNA of this embodiment is not a circular DNA that contains, in this order, within 50 bases, DnaAB consensus sequence 1 or a sequence similar thereto, a sequence similar to DnaAB consensus sequence 2, and a sequence similar to DnaAB consensus sequence 2. Here, the similar sequence is a sequence in which 1 to 2 bases have been deleted, substituted, or added in the original sequence.

[0023] A replication initiation sequence (e.g., oriC) capable of binding to an enzyme having DnaA activity contains a DUE and a DnaA assembly region (DOR). When ATP-bound DnaA binds to the replication initiation sequence, the DnaA-oriC complex undergoes a structural change, cleaving the double-stranded DNA within the DUE region and initiating DNA replication. Thus, in one aspect, the circular DNA used in the method of this embodiment contains (i) a replication initiation sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase-binding sequence, and the replication initiation sequence capable of binding to the enzyme having DnaA activity has one or more of the following characteristics (F1) to (F5): (F1) The DUE has a mutant DUE with a higher AT content compared to wild-type DUE; (F2) The DUE has a repetitive AT sequence in the M element; or (F3) The DUE has four or more consecutive KAK (where K is T or G) motif sequences in the R element. (F4) The R5M sequence in the DOR region has a sequence identical to the consensus sequence of DnaA (TGTGNAWAA). (F5) Compared to wild-type DUE, the number of TT[A / G]T(T) sequence motifs (e.g., TTATT) in the DUE region is increased.

[0024] The circular DNA of this embodiment has a mutant DUE that has a higher AT content than the wild-type DUE.

[0025] As used herein, a mutated sequence having a high AT content refers to the presence of one or more, two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more mutations compared to the wild-type sequence before mutation. The mutations are deletion of C or G, or substitution of C or G with A or T, with the latter being more preferred.

[0026] DUE has three 13-base elements with a high AT content, referred to as the L, M, and R elements, respectively (Katayama et al., Frontiers in Microbiology (2017) Vol. 8: 2496). In one aspect, the circular DNA of this embodiment has a mutant DUE with a high AT content compared to the wild-type DUE, and preferably at least one, and preferably at least two, elements have a mutant DUE with a high AT content compared to the wild-type DUE. The circular DNA of this embodiment is more preferably a mutant DUE in which the M or R element, and even more preferably the M and R elements, have a high AT content compared to that of the wild-type DUE. A mutant DUE with a high AT content compared to the wild-type DUE is, for example, a sequence with an increased number of AT motifs (a sequence of A and T) compared to the wild-type DUE.

[0027] The sequences of the wild-type DUE and the L, M, and R elements of the wild-type DUE are shown below. Each DUE is a 13-mer having the same sequence as the consensus sequence (GATCTnTTnTTTT: SEQ ID NO: 110) (Bramhill, D., & Kornberg, A. (1988) Cell, 52(5), 743-755.). In one aspect, the circular DNA of this embodiment has one or more, two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more mutations in one or more of these sequences, for example, SEQ ID NO: 111. In one aspect, the circular DNA of this embodiment has one or more, preferably two or more, three or more, or four or more mutations in SEQ ID NO: 113 or SEQ ID NO: 114, or in each of SEQ ID NO: 113 and SEQ ID NO: 114. The mutations are deletion of C or G, substitution of C or G with A or T, preferably substitution of C or G with A or T.

[0028]

[0029] In one aspect, the circular DNA of this embodiment preferably has a repetitive AT sequence in the M element of DUE compared to wild-type DUE, for example, a sequence of (AT)n. Here, n is an integer of 2 or greater, preferably an integer of 3 or greater, 4 or greater, or 5 or greater. The upper limit of n is not particularly limited, but may be, for example, an integer of 30 or less, preferably an integer of 25 or less, 20 or less, or 15 or less, and in one aspect, an integer of 12 or less, 10 or less, or 8 or less. For example, n is an integer of 3 to 10, an integer of 3 to 8, an integer of 5 to 8, an integer of 5 to 7, or 7. The repetitive AT sequence in the M element may include a sequence other than the M element, as long as the repetitive sequence includes the sequence of the M element. In one aspect, the circular DNA of this embodiment may have a repetitive AT sequence in the 5'-side sequence of the M element. Furthermore, the repetitive AT sequence in the M element may include multiple repetitive AT sequences that are non-contiguous, as long as the AT sequence is repeated and the sequence of the M element is included in the repetitive sequence. For example, a circular DNA has (AT) in the M element of DUE. ｎ’ Sequence and (AT) ｎ” In this case, the M element of the DUE may have the sequence (AT) ｎ (where n=n′+n″). In a preferred aspect, the circular DNA of this embodiment has, for example, in the M element of DUE (including its surrounding sequences), a sequence selected from GATATGTTCTATT (SEQ ID NO: 50), TATATATTCTATT (SEQ ID NO: 51), TATATATTATATATT (SEQ ID NO: 32), TTATTAATATTAAC (SEQ ID NO: 52), ATATATATATTCTATT (SEQ ID NO: 53), and ATATATATATTATATT (SEQ ID NO: 54), or a sequence in which one or more bases (preferably one or more bases other than AT) of these sequences have been substituted, deleted, or inserted.

[0030] In one aspect, the circular DNA of this embodiment has a mutant DUE in which the R element of the DUE has more consecutive KAK motif sequences (K is T or G; the same applies unless otherwise specified herein) than the wild-type DUE. Here, the consecutive KAK motif sequences in the R element may include sequences other than the R element, as long as the R element sequence is included in the consecutive sequence; that is, it may include peripheral sequences centered around the R element. For example, in a wild-type oriC, there are three consecutive KAK motif sequences in the R element of the DUE, but in the circular DNA of this embodiment, this number is preferably four or more, five or more, six or more, or seven or more. The upper limit of the number of KAK motif sequences in the circular DNA of this embodiment is not particularly limited, but is preferably 15 or less, 10 or less, or 7 or less, for example. For example, the R element of the DUE may have five to seven consecutive KAK motif sequences. In a preferred aspect, the circular DNA of this embodiment has, for example, a sequence selected from GATTATTATTAG (SEQ ID NO: 55), GATTATTATTAGGAT (SEQ ID NO: 56), TATTATTATTAG (SEQ ID NO: 33), and TATTATTATTAGGAT (SEQ ID NO: 57) in the R element of DUE (including its surrounding sequences), a sequence in which T is substituted with G or G is substituted with T in the KAK motif of these sequences, or a sequence in which one or more bases (preferably one or more bases other than the KAK motif sequence) of these sequences have been substituted, deleted, or inserted. Without being bound by theory, it is believed that this strengthens the binding of the DnaA protein to the DUE region.

[0031] In one aspect, the circular DNA of this embodiment preferably has a DUE having an (AT)n sequence in the M element, where n is an integer of 3 or more, and a DUE having five or more consecutive KAK motif sequences in the R element.

[0032] In one aspect, the circular DNA of this embodiment has an increased number of TT[A / G]T(T) sequence motifs (e.g., TTATT) in the DUE compared to wild-type DUE, and preferably has two or more, for example, three or more TT[A / G]T(T) sequences (e.g., TTATT).

[0033] In one aspect, the circular DNA of this embodiment preferably has a DUE sequence of TATATATTATATATT (SEQ ID NO: 32) and TATTATTATTAG (SEQ ID NO: 33), or sequences analogous thereto. These analogous sequences are sequences in which one or more bases, for example, 1, 2, 3, 4, or 5 bases, preferably 1 to 3 bases, more preferably 1 to 2 bases, and particularly preferably 1 base, are deleted, substituted, or added in SEQ ID NO: 32 or 33.

[0034] For DOR, there are 11 DnaA-boxes within the minimal oriC region (Fig. 12B), three of which (R1, R2, and R4) have the same sequence 5'-TGTGnA[T / A]AA-3' and have high affinity for DnaA, whereas the remaining eight (R5M, τ2, l1, l2, C3, C2, l3, and C1) have one to six mutations that confer low affinity for DnaA.

[0035] In one aspect, the circular DNA of this embodiment has an R5M sequence of DOR that is identical to the consensus sequence of DnaA (TGTGNAWAA; N=A, T, G, or C, W=A or T).

[0036] Circular DNA having such a structure (circular DNA having oriC having such a structure) can be efficiently amplified by the PCR method described below, and more specifically, is useful for one or more of amplification with a higher yield, amplification at a higher amplification rate, amplification with a smaller amount of enzyme, and amplification at a lower temperature. Furthermore, circular DNA having such a structure allows for more accurate amplification even when the AT content of the entire circular DNA is high.

[0037] In one aspect, the circular DNA of this embodiment comprises (i) a replication initiation sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence, but does not comprise a DnaA box cluster at a position different from the replication initiation sequence, and the DUE has an (AT)n sequence in the M element, where n is an integer of 3 or greater, and the DUE has five or more consecutive KAK motif sequences in the R element.

[0038] Circular DNA having such a configuration (circular DNA having oriC having such a configuration) can be efficiently amplified by the PCR method described below, and more specifically, is useful for one or more of amplification with a higher yield, amplification at a higher amplification rate, amplification with a smaller amount of enzyme, and amplification at a lower temperature. Furthermore, circular DNA having such a configuration produces fewer by-products such as concatemers, allowing for more accurate amplification.

[0039] The circular DNA of this embodiment may further have a ter sequence inserted outward relative to the replication initiator sequence. In one aspect, the ter sequence is inserted outward relative to the end of the replication initiator sequence far from the DUE. In another aspect, the ter sequence is inserted outward relative to the end of the replication initiator sequence close to the DUE. This suppresses the production of DNA multimers when a plasmid containing the DNA cassette is amplified in a test tube by PCR, allowing an amplified product of the plasmid to be obtained efficiently.

[0040] With regard to the ter sequence, "inserting it outward from the replication initiation sequence" means inserting the ter sequence in a direction that allows replication outward from the replication initiation sequence, such as oriC, but does not allow replication inward toward the replication initiation sequence, thereby terminating it, due to the action of a combination of proteins that have the activity of binding to the ter sequence and inhibiting replication.

[0041] In one aspect, the circular DNA of this embodiment further has a pair of ter sequences, each inserted facing outward relative to the replication initiation sequence. With regard to the ter sequences, "a pair of ter sequences, each inserted facing outward relative to the replication initiation sequence" means that one ter sequence is inserted on the 5' side of the replication initiation sequence, and the other is inserted on the 3' side of the replication initiation sequence.

[0042] Examples of the ter sequence to be inserted on the 5' side of the replication initiation sequence include the sequences shown in SEQ ID NOs: 34 to 49. Examples of the ter sequence to be inserted on the 3' side of the replication initiation sequence include a sequence containing a complementary sequence to the nucleotide sequence inserted on the 5' side of the replication initiation sequence.

[0043] For example, known replication termination systems that utilize a ter sequence and a protein that binds to a ter sequence on DNA to inhibit replication include the Tus-ter system in Escherichia coli and the RTP-ter system in Bacillus bacteria (Patent Document 3). Ter sequences that can be used in these systems or modified sequences thereof can be used as the ter sequence. A modified ter sequence refers to a base sequence in which a mutation has been introduced to substitute, delete, or insert one or more bases in the base sequence before modification while retaining the function of the ter sequence. When the Tus-ter system is used, a Tus protein is used during plasmid replication, and when the RTP-ter system is used, an RTP protein is used during plasmid replication.

[0044] The ter sequence of the circular DNA according to this embodiment may be a wild-type ter sequence, or a mutant ter sequence in which one or more bases of the wild-type ter sequence have been mutated by substitution, deletion, or insertion. Examples of wild-type ter sequences that can be used in the Tus-ter system include 5'-GN[A / G][T / A]GTTGTAAC[T / G]A-3' (SEQ ID NO: 34), 5'-G[T / G]A[T / A]GTTGTAAC[T / G]A-3' (SEQ ID NO: 35), 5'-GTATGTTGTAACTA-3' (SEQ ID NO: 36), and 5'-AGTATGTTGTAACTAAAG-3' (SEQ ID NO: 37). A preferred base sequence includes 5'-GGATGTTGTAACTA-3' (SEQ ID NO: 37), 5'-GTATGTTGTAACGA-3' (SEQ ID NO: 38), 5'-GTATGTTGTAACGA-3' (SEQ ID NO: 39), 5'-GGATGTTGTAACTA-3' (SEQ ID NO: 40), 5'-GGAAGTTGTAACGA-3' (SEQ ID NO: 41), or 5'-GTAAGTTGTAACGA-3' (SEQ ID NO: 42). Furthermore, the ter sequence contained in the circular DNA according to this embodiment is preferably a mutant ter sequence. Examples of mutant ter sequences include base sequences containing a modified sequence in which one or several bases in the nucleotide sequence of SEQ ID NO: 34 are substituted. The mutant ter sequence possessed by the circular DNA according to this embodiment is preferably 5'-GN[A / G][T / A]GTTGTACC[T / G]A-3' (SEQ ID NO: 43) or 5'-GTATGTTGTAcCTA-3' (SEQ ID NO: 44), and an example thereof includes 5'-AGTATGTTGTACCTAAAG-3' (SEQ ID NO: 5) having SEQ ID NO: 44.

[0045] An example of a ter sequence that can be used in the RTP-ter system is a base sequence containing 5'-AC[T / A][A / G]ANNNNN[C / T]NATGTACNAAAT-3' (SEQ ID NO: 45). Preferred ter sequences for use in the RTP-ter system contained in the DNA cassette of the present invention are base sequences containing 5'-ACTAATT[A / G]A[A / T]C[T / C]ATGTACTAAAT-3' (SEQ ID NO: 46), 5'-ACTAATT[A / G]A[A / T]C[T / C]ATGTACTAAATTTTCA-3' (SEQ ID NO: 47), 5'-GAACTAATTAAAACTATGTACTAAATTTTCA-3' (SEQ ID NO: 48), or 5'-ATACTAATTGATCCATGTACTAAATTTTCA-3' (SEQ ID NO: 49).

[0046] The position of the ter sequence is not particularly limited as long as it is inserted facing outward relative to the replication initiation sequence, and it may be located between the replication initiation sequence and the SGS, or may be located on the outside. In one aspect, the ter sequence is preferably not adjacent to the replication initiation sequence. When the circular DNA has a pair of ter sequences, each facing outward relative to the replication initiation sequence, it is preferable that at least one of the ter sequences is not adjacent to the replication initiation sequence. For example, the distance between the 3'-terminal base of at least one ter sequence and the end of the replication initiation sequence (preferably the end of the replication initiation sequence farther from the DUE) is preferably 5 bases or more, e.g., 10 bases or more. Circular DNA having such a configuration can be efficiently amplified by the PCR method described below, and more specifically, is useful for one or more of: amplification with a higher yield, amplification with a smaller amount of enzyme, and amplification with a higher amplification rate.

[0047] In one aspect, the circular DNA of this embodiment may further have one or a pair of ter sequences inserted inward relative to the replication initiation sequence. The term "inserted inward relative to the replication initiation sequence" with respect to the ter sequence means that the ter sequence is inserted in a direction that does not allow replication outward from the replication initiation sequence, such as oriC, and stops replication, while allowing replication inward toward the replication initiation sequence, due to the action of a combination of proteins that bind to the ter sequence and inhibit replication. In this case, it is preferable that the ter sequence is not adjacent to or close to the replication initiation sequence. In one aspect, it is preferable that the one or a pair of ter sequences inserted inward relative to the replication initiation sequence is at least 50 bases, at least 75 bases, at least 100 bases, at least 150 bases, or at least 200 bases away from the replication initiation sequence.

[0048] (a) In one aspect, the circular DNA produced by the method of this embodiment includes (i) a replication origin sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence. (b) In one aspect, the circular DNA produced by the method of this embodiment includes a modified sequence of oriC as the replication origin sequence capable of binding to an enzyme having DnaA activity, in which one to five DnaA boxes selected from C3, C2, I3, C1, and R4 are deleted, and a gyrase binding sequence. (c) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of (a) or (b) above, wherein the replication initiation sequence capable of binding to an enzyme having DnaA activity has one or more of the following characteristics (F1) to (F5): (F1) a mutant DUE with a higher AT content compared to the wild-type; (F2) the DUE has a repetitive AT sequence in the M element; (F3) the DUE has four or more consecutive KAK motif sequences in the R element; (F4) the R5M sequence in the DOR region has a sequence identical to the consensus sequence of DnaA (TGTGNAWAA); or (F5) the number of TT[A / G]T(T) sequence motifs (e.g., TTATT) in the DUE region is increased compared to the wild-type. (d) In one aspect, the circular DNA produced by the method of this embodiment comprises a replication initiator sequence capable of binding to an enzyme having DnaA activity, and the replication initiator sequence has one or more of the following characteristics (F1) to (F5): (F1) a mutant DUE with a higher AT content compared to the wild-type, (F2) the DUE has a repetitive AT sequence in the M element, (F3) the DUE has four or more consecutive KAK motif sequences in the R element, (F4) the R5M sequence in the DOR region has the same sequence as the DnaA consensus sequence (TGTGNAWAA), or (F5) the number of TT[A / G]T(T) sequence motifs (e.g., TTATT) in the DUE region is increased compared to the wild-type. (e) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of any of (a) to (d) above, further comprising one or a pair of ter sequences inserted outward relative to the replication initiator sequence.(f) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of (d) above, wherein the ter sequence is not adjacent to the replication initiation sequence. (g) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of (a) or (b) above, wherein the replication initiation sequence and the gyrase binding sequence are adjacent and further include one or a pair of ter sequences inserted outward relative to these sequences. (h) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of (a) or (b) above, further include one or a pair of ter sequences inserted outward relative to the replication initiation sequence, wherein the distance between the gyrase binding sequence and the replication initiation sequence is greater than the distance between the ter sequence and the replication initiation sequence. (i) In one aspect, the circular DNA produced by the method of this embodiment is the circular DNA of any of (e) to (h) above, wherein the ter sequence is a mutant ter sequence. (j) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (e) to (h) above, wherein the ter sequence comprises the sequence of SEQ ID NO: 5, 43, or 44. (k) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, wherein the circular DNA does not contain a DnaA box cluster at a position different from the replication origin sequence. (l) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, wherein the circular DNA does not contain a DnaA box cluster. Does not contain a DnaA box cluster. (m) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, and does not have a region within 60 bases (e.g., within 50 bases) at a position different from the replication initiation sequence, which region contains DnaAB consensus sequence 1 or a sequence similar thereto, DnaAB consensus sequence 2 or a sequence similar thereto, and DnaAB consensus sequence 2 or a sequence similar thereto, in this order.(n) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, and does not have a region containing DnaAB consensus sequence 1 or a sequence similar thereto, and DnaAB consensus sequence 2 or a sequence similar thereto, in this order, within 40 bases (e.g., within 30 bases) at a position different from the replication origin sequence. (o) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, and does not have a region containing DnaAB consensus sequence 1 or a sequence similar thereto, DnaAB consensus sequence 2 or a sequence similar thereto, and DnaAB consensus sequence 2 or a sequence similar thereto, in this order, within 60 bases (e.g., within 50 bases). (p) In one aspect, the circular DNA produced by the method of this embodiment is any of the circular DNAs (a) to (j) above, and does not have a region within 40 bases (e.g., within 30 bases) that contains DnaAB consensus sequence 1 or a sequence similar thereto and DnaAB consensus sequence 2 or a sequence similar thereto, in this order.

[0049] The circular DNA prepared as described above is amplified by PCR. More specifically, a reaction mixture is formed containing the circular DNA and a reaction solution containing: a first group of enzymes that catalyze the replication of the circular DNA; a second group of enzymes that synthesize two sister circular DNAs that form catenanes; and a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs. The reaction mixture is then incubated at a temperature of 80° C. or less to amplify the circular DNA.

[0050] Examples of the first group of enzymes that catalyze the replication of circular DNA include the enzymes described in Kaguni JM & Kornberg A. Cell. 1984, 38:183-90. Specifically, the first group of enzymes includes the following: an enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or enzymes having DNA gyrase activity, a single-strand binding protein (SSB), an enzyme having DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and DNA polymerase III. * Examples of the enzymes of the first group include one or more enzymes or enzymes selected from the group consisting of enzymes or enzymes having a DnaA activity, single-stranded DNA binding protein (SSB), enzymes having DnaB-type helicase activity, enzymes having DNA helicase loader activity, enzymes having DNA primase activity, enzymes having DNA clamp activity, and DNA polymerase III. * It includes an enzyme or group of enzymes having the activity.

[0051] The biological origin of the enzyme having DnaA activity is not particularly limited, as long as it has initiator activity similar to that of DnaA, an initiator protein of E. coli. For example, DnaA derived from E. coli can be suitably used. DnaA derived from E. coli may be contained in the reaction solution as a monomer in a range of 1.0 nM to 10 μM, preferably 1.0 nM to 5.0 μM, 1.0 nM to 3 μM, 1 nM to 1.5 μM, 1.0 nM to 1.0 μM, 1.0 nM to 500 nM, 50 nM to 200 nM, or 50 nM to 150 nM, but is not limited thereto.

[0052] Nucleoid protein refers to a protein contained in a nucleoid. The one or more nucleoid proteins used in the present invention are not particularly limited in terms of their biological origin, as long as they are enzymes with activity similar to that of E. coli nucleoid proteins. For example, E. coli-derived IHF, i.e., a complex (heterodimer or homodimer) of IhfA and / or IhfB, or E. coli-derived HU, i.e., a complex of hupA and hupB, can be suitably used. E. coli-derived IHF may be contained in the reaction solution as a hetero / homodimer in a range of 5 nM to 400 nM, preferably 5 nM to 200 nM, 5 nM to 100 nM, 5 nM to 50 nM, 10 nM to 50 nM, 10 nM to 40 nM, or 10 nM to 30 nM, but is not limited thereto. HU derived from E. coli may be contained in the reaction solution in a range of 1 nM to 50 nM, preferably 5 nM to 50 nM, or 5 nM to 25 nM, but is not limited thereto.

[0053] The biological origin of the enzyme or enzymes having DNA gyrase activity is not particularly limited, as long as they have activity similar to that of E. coli DNA gyrase. For example, a complex consisting of GyrA and GyrB derived from E. coli can be suitably used. The complex consisting of GyrA and GyrB derived from E. coli may be contained in the reaction solution as a heterotetramer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 200 nM, or 100 nM to 200 nM, but is not limited thereto.

[0054] The single-stranded DNA binding protein (SSB) may be an enzyme having the same activity as the single-stranded DNA binding protein of Escherichia coli, and there are no particular limitations on its biological origin. For example, SSB derived from Escherichia coli can be suitably used. The SSB derived from Escherichia coli may be contained in the reaction solution as a homotetramer in a range of 20 nM to 1000 nM, preferably in a range of 20 nM to 500 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 500 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 50 nM to 150 nM, 100 nM to 500 nM, or 100 nM to 400 nM, but is not limited thereto.

[0055] The biological origin of the enzyme having DnaB-type helicase activity is not particularly limited, as long as it has activity similar to that of E. coli DnaB. For example, DnaB derived from E. coli can be suitably used. DnaB derived from E. coli may be contained in the reaction solution as a homohexamer in a range of 5 nM to 200 nM, preferably 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited thereto.

[0056] The enzyme having DNA helicase loader activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaC. For example, E. coli-derived DnaC can be suitably used. E. coli-derived DnaC may be contained in the reaction solution as a homohexamer in a range of 5 nM to 200 nM, preferably 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited thereto.

[0057] The enzyme having DNA primase activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaG. For example, E. coli-derived DnaG can be suitably used. E. coli-derived DnaG may be contained in the reaction solution as a monomer in a range of 20 nM to 1000 nM, preferably 20 nM to 800 nM, 50 nM to 800 nM, 100 nM to 800 nM, 200 nM to 800 nM, 250 nM to 800 nM, 250 nM to 500 nM, or 300 nM to 500 nM, but is not limited thereto.

[0058] The enzyme having DNA clamping activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaN. For example, E. coli-derived DnaN can be suitably used. E. coli-derived DnaN may be contained in the reaction solution as a homodimer in a range of 10 nM to 1000 nM, preferably 10 nM to 800 nM, 10 nM to 500 nM, 20 nM to 500 nM, 20 nM to 200 nM, 30 nM to 200 nM, or 30 nM to 100 nM, but is not limited thereto.

[0059] DNA polymerase III * As an enzyme or group of enzymes having this activity, DNA polymerase III of Escherichia coli * The biological origin of the enzyme or enzymes is not particularly limited, as long as they have the same activity as the complex. For example, an enzyme group containing any of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli can be used. Preferably, an enzyme group containing a complex of DnaX, HolA, HolB, and DnaE derived from Escherichia coli can be used. More preferably, an enzyme group containing a complex of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli can be used. The E. coli-derived DNA polymerase III* complex may be present in the reaction solution as a heteromultimer in a range of 2 nM to 50 nM, preferably 2 nM to 40 nM, 2 nM to 30 nM, 2 nM to 20 nM, 5 nM to 40 nM, 5 nM to 30 nM, or 5 nM to 20 nM, but is not limited thereto.

[0060] In the present invention, two sister circular DNAs forming a catenane refer to two circular DNAs that are synthesized by a DNA replication reaction and are in a linked state.

[0061] Examples of the second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes include one or more enzymes selected from the group consisting of enzymes having DNA polymerase I activity, enzymes having DNA ligase activity, and enzymes having RNase H activity, or a combination of such enzymes. In one embodiment, the second group of enzymes preferably includes an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity.

[0062] The biological origin of the enzyme having DNA polymerase I activity is not particularly limited, as long as it has activity similar to that of E. coli DNA polymerase I. For example, E. coli-derived DNA polymerase I can be suitably used. E. coli-derived DNA polymerase I may be contained in the reaction solution as a monomer in a range of 10 nM to 200 nM, preferably 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 40 nM to 150 nM, 40 nM to 100 nM, or 40 nM to 80 nM, but is not limited thereto.

[0063] The enzyme having DNA ligase activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DNA ligase. For example, E. coli-derived DNA ligase or T4 phage DNA ligase can be suitably used. The E. coli-derived DNA ligase may be contained in the reaction solution as a monomer in a range of 10 nM to 200 nM, preferably 15 nM to 200 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, or 20 nM to 80 nM, but is not limited thereto.

[0064] The biological origin of the enzyme having RNase H activity is not particularly limited, as long as it has the activity of degrading the RNA strand of an RNA:DNA hybrid. For example, RNase H derived from Escherichia coli can be suitably used. The RNase H derived from Escherichia coli may be contained in the reaction solution as a monomer in a range of 0.2 nM to 200 nM, preferably 0.2 nM to 200 nM, 0.2 nM to 100 nM, 0.2 nM to 50 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 50 nM, or 10 nM to 50 nM, but is not limited thereto.

[0065] Examples of the third enzyme group that catalyzes the separation reaction of the two sister circular DNAs include the enzyme group described in Peng H & Marians KJ. PNAS. 1993, 90: 8571-8575. Specifically, the third enzyme group may include one or more enzymes selected from the group consisting of enzymes having topoisomerase IV activity, enzymes having topoisomerase III activity, and enzymes having RecQ-type helicase activity, or a combination of such enzymes. In one embodiment, the third enzyme group preferably includes an enzyme having topoisomerase IV activity and / or an enzyme having topoisomerase III activity.

[0066] The biological origin of the enzyme having topoisomerase III activity is not particularly limited, as long as it has activity similar to that of Escherichia coli topoisomerase III. For example, Escherichia coli-derived topoisomerase III can be suitably used. Escherichia coli-derived topoisomerase III may be contained in the reaction solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0067] The biological origin of the enzyme having RecQ helicase activity is not particularly limited, as long as it has activity similar to that of E. coli RecQ. For example, RecQ derived from E. coli can be suitably used. E. coli RecQ may be contained in the reaction solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0068] The enzyme having topoisomerase IV activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of Escherichia coli topoisomerase IV. For example, Escherichia coli-derived topoisomerase IV, which is a complex of ParC and ParE, can be suitably used. Escherichia coli-derived topoisomerase IV may be contained in the reaction solution as a heterotetramer in a range of 0.1 nM to 50 nM, preferably 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, 1 nM to 10 nM, or 1 nM to 5 nM, but is not limited thereto.

[0069] The reaction solution may further contain an enzyme. For example, when the circular DNA to be amplified by PCR has the above-mentioned ter sequence, the reaction solution may further contain a protein (e.g., Tus protein derived from Escherichia coli) that has the activity of binding to the ter sequence and inhibiting replication.

[0070] The reaction solution may further contain a linear DNA-specific exonuclease, which has the effect of reducing the amount of linear DNA generated by double-strand cleavage during the amplification reaction and improving the yield of the desired circular DNA.

[0071] A linear DNA-specific exonuclease is an enzyme that sequentially hydrolyzes linear DNA from the 5'-end or the 3'-end. The linear DNA-specific exonuclease is not particularly limited in type or biological origin, as long as it has the activity of sequentially hydrolyzing linear DNA from the 5'-end or the 3'-end. Examples of usable exonucleases include RecBCD (exonuclease V), λ exonuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, and Plasmid-Safe (registered trademark) ATP-Dependent DNase (epicentre). The linear DNA exonuclease may be contained in the reaction solution at a concentration of 0.001 to 1.0 U / μL, preferably 0.005 U to 1.0 U / μL, 0.01 to 1.0 U / μL, 0.05 to 1.0 U / μL, or 0.1 to 1.0 U / μL, but is not limited thereto. The enzyme activity unit (U) for linear DNA exonuclease is defined as the amount of enzyme required to acid-soluble 1 nmol of deoxyribonucleotides in linear DNA in a 30-minute reaction at 37°C.

[0072] The reaction solution may further contain a single-stranded DNA-specific exonuclease, which has the effect of reducing the amount of low-molecular-weight by-products generated during the amplification reaction and improving the yield of the desired circular DNA.

[0073] A single-stranded DNA-specific exonuclease is an enzyme that sequentially hydrolyzes nucleotides at the 5' or 3' end of single-stranded DNA. The single-stranded DNA-specific exonuclease is not particularly limited in type or biological origin, as long as it has the activity of sequentially hydrolyzing nucleotides at the 5' or 3' end of single-stranded DNA. For example, exonuclease I (exo I), RecJ, exonuclease T, etc. can be used. A preferred single-stranded DNA-specific exonuclease is exo I. The single-stranded DNA-specific exonuclease may be contained in the reaction solution in a range of 0.1 to 1.0 U / μL, preferably 0.15 to 1.0 U / μL, 0.2 to 1.0 U / μL, or 0.2 to 0.5 U / μL, but is not limited thereto. The enzyme activity unit (U) for exo I is defined as the amount of enzyme required to render 10 nmol of deoxyribonucleotides in single-stranded DNA acid-soluble in a 30-minute reaction at 37°C. The enzyme activity unit (U) for RecJ is defined as the amount of enzyme required to render 0.05 nmol of deoxyribonucleotides in single-stranded DNA acid-soluble in a 30-minute reaction at 37°C.

[0074] The enzymes contained in the reaction solution containing the first, second, and third enzyme groups may be commercially available enzymes, or may be extracted from microorganisms and purified as necessary. Extraction and purification of enzymes from microorganisms can be carried out appropriately using techniques available to those skilled in the art.

[0075] When enzymes other than those derived from Escherichia coli are used as the first, second, and third enzyme groups, they can be used in a concentration range corresponding in terms of enzyme activity units to the concentration range specified for the enzymes derived from Escherichia coli.

[0076] The reaction solution may contain a buffer, ATP, GTP, CTP, UTP, dNTP, a magnesium ion source, and an alkali metal ion source.

[0077] The buffer solution contained in the reaction solution is not particularly limited as long as it is suitable for use at pH 7 to 9, preferably pH 8. Examples include Tris-HCl, Tris-OAc, Hepes-KOH, phosphate buffer, MOPS-NaOH, and Tricine-HCl. Preferred buffers are Tris-HCl and Tris-OAc. The concentration of the buffer can be appropriately selected by those skilled in the art and is not particularly limited. In the case of Tris-HCl or Tris-OAc, the concentration can be selected from, for example, 10 mM to 100 mM, 10 mM to 50 mM, or 20 mM.

[0078] ATP means adenosine triphosphate. The concentration of ATP contained in the reaction solution at the start of the reaction may be, for example, in the range of 0.1 mM to 3 mM, and preferably in the range of 0.1 mM to 2 mM, 0.1 mM to 1.5 mM, or 0.5 mM to 1.5 mM.

[0079] GTP, CTP, and UTP mean guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, respectively. The concentrations of GTP, CTP, and UTP contained in the reaction solution at the start of the reaction may each independently be in the range of, for example, 0.1 mM to 3.0 mM, preferably 0.5 mM to 3.0 mM, or 0.5 mM to 2.0 mM.

[0080] dNTP is a collective term for deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The concentration of dNTP contained in the reaction solution at the start of the reaction may be, for example, in the range of 0.01 to 1 mM, preferably 0.05 to 1 mM, or 0.1 to 1 mM.

[0081] The magnesium ion source is a substance that provides magnesium ions (Mg2+) in the reaction solution. Examples include Mg(OAc)2, MgCl2, and MgSO4. A preferred magnesium ion source is Mg(OAc)2. The concentration of the magnesium ion source contained in the reaction solution at the start of the reaction may be, for example, a concentration that provides magnesium ions in the range of 5 to 50 mM in the reaction solution.

[0082] The alkali metal ion source is a substance that provides alkali metal ions in the reaction solution. Examples of alkali metal ions include sodium ions (Na+) and potassium ions (K+). Examples of alkali metal ion sources include potassium glutamate, potassium aspartate, potassium chloride, potassium acetate, sodium glutamate, sodium aspartate, sodium chloride, and sodium acetate. A preferred alkali metal ion source is potassium glutamate or potassium acetate. The concentration of the alkali metal ion source contained in the reaction solution at the start of the reaction may be a concentration that provides alkali metal ions in the reaction solution at 100 mM or more, preferably in the range of 100 mM to 300 mM, but is not limited thereto.

[0083] The reaction solution may further contain a non-specific adsorption inhibitor for proteins (bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.), a non-specific adsorption inhibitor for nucleic acids (tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), glycogen, heparin, oligo-DNA, poly(IC) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyinosine-polydeoxycytidine), poly(A) (polyadenine), and poly(dA) (polydeoxyadenine), etc.), a linear DNA-specific exonuclease (RecBCD, λ exonuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, Plasmid-Safe® ATP-Dependent The reaction solution may contain a DNA ligase derived from Escherichia coli, such as DNase (epicentre), a RecG-type helicase (such as RecG derived from Escherichia coli), an ammonium salt (ammonium sulfate, ammonium chloride, ammonium acetate), or a reducing agent (DTT, β-mercaptoethanol, glutathione, etc.), and when the reaction solution contains DNA ligase derived from Escherichia coli, it may also contain its cofactor, NAD (nicotinamide adenine dinucleotide).

[0084] The reaction mixture containing the enzyme and the cell-free protein expression system may be mixed directly with circular DNA as a template to form a reaction mixture for amplifying the circular DNA. The cell-free protein expression system may be a cell-free translation system using total RNA, mRNA, an in vitro transcription product, or the like, which contains RNA consisting of a sequence complementary to the base sequence of the gene encoding the enzyme, as template RNA, or may be a cell-free transcription / translation system using a gene encoding each enzyme or an expression vector containing a gene encoding each enzyme as template DNA.

[0085] The reaction solution is mixed with the circular DNA to form a reaction mixture, and the reaction mixture is incubated at a temperature in the range of 80° C. or less, thereby obtaining a reaction product containing the amplified circular DNA. The amplification may be isothermal or non-isothermal.

[0086] In the case of isothermal amplification, the isothermal conditions are not particularly limited as long as they allow the DNA amplification reaction or DNA replication reaction to proceed. For example, a certain temperature within the optimal temperature range of the DNA polymerase can be used. Examples of isothermal conditions include a certain temperature of 15°C or higher, 16°C or higher, 18°C ​​or higher, 20°C or higher, 23°C or higher, 24°C or higher, 25°C or higher, 26°C or higher, 27°C or higher, or 30°C or higher, and a certain temperature of 80°C or lower, 75°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, 50°C or lower, 45°C or lower, 40°C or lower, 35°C or lower, or 33°C or lower. Further, isothermal conditions include, for example, a temperature in the range of 15 ° C. to 80 ° C., 16 ° C. to 80 ° C., 18 ° C. to 80 ° C., 20 ° C. to 80 ° C., or 25 ° C. to 80 ° C., 15 ° C. to 75 ° C., 16 ° C. to 75 ° C., 18 ° C. to 75 ° C., 20 ° C. to 75 ° C., or 25 ° C. to 75 ° C., 15 ° C. to 70 ° C., 16 ° C. to 70 ° C., 18 ° C. to 70 ° C., 20 ° C. to 70 ° C., or 25 ° C. to 70 ° C., 15 ° C. to 65 ° C., 16 ° C. to 65 ° C., 18 ° C. to 65 ° C., 20 ° C. to 65 ° C., or 25 ° C. to 65 ° C., a temperature in the range of 25 ° C. to 50 ° C., a temperature in the range of 25 ° C. to 40 ° C., a temperature in the range of 30 ° C. to 33 ° C., or about 30 ° C. As used herein, "isothermal" in isothermal amplification means maintaining the temperature within a range of ±7°C, ±5°C, ±3°C, or ±1°C relative to the temperature set during the reaction. The reaction time for isothermal amplification can be set appropriately depending on the amount of the amplification product of the desired circular DNA, and can be, for example, 1 hour to 30 hours, preferably 2 hours to 25 hours, more preferably 3 hours to 20 hours, and even more preferably 5 hours to 15 hours. In one aspect, the circular DNA of this embodiment can achieve a high amplified concentration even in a short reaction time.

[0087] When circular DNA is amplified non-isothermally, it can be amplified under temperature cycle conditions in which incubation is repeated at two temperatures below 80°C, preferably below 65°C. The first temperature in the temperature cycle is a temperature at which replication initiation of double-stranded DNA can be initiated, and the second temperature is a temperature at which replication initiation is suppressed and DNA extension reaction proceeds. The first temperature can be 30°C or higher, for example, 30°C to 80°C, 30°C to 50°C, 30°C to 40°C, or 37°C. The incubation time at the first temperature is not particularly limited, but may be 10 seconds to 10 minutes per cycle, preferably 1 minute. The second temperature can be 27°C or lower, for example, 10°C to 27°C, 16°C to 25°C, or 24°C. The incubation time at the second temperature is not particularly limited, but is preferably set according to the length of the circular DNA to be amplified, and may be, for example, 1 second to 10 seconds per 1,000 bases per cycle. The number of temperature cycles is not particularly limited, but may be 10 to 50 cycles, 20 to 45 cycles, 25 to 45 cycles, or 40 cycles.

[0088] The method of this embodiment may further include incubating at a temperature in the range of 80°C or less to obtain a reaction product containing the amplified circular DNA, diluting the reaction product by at least 2-fold, preferably 3-fold, 4-fold, or 5-fold, with a solution that does not contain the first to third enzyme groups, and then maintaining (re-incubating). Without being bound by theory, dilution of the enzyme groups suppresses new replication initiation, while ongoing replication extension, catenation, and separation reactions continue due to the effects of residual enzymes. Furthermore, by-products generated by nicks or other defects during the reaction can be repaired during this process by the effects of residual ligase, etc. Therefore, the transition from amplification intermediates and by-products to the final product is specifically guided, and an improved yield of the desired circular DNA can be expected.

[0089] The dilution can be maintained under conditions in which the second enzyme group and the third enzyme group can act, for example, at 15 to 50°C, preferably 20 to 40°C, more preferably 25 to 35°C, for example, 10 minutes to 3 hours, preferably 15 minutes to 2 hours, more preferably 20 minutes to 1 hour.

[0090] The composition of the solution that does not contain the first to third enzyme groups is not particularly limited as long as it is a solution in which the second and third enzyme groups can act, and it is convenient to use a solution that has the same composition as the reaction solution except that it does not contain the first, second, and third enzyme groups. Such a solution contains a buffer solution, ATP, GTP, CTP, UTP, dNTP, a magnesium ion source, and an alkali metal ion source. The solution may further contain one or more selected from the above-mentioned inhibitors of nonspecific adsorption of proteins, inhibitors of nonspecific adsorption of nucleic acids, ammonium salts, reducing agents, and NAD.

[0091] In one aspect, when the replication initiation sequence in the circular DNA capable of binding to an enzyme having DnaA activity has a mutant DUE with a higher AT content compared to the wild-type, it is preferable that the method further comprises diluting the circular DNA by at least two-fold with a solution that does not contain the first to third enzyme groups, and then maintaining (re-incubating) the DNA.

[0092] This embodiment also relates to a circular DNA that includes a replication origin sequence capable of binding to an enzyme having DnaA activity and a gyrase binding sequence, but does not include a DnaA box cluster at a position different from the replication origin sequence. This embodiment also relates to a circular DNA that includes a replication origin sequence capable of binding to an enzyme having DnaA activity, a gyrase binding sequence, and a plasmid replication origin, but does not include a DnaA box cluster. The circular DNA may have one or a pair of ter sequences. The replication origin sequence capable of binding to an enzyme having DnaA activity in the circular DNA may be a mutant DUE. For each sequence, see the description of the method for producing circular DNA above. Such a circular DNA can be efficiently amplified by PCR.

[0093] In one aspect of this embodiment, the circular DNA that serves as a template for the method of this embodiment can be simply prepared by preparing a DNA cassette containing (i) a replication initiation sequence that can bind to an enzyme having DnaA activity and (ii) a gyrase binding sequence, a DNA cassette containing (i) a replication initiation sequence that can bind to an enzyme having DnaA activity and having a mutant DUE that has a higher AT content than the wild-type, a DNA cassette containing (i) a replication initiation sequence that can bind to an enzyme having DnaA activity and (ii) a gyrase binding sequence and having a mutant DUE that has a higher AT content than the wild-type, or any of these DNA cassettes that have one or a pair of ter sequences, and linking the DNA cassette to a DNA fragment or circular DNA that does not contain a DnaA box cluster.

[0094] The DNA fragment or circular DNA not containing a DnaA box cluster may be derived from a commercially available plasmid, for example, a plasmid having p15A or fori as the origin of plasmid replication, or linear DNA obtained by enzymatic cleavage of such a plasmid.

[0095] The method for ligating a DNA fragment or circular DNA that does not contain a DnaA box cluster with the DNA cassette is not particularly limited. As a ligation reaction between linear DNA fragments, for example, the infusion method, the Gibson Assembly method, the Golden Gate Assembly method, a method using a commercially available kit such as USER (registered trademark) Cloning (manufactured by NEB), and the Recombination Assembly method (WO 2019 / 009361) are known, and such known techniques can be used.

[0096] As a ligation reaction between linear DNA and circular DNA, for example, Gateway cloning using a site-specific recombination mechanism is known, and a reagent kit is commercially available (manufactured by Thermo Fisher). Alternatively, a method using a homologous recombination reaction using a RecA family recombinase and, if necessary, an exonuclease (WO 2023 / 028145) may be used.

[0097] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0098] Preparation Example 1 The following sequence, which has ter sequences flanking both ends of a portion (264 bases) of the oriC sequence derived from the Escherichia coli chromosome, was prepared as the OriC1.0 cassette (SEQ ID NO: 3). The underlined portion is the ter sequence (SEQ ID NO: 1), and the portion sandwiched between the ter sequences is the oriC sequence (SEQ ID NO: 2).

[0099]

[0100] Preparation Example 2 A mutant ter sequence (terG16 sequence, SEQ ID NO: 5) was linked adjacent to the 5' side of the oriC sequence (SEQ ID NO: 4) derived from the E. coli chromosome, a complementary sequence of the terG16 sequence was linked near the 3' side of the oriC, a Tac promoter sequence (SEQ ID NO: 6) was located further 5' near the terG16 sequence, and a terminator sequence of the E. coli fdhF gene (SEQ ID NO: 7) was located further 3' near the complementary sequence of the terG16 sequence. This DNA fragment was prepared as the OriC2.0 cassette (426 bp, SEQ ID NO: 8). The terG16 sequence is a single-nucleotide mutation of the nucleotide sequence of SEQ ID NO: 37. The underlined portion represents the terG16 sequence, and the portion of the region between the underlined portions indicated in capital letters is the oriC sequence (286 bp).

[0101]

[0102] Preparation Example 3 A DNA fragment in which SGS (SEQ ID NO: 9) was linked to the 5' side of oriC in the OriC2.0 cassette sequence was prepared as the OriCsg cassette (582 bp, SEQ ID NO: 10). The base sequence is shown in Table 5. In the table, the region indicated in underlined capital letters represents SGS (bold represents the SGS consensus sequence), and the region indicated in capital letters near the center (not underlined) represents the oriC sequence. The region enclosed in a box represents the terG16 sequence and its complementary sequence.

[0103]

[0104] Preparation Example 4 A DNA fragment in which an E. coli -derived Stuffer sequence (a sequence located outside oriC in the E. coli genome and having no function) was linked to the 5' side of oriC in the OriC2.0 cassette sequence was prepared as the OriCsg_Stuffer cassette (582 bp, SEQ ID NO: 11). The base sequence is shown in Table 6. In the table, the region shown in underlined capital letters represents the Stuffer sequence, and the region shown in capital letters near the center (ununderlined) represents the oriC sequence. Additionally, the region enclosed in a box represents the terG16 sequence and its complementary sequence.

[0105]

[0106] Preparation Example 5 A DNA fragment in which the M and R elements of the L, M, and R elements constituting the oriC DUE in the OriC2.0 cassette sequence are mutant DUEs with a higher AT content compared to the wild-type was prepared as the OriC5.6AT cassette (427 bp, SEQ ID NO: 12). The base sequence is shown in Table 7. In the table, the bolded part is the DUE part, and the boxed part in the bolded part is the mutated part in the mutant DUE. The underlined parts are the M element (underlined part on the 5'-end) and the R element (underlined part on the 3'-end, with a single-base deletion mutation) of the DUE.

[0107]

[0108] [Preparation Example 6] The following four types of plasmids were prepared. E1-p15A plasmid: An E1 sequence (579 bp) containing a marker gene was ligated to pACYC plasmid (Nippon Gene Co., Ltd.) using the USER method described below, and the resulting plasmid was amplified by PCR amplification to create a plasmid with a total length of 3.5 kbp. H2-p15A plasmid: An H2 sequence (a sequence encoding a part of a known virus, 3.8 kbp) was ligated to pACYC plasmid (Nippon Gene Co., Ltd.) using the USER method described below, and the resulting plasmid was amplified by PCR amplification to create a plasmid with a total length of 6.8 kbp. pUC plasmid: An E1 sequence (579 bp) was ligated to plasmid pUC4K (GenBank accession number: X06404, total length 3.9 kbp) using the USER method described below, and the resulting plasmid was amplified by PCR amplification to create a plasmid with a total length of 3.5 kbp. pBeloBAC plasmid: manufactured by NEB (GenBank accession number: U51113, total length 7.5 kb)

[0109] [Example 1] Ligation and amplification of p15A plasmid and OriC cassette Each of the OriC cassette fragments from Preparation Examples 1 to 5 and partial fragments of the two p15A plasmids from Preparation Example 6 were prepared by PCR using primers containing dUTP as shown in Table 8. In Table 8, u represents deoxyuridine (dU). The prepared DNA fragments were mixed and added to 5 μL of a reaction solution (1×rCutSmart buffer (NEB), 7.5% by mass PEG8000, 10 mU / μL Thermolabile USER II Enzyme (NEB; an enzyme containing uracil DNA glycosylase and endonuclease VIII that causes one-base gap formation at the dUTP moiety)), and ligated by treating at 37° C. for 15 minutes and at 60° C. for 5 minutes (USER method). Thereafter, a PCR amplification reaction was carried out using the ligated DNA as a template, and the desired circular DNA was amplified.

[0110]

[0111] The PCR amplification reaction was carried out as follows: 0.5 μL of a solution containing a template was mixed with 4.5 μL of a reaction mixture (appropriately diluted) having the composition shown in Table 9 to prepare a PCR amplification reaction solution (5 μL), which was then incubated at 33°C for 10 hours to carry out the PCR amplification reaction, and then cooled to 4°C.

[0112]

[0113] In Table 9, SSB is SSB derived from E. coli, IHF is a complex of IhfA and IhfB derived from E. coli, DnaG is DnaG derived from E. coli, DnaN is DnaN derived from E. coli, Pol III* is a DNA polymerase III* complex which is a complex consisting of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from E. coli, DnaB is DnaB derived from E. coli, DnaC is DnaC derived from E. coli, DnaA is DnaA derived from E. coli, RNaseH is RNaseH derived from E. coli, Ligase is DNA ligase derived from E. coli, Pol I is DNA polymerase I derived from E. coli, GyrA is GyrA derived from E. coli, GyrB is GyrB derived from E. coli, Topo IV represents a complex of ParC and ParE derived from E. coli, Topo III represents topoisomerase III derived from E. coli, and RecQ represents RecQ derived from E. coli.

[0114] SSB was purified and prepared from an E. coli strain expressing SSB using a process including ammonium sulfate precipitation and ion exchange column chromatography. IHF was purified and prepared from an E. coli strain co-expressing IhfA and IhfB using a process including ammonium sulfate precipitation and affinity column chromatography. DnaG was purified and prepared from an E. coli strain expressing DnaG using a process including ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography. DnaN was purified and prepared from an E. coli strain expressing DnaN using a process including ammonium sulfate precipitation and anion exchange column chromatography. Pol III* was purified and prepared from an E. coli strain co-expressing DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaB and DnaC were purified and prepared from an E. coli strain co-expressing DnaB and DnaC using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaA was purified and prepared from an E. coli strain expressing DnaA using a process including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography. GyrA and GyrB were purified and prepared from a mixture of an E. coli strain expressing GyrA and an E. coli strain expressing GyrB using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo IV was purified and prepared from a mixture of an E. coli strain expressing ParC and an E. coli strain expressing ParE using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo III was purified and prepared from a Topo III-expressing E. coli strain through a process including ammonium sulfate precipitation and affinity column chromatography. RecQ was purified and prepared from a RecQ-expressing E. coli strain through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. RNase H, Ligase, and Pol I were commercially available enzymes derived from E. coli (Takara Bio).Tus was purified and prepared from an E. coli expression strain of Tus through a process including affinity column chromatography and gel filtration column chromatography.

[0115] Example 2 Ligation and Amplification of pUC Plasmid and OriC Cassette Each of the OriC cassette fragments from Preparation Examples 1 to 3 and a partial fragment of the pUC plasmid from Preparation Example 6 were prepared by PCR using primers containing dUTP. The primers used were those shown in Example 1 and those listed in Table 10. The prepared DNA fragments were mixed and ligated using the same method as in Example 1. Using the ligated DNA as a template, a PCR amplification reaction was carried out using the same method as in Example 1 to amplify the desired circular DNA.

[0116]

[0117] [Example 3] Ligation and amplification of pBeloBAC plasmid and OriC cassette Each of the OriC cassette fragments of Preparation Examples 1 to 3 and 5 and a partial fragment of the pBeloBAC plasmid of Preparation Example 6 were prepared by PCR using the primers listed in Table 11. For the homologous recombination reaction, 200 pM of the plasmid and 200 pM of each OriC cassette fragment to which an overlapping sequence had been added were added to 5 μL of a reaction solution (1 μM RecA, 80 mU / μL exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng / μL creatine kinase, 50 mM potassium glutamate, 150 mM TMAC, 5% by mass PEG 8000, and 10% by volume DMSO), and the mixture was incubated at 42°C for 30 minutes and at 65°C for 2 minutes to ligate the plasmid and each OriC cassette. Using the same method as in Example 1, a PCR amplification reaction was performed using the ligated DNA as a template, and the desired circular DNA was amplified.

[0118]

[0119] Example 4: Cloning of E. coli Circular DNA Incorporating an OriC Cassette Example 4-1: The circular DNA obtained in Examples 1 and 2 was introduced into E. coli NEB stable strain (manufactured by NEB) by chemical methods. A portion of the DNA was then cultured at 30°C for 24 hours on LB agar medium containing 33 μg / mL kanamycin. Multiple E. coli colonies were selected from the agar plate, and each transformed E. coli was cultured with shaking at 30°C for 24 hours in 5 mL of liquid medium containing 33 μg / mL kanamycin. LB liquid medium was used as the liquid medium. Plasmid extraction was performed using a commercially available DNA extraction kit (product name "QIAprep Spin Miniprep Kit", manufactured by QIAGEN). A portion of the plasmid DNA solution eluted with 50 μL of 10 mM Tris-Cl (pH 8.5) solution was treated with the restriction enzyme XbaI, and the concentration was measured using a fluorometer (product name "Quantus (registered trademark) Fluorometer", manufactured by Promega).

[0120] [Example 4-2] The circular DNA obtained in Example 3 was introduced into Escherichia coli DH5α strain (manufactured by Takara Bio) by chemical method. A portion of the culture was then cultured at 37°C for 16 hours on LB agar medium containing 20 μg / mL chloramphenicol. Multiple E. coli colonies were selected from the agar plate, and each transformed E. coli was cultured with shaking at 37°C for 16 hours in 5 mL of liquid medium containing 20 μg / mL chloramphenicol. LB liquid medium was used as the liquid medium. Plasmid extraction was performed using a commercially available DNA extraction kit (product name "QIAprep Spin Miniprep Kit", manufactured by QIAGEN). A portion of the plasmid DNA solution eluted with 50 μL of 10 mM Tris-Cl (pH 8.5) solution was treated with the restriction enzyme XbaI, and the concentration was measured using a fluorometer (product name "Quantus (registered trademark) Fluorometer", manufactured by Promega).

[0121] Example 5 An PCR amplification reaction solution was prepared by mixing 1 μL (200 pg / μL) of each of the solutions of the plasmids (6 types) in which three types of OriC cassettes (OriC1.0, OriC2.0, OriCsg) were incorporated into two types of p15A plasmids and the plasmids (2 types) in which two types of OriC cassettes (OriC2.0, OriCsg) were incorporated into pUC plasmids, obtained in Example 4, with 99 μL of a reaction mixture having the composition shown in Table 9. The PCR amplification reaction solution was incubated at 33°C for 10 hours to carry out an PCR amplification reaction, and then cooled to 4°C.

[0122] Thereafter, a portion of the reaction mixture was subjected to agarose gel electrophoresis together with a marker, and the separated bands were stained with dsGreen.

[0123] In addition, a portion of the reaction solution was treated with the restriction enzyme XbaI, and the DNA concentration was measured using a Quantus-PicoGreen system (Promega). The results are shown in Figure 1. When the p15A plasmid and OriCsg were combined, the DNA concentration after amplification was higher than when other OriC cassettes were used. When the pUC plasmid and OriCsg were combined, no significant increase in the DNA concentration after amplification was observed compared to other OriC cassettes.

[0124] [Example 6] Of the eight plasmids used in Example 5, five plasmids having E1 sequences were used, and an experiment similar to that in Example 5 was performed three times for each sample. As shown in Figure 2, similar results to those in Example 5 were obtained, and when p15A plasmid and OriCsg were combined, the DNA concentration after amplification was higher than when other OriC cassettes were used. Also, as in Example 5, when pUC plasmid and OriCsg were combined, no significant increase in DNA concentration after amplification was observed compared to other OriC cassettes. The average values ​​of the three experiments are shown in Table 12.

[0125]

[0126] [Example 7] Experiments similar to those in Example 5 were performed four times for each sample using three plasmids obtained in Example 4, each incorporating three OriC cassettes (OriC1.0, OriC2.0, and OriCsg) into pBeloBAC. As shown in Figure 3, when the pBeloBAC plasmid was used, as with the p15A plasmid, the DNA concentration after amplification was higher when combined with OriCsg than when other OriC cassettes were used. The average values ​​of the experiments are shown in Table 13 (for experiments using the OriC1.0 cassette, the average value was calculated from the results of three experiments).

[0127]

[0128] [Example 8] Using the four plasmids obtained in Example 4 in which four types of OriC cassettes (OriC1.0, OriC2.0, OriCsg, OriC_Stuffer) were incorporated into the E1-p15A plasmid, an experiment similar to that in Example 5 was performed three times for each sample. As shown in Figure 4, when the pBeloBAC plasmid was used, as with the p15A plasmid, when OriCsg was combined, the DNA concentration after amplification was higher compared to other OriC cassettes. On the other hand, when the MuSGS sequence of OriCsg was changed to the Stuffer sequence, the DNA concentration after amplification was lower compared to OriCsg. The average values ​​of the experiment are shown in Table 14.

[0129]

[0130] [Example 9] [Example 9-1] Using the four plasmids obtained in Example 4 in which four types of OriC cassettes (OriC1.0, OriC2.0, OriCsg, OriC5.6AT) were incorporated into pBeloBAC, an experiment similar to that in Example 5 was performed three times for each sample. As shown in Figure 5, when the pBeloBAC plasmid was used, the DNA concentration after amplification was highest in the order of OriC5.6AT > OriCsg > OriC2.0 > OriC1.0. When the OriC5.6AT cassette was used, there was more open circular DNA (OC) after amplification compared to the other OriC cassettes.

[0131] Example 9-2 After amplifying circular DNA in the same manner as in Example 9-1, a portion of the PCR amplification reaction solution (1 μL) was diluted 1:5 with the PCR reaction buffer shown in Table 9. The diluted solution was incubated at 30°C for 30 minutes and then cooled on ice (finalization treatment). Thereafter, a portion of each reaction solution was subjected to agarose gel electrophoresis together with a marker in the same manner as in Example 9-1, and the separated bands were stained with dsGreen. The results are shown in Figure 6. Compared to Example 9-1, the amount of OC was reduced when the OriC5.6AT cassette was used.

[0132] Example 10 An experiment similar to that of Example 5 was carried out using the four plasmids used in Example 9, except that the PCR amplification reaction conditions were 24°C, 27°C, 30°C, or 33°C for 10 hours. The results are shown in Figure 7. As shown in Figure 7, circular DNA containing OriC1.0 as the OriC cassette was hardly amplified at 27°C or below. Circular DNA containing OriC2.0 or OriCsg showed a small amount of amplification at 27°C. Circular DNA containing OriC5.6AT was amplified even at 24°C, demonstrating that amplification at lower temperatures was possible compared to when other OriC cassettes were used.

[0133] Example 11 An experiment similar to that of Example 5 was carried out using four types of plasmids obtained in Example 4, each having four types of OriC cassettes (OriC1.0, OriC2.0, OriCsg, and OriC5.6AT) integrated into the E1-p15A plasmid, except that the PCR amplification reaction conditions were 33°C for 3 hours, 6 hours, or 10 hours. The results are shown in Figure 8. The change in DNA concentration with respect to reaction time is shown in the graph of Figure 9 and Table 15.

[0134]

[0135] 8 and 9 , the amount of DNA amplified was greatest when OriC5.6AT was used as the OriC cassette, and the amplification rate was also faster than when the other cassettes were used. On the other hand, an increase in OC was observed as the amplification reaction time increased.

[0136] [Example 12] A partial fragment of the commercially available pACYC184_DNA plasmid (Nippon Gene Co., Ltd.) having a replication origin region derived from the p15A plasmid and the OriC cassette fragments (OriC2.0 and OriCsg) of Preparation Examples 2 and 3 were prepared by PCR using the primers listed in Table 16.

[0137]

[0138] Ligation was performed using homologous recombination in the same manner as in Example 3, and then PCR amplification was performed using the ligated DNA as a template to amplify circular DNA in which OriC2.0 or OriCsg was incorporated into the pACYC184 plasmid. E. coli cloning was then performed in the same manner as in Example 4-2.

[0139] Using 200 pg of each of the two resulting plasmids (plasmid containing OriC2.0: 4671 bp, plasmid containing OriCsg: 4827 bp) and 100 μL of the PCR reaction mixture, PCR amplification reactions, agarose gel electrophoresis, and DNA concentration measurements were performed in the same manner as in Example 5. For PCR amplification, the reaction mixture with the composition shown in Table 9 was appropriately diluted and used. During the reaction, 0.05% Tween 20 (Wako) was added. The results are shown in Figure 10. The average values ​​of three experiments are also shown in Table 17.

[0140]

[0141] Even when a p15A plasmid different from that used in Example 5 and the like was used, it was confirmed that when OriCsg was combined, the DNA concentration after amplification was higher than when OriC2.0 was combined.

[0142] [Example 13] Linear DNA lacking the E. coli ori sequence was prepared from the pACYC184 plasmid used in Example 12. Each OriC cassette fragment and a partial fragment of the pACYC184 plasmid of Example 12 were prepared by PCR using the primers listed in Table 18.

[0143]

[0144] The prepared linear DNA was ligated to the OriC cassette fragments (OriC2.0 and OriCsg) using a homologous recombination reaction in the same manner as in Example 12, and then a PCR amplification reaction was performed using the ligated DNA as a template to amplify circular DNA in which OriC2.0 or OriCsg had been incorporated into the pACYC184 plasmid, which does not have an on.

[0145] Using 200 pg of the two resulting circular DNAs (circular DNA with OriC2.0: 3901 bp, circular DNA with OriCsg: 4057 bp) and 100 μL of the PCR reaction mixture, PCR amplification reactions, agarose gel electrophoresis, and DNA concentration measurements were performed in the same manner as in Example 5. The same reaction mixture as in Example 12 was used for PCR amplification. The results are shown in Figure 11. Two lots of OriCsg were tested. The average values ​​of three experiments are shown in Table 19.

[0146]

[0147] Even when a p15A plasmid not having an E. coli ori was used, it was confirmed that the DNA concentration after amplification was higher when combined with OriCsg than when combined with OriC2.0.

[0148] Example 14 In Example 14, the following method was used.

[0149] <Reaction PCR Amplification> Reaction PCR amplification was performed using a previously published method (Non-Patent Documents 1 and 2) with minor modifications. A 5 μL reaction mixture containing 1× Reaction PCR Buffer I, 1× Reaction PCR Buffer II, 1× Enzyme Mix, and 3 ng / μL λ phage DNA was used and incubated at 33°C for 12 hours. The reaction mixture was diluted with 1× Reaction PCR Buffer and incubated at 30°C for 30 minutes. When the template was AT-rich circular DNA, AT-PCR was performed using the previously published reaction mixture supplemented with 300 nM HU and 100 nM H-NS to enhance amplification. For low-temperature PCR amplification experiments, a 10 μL reaction mixture containing 1× Reaction PCR Buffer I and II, 0.05% Tween 20, and 0.5× Enzyme Mix was used. The PCR reaction mixture was diluted with 1x PCR buffer and incubated at 30°C for 30 minutes. A portion was mixed with stop buffer (Non-Patent Document 2) and analyzed by agarose gel electrophoresis. Multiple DNA assemblies were performed using the RA method (WO 2019 / 009361). Agarose gel electrophoresis was performed as previously reported (Nara and Su'etsugu, Biotechniques, 2021, vol. 71, pp. 528-533).

[0150] <Construction of OriC Mutants> The construction of each mutant and the circular DNA incorporating it are described in detail below. PCR amplification was performed using KOD One polymerase (TOYOBO). The resulting fragment was gel extracted and purified by column purification (Macherey-Nagel). The resulting oriC mutant fragment was inserted into a pSV-β-galactosidase (pSVβ; Promega) vector (7.2 kb) using Escherichia coli RecA and exonuclease III according to a previously reported method (WO 2023 / 038145, Example 1), and amplified by PCR. The resulting amplified product was purified by column purification (Macherey-Nagel).

[0151] Real-time PCR: Each reaction was performed using 10 μL of a reaction mixture containing 1× PCR buffer I and II, 0.05% Tween 20, 300 nM YO-PRO-1 iodine (Ex / Em = 491 / 509 nm) (Thermo Fisher Scientific), and 0.5× RE enzyme mixture. Nine μL of the reaction mixture was preincubated at 20°C for 15 minutes and then transferred to ice to prevent PCR reaction initiation. Next, 1 μL of oriC DNA was added to a final concentration of 1 pM, and the PCR reaction was initiated at 30°C using a real-time system (Thermal Cycler Dice Real-time system, Takara). Amplified DNA was detected every 10 minutes using a SYBR filter. The doubling time of the amplified DNA in the PCR system was calculated from the slope of the logarithmic phase of the amplification curve. To standardize the experimental conditions for each oriC mutant, all mutants were cloned into the pSVβ plasmid with a GC content of 52%.

[0152] Competitive Amplification Assay: A 13-kb minichromosome containing oriCwt was used as a competitor and mixed with a 7-kb minichromosome containing an oriC mutant at a ratio of 200 pM:100 pM. The DNA mixture was amplified using the PCR amplification method described above with minor modifications. Each PCR reaction was performed using a 10-μL reaction mixture containing 1× PCR buffer I and II, 0.05% Tween 20, and 0.5× RE enzyme mixture as final concentrations. The PCR reaction mixture was preincubated at 20°C for 15 minutes and then mixed with the DNA mixture on ice to prevent initiation of the PCR reaction. After addition of the DNA mixture, PCR amplification was carried out at 30°C for 12 hours. The PCR reaction mixture was diluted with 1× PCR buffer, incubated at 30°C for 30 minutes, and mixed with stop buffer. An aliquot was then mixed with stop buffer and analyzed by agarose gel electrophoresis.

[0153] [Example 14-1] Construction of circular DNA incorporating OriC mutant

[0154] <Circular DNA incorporating OriCwt> Using the primer pair P1 / P2 shown in Table 21, wild-type oriCwt having the sequence shown in Table 20 was amplified by PCR.

[0155]

[0156] The resulting amplification product was introduced into a pSV-β-galactosidase vector (pSVβ) (6.8 kb, 53% GC) (Promega) using the method described in Example 1 of WO 2023 / 038145, and amplified to obtain a minichromosome carrying oriCwt (pSVβ-oriCwt).

[0157] <Circular DNA incorporating oriC2.0AT, oriC3.0AT, oriC3.4AT, or oriC3.6AT> Minichromosomes containing oriC2.0AT, oriC3.0AT, or oriC3.4AT were constructed using the primer pairs listed in Table 21. Specifically, of the primer pairs listed in Table 21, P1 / P4 and P2 / P3, P1 / P6 and P2 / P5, and P1 / P8 and P2 / P7 were used. These two primer pairs were then used separately to amplify the oriC region of the template pSVβ-oriCwt DNA in two fragments by PCR. The resulting two fragments were then assembled by overlap PCR using the P1 / P2 primers and subsequently inserted into the pSVβ vector using the method described in Example 1 of WO 2023 / 038145.

[0158] A minichromosome carrying oriC3.6AT was constructed using the following primer pairs. The oriC region of the template pSVβ-oriC3.0AT DNA was split into two fragments and PCR-amplified using primers P1 / P10 and P2 / P9, among the primer pairs shown in Table 21. The resulting two fragments were then assembled by overlap PCR using primers P1 / P2 and subsequently introduced into the pSVβ vector using the method described in Example 1 of WO 2023 / 038145.

[0159] <Circular DNA incorporating oriC4.0AT, oriC5.4AT, or oriC5.6AT> oriC4.0AT was constructed as follows. The primer pairs P1 / P12 and P2 / P7 listed in Table 21 were used to PCR amplify oriC3.6AT as a template, resulting in two fragments. These fragments were then assembled by overlap PCR using P1 / P2 and then inserted into a pSVβ vector using the method described in Example 1 of WO 2023 / 038145. oriC5.4AT and oriC5.6AT were constructed using primer pairs P1 / P12 and P2 / P11, and P1 / P14 and P2 / P13, respectively. These primer pairs were used for PCR amplification using oriC3.4AT and oriC3.6AT as templates. The assembly fragment generated by overlap PCR using P1 / P2 was introduced into the pSVβ vector using the method described in Example 1 of WO 2023 / 038145 to obtain a circular DNA incorporating oriC5.4AT and oriC5.6AT.

[0160] <Circular DNA incorporating oriC6.0AT> oriC6.0AT was constructed as follows. Fragments obtained using oriC4.0AT as a template were assembled by overlap PCR using primer pairs P1 / P14, P12 / P13, and P2 / P11 listed in Table 21 and P1 / P2. The resulting assembly product was introduced into a pSVβ vector using the method described in Example 1 of WO 2023 / 038145.

[0161] Minichromosomes containing oriC mutants, which were prepared and amplified using the method described in Example 1 of WO 2023 / 038145, were purified using a spin column (product name "QIAprep Spin Miniprep Kit", manufactured by Qiagen). The sequences of the oriC mutants were confirmed by Sanger sequencing.

[0162] <Circular DNA incorporating oriCdueM9T, oriCdueM9A, oriCdueM13T, or oriCdueM13A> A 7-kb minichromosome containing a poly(A) or T sequence in the DUE-M or DUE-R region was constructed. oriCdueM9T, oriCdueM9A, oriCdueM13T, and oriCdueM13A were designed with the primer pairs P15 / P16, P17 / P18, P19 / P20, and P21 / P22, respectively, listed in Table 21. These primers were subsequently used in inverse PCR amplification using pSVβ-oriCwt DNA as a template.

[0163] Subsequently, 100 μL of competent E. coli DH5α cells (cell name "DH5α High Champion", manufactured by SMOBIO) were directly transformed with 1 μL of the resulting fragment. The resulting transformants were selected on LB plates containing 50 μg / mL carbenicillin at 30°C. After culturing these transformants for 16 hours at 30°C, minichromosome DNA containing oriCdueM9T, oriCdueM9A, oriCdueM13T, and oriCdueM13A was purified from the transformed cells using a spin column (product name "QIAprep Spin Miniprep Kit", manufactured by Qiagen). The sequences of the oriC mutants were confirmed by Sanger sequencing.

[0164]

[0165] [Example 14-2] PCR amplification using an OriC mutant with a mutation in DUE Figure 12A is a schematic diagram of the PCR amplification cycle. The amplification process starts from the replication initiation sequence (oriC) and exponentially amplifies circular DNA through bidirectional amplification, resulting in supercoiled DNA. The initiation step proceeds only when double-stranded DNA is cleaved at oriC.

[0166] Figure 12B shows a schematic diagram of the initiation process at oriC. oriC contains an AT-rich double-stranded cleavage domain (DUE) and an initiation protein DnaA assembly domain (DOR). ATP-bound DnaA (ATP-DnaA) binds to the DOR and forms a homomultimeric complex, which cleaves the AT-rich DUE and allows entry of the DnaB helicase. The dark arrows (R1, R5M, R2, and R4) indicate the DnaA-ADP binding site. The light arrows (τ2, l1, l2, C3, C2, l3, and C1) indicate the DnaA-ATP binding site.

[0167] The effect of OriC mutants with DUE mutations on PCR amplification efficiency was investigated. A schematic diagram of the oriC2.0AT and oriC3.0AT sequences prepared in Example 14-1 is shown in Figure 13A. DUE L, M, and R represent 13-mer repeat sequences. R1 represents the DnaA-box R1 sequence. The dotted box indicates the region (1-60 bp) used to calculate the GC content shown to the right of the sequence. The sequences in Figure 13A are shown in Table 22. In Table 22, the underlined wild-type sequence indicates the DnaA ssDUE binding sequence (TT[A / G]T(T)), the boxed letters indicate bases that differ from the wild-type sequence, and "-" indicates a base deletion. Each base number is assigned starting from the region 70 bp upstream from just before the R1 DnaA-box base.

[0168]

[0169] 10-kb fragments with GC contents of 56%, 21%, or 23% were assembled with oriC2.0AT (2.0AT), oriC3.0AT (3.0AT), or oriCwt (wt) and amplified using PCR. The resulting PCR products were electrophoresed on a 1% agarose gel in 0.5x TBE and stained with SYBR green I. The results are shown in Figure 13B. As shown in Figure 13B, compared to the wild-type, the OriC2.0AT and OriC3.0AT cassettes increased the amplification efficiency of circular DNA with a low GC content (i.e., circular DNA with a high AT content), which cannot be amplified with the wild-type oriC. It was presumed that the 10 kb fragment with a GC content of 23% contained a region where the double strand derived from the DUE of oriC2.0AT was easily cleaved, making it difficult for the DNA structure (negative supercoil) necessary for the initiation of replication to form, and therefore the 10 kb fragment with a GC content of 21% was amplified, but the 10 kb fragment with a GC content of 23% was not amplified.

[0170] Real-time PCR was performed on oriCwt, oriC2.0AT, and oriC3.0AT. Amplification of 7 kb circular DNA (pSVβ, 52% GC) containing each oriC was initiated at a DNA concentration of 1 pM in the presence of YO-PRO-I intercalator at 30°C. The results are shown in Figure 13C. The values ​​accompanying each graph represent the doubling time and standard deviation. As shown in Figure 13C, the rise time of the amplification curves was enhanced for oriC2.0AT and oriC3.0AT, suggesting improved amplification efficiency in the initial stage of PCR amplification.

[0171] The amplification rate was calculated from the doubling time calculated from the slope of the logarithmic plot of amplification, shown in Figure 13C. Real-time PCR analysis showed that the amplification rates of oriC2.0AT (29.5 min) and oriC3.0AT (24.7 min) increased by 1.1-fold and 1.3-fold, respectively, compared with oriCwt (31.9 min). Despite the low GC content of the DUE, oriC2.0AT (10%) showed a slower amplification rate than oriC3.0AT (20%) (high GC content), suggesting that the amplification rate was enhanced not only by the reduced GC content of the DUE region but also by sequence specificity.

[0172] In the DUE of oriC2.0AT, three nine-base repeats of the 5'-TATATTTAT-3' sequence were observed, spaced 5 and 9 bases apart (Fig. 13A). The DUE mutations in oriC3.0AT included two C-to-A substitutions and a single-base deletion (Fig. 13A). These mutations reduced the GC content by 5% compared to oriCwt. Furthermore, the number of KAK sequences (the number of consecutive KAK motifs) in DUE-R increased from 3 to 5. These DUE mutations also reduced the GC content and increased the number of TAT sequence motifs (e.g., TTATT) that resemble the DnaA ssDUE binding sequence TT[A / G]T(T).

[0173] Example 14-3: PCR amplification by OriC mutants with mutations in DUE and DOR 1 The effect of OriC mutants with mutations in DUE and DOR on PCR amplification efficiency was investigated. A schematic diagram of the oriC3.0AT and oriC4.0AT sequences prepared in Example 14-1 is shown in Figure 14A. In Figure 14A, mutations in oriC4.0AT compared to oriC3.0AT are shown above the SNPs line. A "-" in the sequence indicates a base deletion, and lowercase letters indicate sequences that do not match the DnaA consensus sequence in the DOR region. As with Figure 13, each base number is assigned starting from the region 70 bp upstream from just before the base of the R1 DnaA-box.

[0174] The three sequences in Figure 14A are shown in Tables 23 and 24. Boxed letters indicate bases that differ from the wild-type sequence. Lowercase letters indicate sequences that do not match the DnaA consensus sequence in the DOR region. In Table 23, the underlined regions indicate the M and R elements of DUE from the 5' side, and "." indicates a base deletion. In Table 24, the underlined regions indicate the τ2, I1, and I2 elements of DOR from the 5' side.

[0175]

[0176]

[0177] Figure 14B shows a schematic diagram of mutations on oriC3.0AT, oriC3.4AT, oriC3.6AT, and oriC4.0AT. Asterisks indicate the positions of mutated bases that differ from the wild-type. oriC3.4AT is a cassette having only a mutation in the DOR of oriC4.0AT. oriC3.6AT is a cassette having only a mutation in the DUE of oriC4.0AT. Using these mutant oriC cassettes, circular DNA amplification was performed using a method similar to the real-time PCR method of Example 14-2, and the calculated doubling times (minutes) and standard deviations of DNA amplification are shown on the far right of Figure 14B.

[0178] FIG. 14C shows the results of real-time PCR performed using oriCwt, oriC3.0AT, oriC3.4AT, oriC3.6AT, and oriC4.0AT in the same manner as in Example 14-2.

[0179] As shown in Figure 14A, oriC4.0AT has three mutations in the DOR region compared to oriC3.0AT: G137T at τ2, C150T at I1, and G161T at I2. As shown in Figures 14B and 14C, real-time PCR analysis revealed that the amplification rate of oriC4.0AT (23.4 min) was 1.4-fold and 1.05-fold faster than oriCwt (31.9 min) and oriC3.0AT (24.7 min), respectively. Furthermore, oriC3.6AT (20.7 min), a cassette containing only the mutation in the DUE of oriC4.0AT, had an even higher amplification rate (1.1-fold) and a faster rise in the amplification curve compared to oriC4.0AT. In contrast, oriC3.4AT (34.1 min), a cassette containing only a mutation in the DOR of oriC4.0AT, showed a 0.7-fold decrease in amplification rate compared to oriC4.0AT and an increase in the amplification curve similar to that of oriCwt. These results suggest that the DUE mutation significantly improves the amplification rate. Compared to wild-type oriC, oriC cassettes that showed increased amplification rates contained a continuous KAK sequence in the R element of the DUE region, and oriC cassettes with particularly high amplification rates also contained a repetitive AT sequence on the left (5') side of the M element of the DUE region.

[0180] Figure 14D shows the results of amplifying circular DNAs with different GC contents using oriCwt, oriC3.0AT, oriC3.6AT, and oriC4.0AT, as in Example 14-2. 10-kb DNA fragments with 23% and 56% GC content were assembled with each oriC cassette to obtain circular DNAs. The resulting DNAs were amplified by PCR, electrophoresed on a 1% agarose gel in 0.5x TBE, and stained with SYBR Green I.

[0181] As shown in Figure 14D, circular DNA with a 23% GC content, which could not be amplified when oriC3.0AT was used in Example 14-2, could be amplified when oriC3.6AT and oriC4.0AT were used. As shown in Figure 14A, compared to oriC3.0AT, oriC3.6AT and oriC4.0AT contain a repetitive AT sequence to the left of the M element in the DUE region and have a continuous KAK sequence in the R element in the DUE region. This is thought to enable amplification of DNA with a high AT content.

[0182] Example 14-4: PCR amplification 2 using an OriC mutant with mutations in DUE and DOR. Compared to Example 14-3, we confirmed whether the DNA amplification rate in the PCR method would change by further reducing the GC content of DUE and increasing the number of KAK sequences. Furthermore, we introduced an additional mutation into R5M of DOR, which is particularly important for initiating DnaA homo-oligomer formation, and confirmed whether the DNA amplification rate in the PCR method would change.

[0183] The effect of OriC mutants with mutations in DUE and DOR on PCR amplification efficiency was investigated. A schematic diagram of the oriC4.0AT and oriC6.0AT sequences prepared in Example 14-1 is shown in Figure 15A. In Figure 15A, "-" in the sequence indicates a base deletion, and lowercase letters indicate sequences that do not match the DnaA consensus sequence in the DOR region. The underlined sequences indicate consecutive KAK sequences. As in Figure 13, the base numbers are assigned starting from the region 70 bp upstream from just before the base of the R1 DnaA-box.

[0184] The three sequences in Figure 15A are shown in Tables 25 and 26. Boxed letters indicate bases that differ from the wild-type sequence, lowercase letters indicate sequences in the DOR region that do not match the DnaA consensus sequence, and shaded letters indicate consecutive KAK sequences. In Table 25, the underlined region indicates the M and R elements of DUE from the 5' side, and a period (.) indicates a base deletion. In Table 26, the underlined region indicates the R5M, τ2, I1, and I2 elements of DOR from the 5' side. As shown in Table 25, oriC4.0AT and oriC6.0AT contain five and seven KAK motifs, respectively, in DUE-R, and the GC content is reduced to 10%. Furthermore, as shown in Table 26, the R5M sequence in the DOR region matches the DnaA consensus sequence (Figure 15A).

[0185]

[0186]

[0187] Figure 15B shows a schematic diagram of mutations on oriC5.4AT, oriC5.6AT, and oriC6.0AT. Asterisks indicate the positions of mutated bases that differ from the wild-type. oriC5.4AT is a cassette with the same mutation as in the DOR of oriC6.0AT. oriC5.6AT is a cassette with the sequence shown in Table 7, and in particular, has the same mutations as in the DUE (M and R) of oriC6.0AT. Using these mutant oriC cassettes, circular DNA amplification was performed using a method similar to the real-time PCR method of Example 14-2, and the calculated doubling times (minutes) and standard deviations of DNA amplification are shown on the far right of Figure 15B.

[0188] FIG. 15C shows the results of real-time PCR performed using oriCwt, oriC5.4AT, oriC5.6AT, and oriC6.0AT in the same manner as in Example 14-2.

[0189] As shown in Figures 15B and 15C, the doubling time of oriC6.0AT (25.8 min) was slower than that of oriC4.0AT (23.4 min), decreasing by 0.9-fold. On the other hand, when oriC5.6AT (20.3 min), which has only the DUE mutation of oriC6.0AT, was used, the DNA amplification rate increased, being slightly faster than that of oriC3.6AT (20.7 min; Figures 14B and 14C). The number of KAK sequences in DUE-R was seven for oriC5.6AT and five for oriC3.6AT.

[0190] On the other hand, when oriC5.4AT, which has only the DOR mutation of oriC6.0AT, was used (24.5 minutes), the DNA amplification rate increased by 1.05-fold compared to oriC6.0AT. When oriC5.4AT was used, the DNA amplification rate in the PCR method was higher (1.3-fold) than when oriCwt was used. However, the rise of the amplification curve was only slightly faster than when oriCwt was used. The end of replication was also earlier than when oriCwt was used.

[0191] The effect of increasing the A / T sequence in the DUE-MR region on PCR amplification was investigated. Schematic diagrams of the oriCdueM9T, oriCdueM9A, oriCdueR13T, and oriCdueR13A sequences prepared in Example 14-1 are shown in Figure 15D. These are DUE-M or DUE-R mutants containing poly(A) or poly(T) sequences. The underlining is the same as in Figure 15A. As in Figure 13, the base numbers for each are assigned based on the region 70 bp upstream from just before the R1 DnaA-box base, with the region being the starting point. Furthermore, using these mutant oriC cassettes, circular DNA amplification was performed using a method similar to the real-time PCR method in Example 14-2. The calculated DNA amplification doubling times (minutes) and standard deviations are shown at the far right of Figure 15D. "N.D." indicates that no DNA amplification was detected.

[0192] The five sequences in Figure 15D are shown in Table 27. In Table 27, boxed letters indicate bases that differ from the wild-type sequence, and shaded letters indicate the continuous KAK sequence. The underlined regions indicate the M and R elements of DUE from the 5' side.

[0193]

[0194] As shown in Figure 15D, DNA amplification by PCR was possible when sequences in which DUE-M was mutated to polyA (oriCdueM9A, 42.8 min) and polyT (oriCdueM9T, 35.3 min) were used. However, the doubling times of both DUE-M mutants were slower than oriCwt (31.9 min), being 0.7-fold and 0.9-fold slower, respectively. On the other hand, the amplification rate of the DUE-R mutant with polyT (oriCdueR13T, 67.3 min) was much lower than that of the DUE-M mutants with polyA or T, and no DNA amplification was observed with the DUE-R mutant with polyA (oriCdueR13A). Reducing the GC content by adding polyA or T to the DUE-MR region appears to significantly suppress PCR amplification without increasing the amplification rate. These results also suggest that the repeated AT motif of DUE-M and the KAK motif of DUE-R contribute to the enhancement of PCR amplification.

[0195] Furthermore, when a similar experiment was performed using a sequence in which the KAK motif of the R element was increased (GTATTATTATTAGgat (SEQ ID NO: 115); uppercase letters indicate DUE-R, lowercase letters indicate the 3'-side sequence, and underlines indicate mutations from the wild-type), the doubling time of this DUE-R mutant was 29.2 ± 2.0 minutes, suggesting that the KAK motif of DUE-R contributes to enhanced PCR amplification.

[0196] In addition, when a similar experiment was performed using a sequence in which an AT motif was added to DUE-R (GTATATATATTAGgat (SEQ ID NO: 116); capital letters indicate DUE-R, lowercase letters indicate the 3'-side sequence, and underlines indicate mutations from the wild-type), the doubling time of this DUE-R mutant was increased compared to the wild-type.

[0197] [Example 14-5] We investigated whether the oriC cassette mutants enable PCR amplification at low temperatures. 1 pM of the 7 kb circular DNA containing oriCwt, oriC3.4AT, oriC3.6AT, oriC4.0AT, oriC5.4AT, oriC5.6AT, and oriC6.0AT prepared in Example 14-1 was amplified by PCR at 16°C, 18°C, 20°C, or 33°C for 12 hours. The results of 1% agarose gel electrophoresis (AGE) of the amplified products are shown in Figure 16A.

[0198] As shown in Figure 16A, when oriC5.6AT and oriC6.0AT were used, high amplification was observed even at 18°C. When oriC3.6AT and oriC4.0AT were used, slight amplification was observed at 18°C. When oriC5.6AT and oriC6.0AT were used, slight amplification was observed even at 16°C. Mutations in DUE have the effect of promoting PCR amplification at low temperatures, and the mutation resulting in consecutive KAK sequences in DUE-R was particularly effective. It is thought that the consecutive KAK sequences may enhance cleavage of double-stranded DNA and improve amplification efficiency at low temperatures compared to oriC3.6AT.

[0199] Next, a competition test was performed using oriCwt, oriC3.4AT, oriC5.4AT, oriC5.6AT, and oriC6.0AT prepared in Example 14-1. Amplification of 7 kb circular DNA containing each oriC was performed using 13 kb circular DNA containing oriCwt as a competitor. The 7 kb DNA and 13 kb DNA were mixed at a ratio of 100 pM:200 pM and amplified using an PCR reaction mixture containing an enzyme mixture (RE mix) for PCR amplification at a final concentration of 0.25x, 0.5x, or 1.0x. The amplified products were separated by 1.0% agarose gel electrophoresis, and the results are shown in Figure 16B. The lower graph in Figure 16B shows the abundance (%) of the 13-kb DNA, calculated from band intensity using NIH ImageJ software (Schneider, CA, Rasband, WS, and Eliceiri, KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature methods, vol. 9(7), pp. 671-675.).

[0200] As shown in Figure 16B, oriC3.4AT and oriC5.4AT competed for oriCwt, suggesting that increased binding of DnaA to the DnaA box increased the tendency for binding to the DnaA mutants over that of the wild-type. These mutants appeared to be more competitive under conditions with low DnaA levels without increasing the amplification rate. We also confirmed that oriC5.6AT opened more easily at low concentrations of DnaA than the wild-type sequence (data not shown).

[0201] Example 15 An experiment similar to that of Example 5 was carried out using the plasmid obtained in Example 4 in which OriCsg was incorporated into the E1-p15A plasmid, and the plasmid in which OriC5.6ATsg was incorporated instead of OriCsg. The PCR amplification reaction conditions were set at 30°C for 6 hours, 10 hours, 15 hours, or 20 hours. For PCR amplification, a reaction mixture having the composition shown in Table 9 was used after appropriate dilution. 0.05% Tween 20 (Wako) was added during the reaction.

[0202] In the OriCsg cassette sequence (SEQ ID NO: 10), oriC is the same as oriC of OriC5.6ATsg. The base sequence is shown in Table 28.

[0203]

[0204] In the table, the underlined uppercase region represents SGS, as in OriCsg. The region in uppercase near the center (not underlined) represents the oriC sequence, as in OriC5.6AT. In the table, the bolded portion represents the DUE portion, and the boxed portion within the bolded portion represents the mutated portion in the mutant DUE. The underlined portions are the M element (underlined portion on the 5'-end) and R element (underlined portion on the 3'-end, having a one-base deletion mutation) of DUE. Furthermore, the boxed region represents the terG16 sequence and its complementary sequence.

[0205] The OriC5.6ATsg cassette was integrated into the p15A plasmid using the same method as in Example 1. The primers used were OriC_FW_1 and OriC_RV_1 in Table 8. Thereafter, cloning was performed using the same method as in Example 4-1 to obtain a plasmid in which OriC5.6ATsg was integrated into the E1-p15A plasmid.

[0206] The results are shown in Figure 17. The change in DNA concentration with reaction time is shown in Figure 18. When the OriC5.6ATsg cassette, which has the characteristics of both OriCsg and OriC5.6AT, was used, the amplification rate was faster and the DNA concentration after amplification was higher than when the OriCsg cassette was used.

[0207] Example 16 An experiment similar to that of Example 5 was carried out using two types of plasmids in which OriCsg or OriC5.6ATsg was integrated into the E1-p15A plasmid, as in Example 15, except that the PCR amplification reaction conditions were 20°C, 25°C, or 30°C for 5 hours, 10 hours, or 20 hours. For PCR amplification, a reaction mixture having the composition shown in Table 9 was used after appropriate dilution. 0.05% Tween 20 (Wako) was added during the reaction.

[0208] The results are shown in Figure 19. When the OriC5.6ATsg cassette, which has the characteristics of both OriCsg and OriC5.6AT, was used, DNA amplification was observed in a shorter time even at 25°C, and DNA was amplified more quickly even at lower temperatures than when the OriCsg cassette was used.

[0209] Example 17 An experiment similar to that of Example 5 was carried out using the plasmid obtained in Example 4 in which OriC5.6AT was incorporated into the E1-p15A plasmid, and the plasmid obtained in Example 15 in which OriC5.6ATsg was incorporated into the E1-p15A plasmid. Except for the PCR amplification reaction conditions, the PCR amplification reaction was carried out at 30°C for 6, 10, or 20 hours. For PCR amplification, a reaction mixture having the composition shown in Table 9 was used after appropriate dilution. During the reaction, 0.05% Tween 20 (Wako) was added.

[0210] The results are shown in Figure 20. When the OriC5.6ATsg cassette, which has the characteristics of both OriCsg and OriC5.6AT, was used, fewer concatemers were formed and more accurate amplification was considered possible compared to the OriC5.6AT cassette.

[0211] [Example 18] A cassette was prepared which had a terG16 sequence and its complementary sequence adjacent to both ends of an oriC sequence having a mutated DUE portion similar to that of the OriC5.6ATsg cassette shown in Example 15, and which had an SGS similar to that of the OriC5.6ATsg cassette further 5' from the oriC sequence and the terG16 sequence, and which was designated OriC7.0 cassette.

[0212] The following plasmids were prepared: A plasmid obtained in Example 15 in which OriC5.6ATsg was integrated into the E1-p15A plasmid. A plasmid obtained in the same manner in which OriC7.0 was integrated into the E1-p15A plasmid. Two types of plasmids obtained in Example 4 in which two types of OriC cassettes (OriC2.0, OriCsg) were integrated into the E1-p15A plasmid.

[0213] An experiment similar to that of Example 5 was carried out, except that the PCR amplification reaction conditions were set at 30°C for 5, 10, 15, or 20 hours. For PCR amplification, a reaction mixture having the composition shown in Table 9 was used after appropriate dilution. During the reaction, 0.05% Tween 20 (Wako) was added.

[0214] The change in DNA concentration with reaction time is shown in Figure 21. Similar to the results of Example 15, when the OriC5.6ATsg cassette and the OriC7.0 cassette, which have the characteristics of both OriCsg and OriC5.6AT, were used, the amplification rate was faster and the DNA concentration after amplification was higher than when the OriCsg cassette was used.

[0215] Example 19 An experiment similar to that of Example 18 was carried out, except that the PCR amplification reaction conditions were set to 37°C for 2 hours, 5 hours, or 10 hours. The change in DNA concentration with reaction time is shown in Figure 22. Results similar to those of Example 18 were obtained, demonstrating that the DNA amplification rate was higher at a reaction temperature of 37°C compared to 30°C (Example 18).

Claims

1. A method for producing circular DNA comprising: (1) preparing a template circular DNA, the circular DNA comprising (i) a replication origin sequence capable of binding to an enzyme having DnaA activity, and (ii) a gyrase binding sequence, and the circular DNA does not comprise a DnaA box cluster at a position different from that of the replication origin sequence; (2) forming a reaction mixture with the circular DNA prepared in (1) above and a reaction solution comprising: a first group of enzymes that catalyze the replication of the circular DNA; a second group of enzymes that synthesize two sister circular DNAs that form catenanes; and a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) above at a temperature of 80°C or less, thereby obtaining a reaction product comprising the amplified circular DNA.

2. The method of claim 1, wherein the replication initiation sequence of (i) has a mutant double-strand cleavage region that has a higher AT content than the double-strand cleavage region of a wild-type replication initiation sequence, and the double-strand cleavage region has L, M and R elements.

3. The method of claim 1, wherein the gyrase binding sequence of (ii) is a sequence derived from bacteriophage Mu.

4. A method for producing circular DNA comprising: (1) preparing a template circular DNA, the circular DNA (i) comprising a replication origin capable of binding to an enzyme having DnaA activity, and the replication origin having a mutant double-strand cleavage region having a higher AT content than the double-strand cleavage region of a wild-type replication origin, the double-strand cleavage region having L, M and R elements; (2) forming a reaction mixture with the circular DNA prepared in (1) above and a reaction solution comprising: a first group of enzymes that catalyze the replication of the circular DNA; a second group of enzymes that synthesize two sister circular DNAs that form catenanes; and a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) above at a temperature of 80°C or less, thereby obtaining a reaction product containing the amplified circular DNA.

5. The method according to claim 4, further comprising, following (3), (4) diluting the reaction mixture by at least two-fold with a solution that does not contain the first to third enzyme groups, and, after the dilution, maintaining the resulting dilution under conditions in which the second enzyme group and the third enzyme group can act.

6. The method of claim 2 or 4, wherein the mutant double-strand cleavage region is a mutant double-strand cleavage region in which the M and / or R elements of the L, M and R elements constituting the double-strand cleavage region have a higher AT content compared to the wild-type M and / or R elements, the M element constituting the double-strand cleavage region is a mutant double-strand cleavage region having a repetitive AT sequence, and / or the R element constituting the double-strand cleavage region is a mutant double-strand cleavage region having four or more consecutive KAK (K is T or G) motif sequences.

7. The method according to claim 1 or 4, wherein the circular DNA further comprises a ter sequence inserted outward relative to the replication origin sequence, the ter sequence is not adjacent to the replication origin sequence, and the reaction solution (2) further comprises a protein having activity of binding to the ter sequence and inhibiting replication.

8. The method of claim 1 or 4, wherein the circular DNA further comprises a ter sequence inserted outward relative to the replication origin sequence capable of binding to an enzyme having DnaA activity, the ter sequence is a ter sequence into which a mutation has been introduced to substitute, delete or insert one or more bases of a wild-type ter sequence, and the reaction solution of (2) further comprises a protein having an activity of binding to the ter sequence and inhibiting replication.

9. The method of claim 1 or 4, wherein the incubation in (3) comprises isothermal incubation.

10. The method according to claim 1 or 4, wherein the incubation in (3) includes incubation under temperature cycles in which incubation at two temperatures not exceeding 65°C is repeated.

11. The method of claim 1 or 4, wherein the first group of enzymes comprises an enzyme having DnaA activity, an enzyme having SSB, DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and an enzyme or group of enzymes having DNA polymerase III activity; the second group of enzymes comprises an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity; and the third group of enzymes comprises an enzyme having topoisomerase III activity or an enzyme having topoisomerase IV activity.

12. The method of claim 1 or 4, wherein the reaction solution further comprises a linear DNA-specific exonuclease.

13. The method according to claim 1 or 4, wherein the reaction solution further comprises a single-stranded DNA-specific exonuclease.

14. A circular DNA comprising (i) a replication origin sequence capable of binding to an enzyme having DnaA activity and (ii) a gyrase binding sequence, wherein the circular DNA does not contain a DnaA box cluster at a position different from that of the replication origin sequence.

15. A circular DNA comprising (i) an origin of replication capable of binding to an enzyme having DnaA activity, (ii) a gyrase binding sequence, and (iii) a plasmid origin of replication, wherein the circular DNA does not contain a DnaA box cluster at a position different from that of the origin of replication.

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