Method for producing double-stranded DNA

Abstract

The present invention provides a method for producing a linear or circular double-stranded DNA by assembling a plurality of single-stranded DNA fragments. The present invention relates to, for example, a method for producing a double-stranded DNA, the method involving: (1) preparing a set of two or more single-stranded DNA fragments, wherein at least part of each single-stranded DNA fragment has a sequence complementary to at least one of other single-stranded DNA fragments in the set, and each single-stranded DNA fragment corresponds to part of one strand of the double-stranded DNA; (2) preparing a reaction liquid for assembly including the set of single-stranded DNA fragments and a cationic salt or a cationic substance; and (3) incubating the reaction liquid at an incubation temperature higher than 40°C, thereby assembling the set of single-stranded DNA fragments into a double-stranded DNA.

Description

Method for producing double-stranded DNA

[0001] The present invention relates to a method for obtaining linear or circular double-stranded DNA by assembling multiple single-stranded DNA fragments at very low concentrations in a cell-free system at regions where their base sequences are complementary, and a method for amplifying the obtained DNA.This application claims priority based on Japanese Patent Application No. 2023-125149, filed on July 31, 2023, the contents of which are incorporated herein by reference.

[0002] Artificial genes are synthesized by annealing the homologous regions of multiple chemically produced oligo-DNAs (single-stranded DNAs) to generate double-stranded DNA. Multiple linear single-stranded DNA fragments are assembled by linking their complementary regions to produce linear or circular double-stranded DNA, allowing for the production of longer double-stranded DNAs that are difficult to synthesize chemically. However, conventional methods require the preparation of multiple single-stranded DNA fragments at high concentrations, especially when assembling a large number of linear single-stranded DNA fragments.

[0003] For example, Non-Patent Document 1 discloses a method in which 28 types of single-stranded DNA fragments (each 57 to 60 bases long) having complementary strands are prepared at approximately 140 nM each, for a total of 4.0 μM, and then assembled by slow thermal annealing at 96°C to 30°C for 2 hours to obtain a linear double-stranded DNA of 836 base pairs.

[0004] Non-Patent Document 2 discloses a method in which 2.2 μM of each of nine overlapping single-stranded DNA fragments (mainly each 58 bases long) is prepared and assembled by ligase chain reaction (LCR) using a heat-resistant ligase to obtain a linear double-stranded DNA of 441 base pairs.

[0005] Non-Patent Document 3 discloses that 56 types of single-stranded DNA fragments (each 40 bases long) having complementary strands were prepared at 22 nM each, for a total of 2.5 μM, and linear double-stranded DNA was obtained using the polymerase cycling assembly (PCA) method; and that 134 types of single-stranded DNA fragments (each 40 bases long) having complementary strands were prepared at 7.5 nM each, for a total of 1 μM, and the fragments were linked using the PCA method, followed by repeated PCR reactions to obtain double-stranded DNA.

[0006] Non-Patent Document 4 discloses that a total of 15 μM of 48 types of single-stranded DNA fragments (each 50 bases long) having complementary strands were prepared (approximately 0.3 μM each), and after annealing and ligation under stringent conditions using the circular assembly amplification method, error removal was carried out using an exonuclease and an endonuclease to obtain circular double-stranded DNA, which was then cut into linear form and amplified by PCR.

[0007] Patent Document 1 discloses a method for efficiently ligating a large number of nucleic acid fragments, each at a concentration of about 1 nM, isothermally using RecA and, if necessary, a linear double-stranded DNA-specific exonuclease.

[0008] Patent Document 2 relates to a method for producing double-stranded DNA by duplex asymmetric PCR (DA-PCR), and discloses a method comprising the steps of: (1) preparing a reaction mixture by preparing multiple types of oligonucleotides derived from a portion of the sense strand and multiple types of oligonucleotides corresponding to a portion of the antisense strand, and mixing equal concentrations of the various oligonucleotides with DNA polymerase and dNTPs; (2) performing PCR using the reaction mixture from step (1); (3) adding a primer set capable of amplifying the full length of the double-stranded DNA fragment to the reaction mixture from step (2); and (4) performing PCR using the reaction mixture from step (3).

[0009] Patent Document 3 discloses a method for assembling a DNA molecule having a desired sequence, in which seven overlapping pools of oligo DNA (14 oligos x 8 mers) with 4 bp overlaps are ligated using DNA ligase to form seven pools of 60 mer DNA, and then these seven pools are combined into one pool, and a 240 bp product is assembled by polymerase chain assembly reaction.

[0010] In recent years, services have emerged that provide mixtures of up to one million sequences, known as oligo pools, and are expected to increase the throughput and reduce the cost of artificial gene synthesis. One method for obtaining double-stranded DNA by assembling a set of low-concentration single-stranded DNA fragments, such as oligo pools, is the Dropsynth method, in which the assembly reaction is carried out in water-in-oil droplets (Non-Patent Document 5). This method uses a set of barcoded beads, each of which recognizes the single-stranded DNA fragment required for the assembly of a specific gene, and assembly proceeds in separate water droplets, allowing multiple types of double-stranded DNA to be obtained simultaneously. However, this method focuses on obtaining multiple types of double-stranded DNA, such as a library, and involves multiple steps.

[0011] International Publication No. 2019 / 009361 International Publication No. 2019 / 044820 International Publication No. 2021 / 178210

[0012] Zampini et al., Scientific Reports, 2015, vol.5, 11302.Au et al., Biochem. Biophys. Res. Commun., 1998, vol.248(1), p.200-203.Stemmer et al., Gene, 1995, vol.164, p.49-53.Bang and Church, Nature Methods, 2008, vol.5(1), p.37-39.Sidore et al., Nucleic Acids Research, 2020, vol. 48(16), e95.

[0013] Conventional single-stranded DNA assembly methods require the preparation of high-concentration single-stranded DNA and the repeated, laborious process multiple times. While the preparation of high-concentration single-stranded DNA is expensive, low-concentration mixtures of single-stranded DNA, such as oligo pools, are relatively inexpensive. However, when accumulating double-stranded DNA from such low-concentration mixtures, there is a problem in that the sequence error rate of the resulting double-stranded DNA is high due to misannealing.

[0014] Under these circumstances, a main object of the present invention is to provide a method for producing linear or circular double-stranded DNA by assembling a plurality of single-stranded DNA fragments with high accuracy.

[0015] The present inventors have found that the accumulation efficiency of single-stranded DNA fragments can be increased by carrying out the accumulation in the presence of a cationic salt or a cationic substance, and have also found that when accumulating low concentrations of single-stranded DNA fragments, the addition of a polypeptide having specific properties can promote the accumulation of single-stranded DNA and result in double-stranded DNA, thereby completing the present invention.

[0016] That is, the methods etc. according to the present invention are the following [1] to

[17] : [1] A method for producing double-stranded DNA, comprising: (1) preparing a set of two or more single-stranded DNA fragments, wherein at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of one of the strands of the double-stranded DNA, (2) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments and a cationic salt or a cationic substance, and (3) incubating the reaction solution at an incubation temperature higher than 40°C, thereby accumulating the set of single-stranded DNA fragments into double-stranded DNA. [2] The method of [1] above, wherein the reaction solution further contains an accumulation-promoting polypeptide, wherein the accumulation-promoting polypeptide: (i) is a polypeptide that forms aggregates when the accumulation-promoting polypeptide and any DNA fragment are heated in solution at a temperature higher than 40° C., and (ii) is a polypeptide that reduces the amount of aggregates formed when the accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in solution at a temperature higher than 40° C. compared to a solution containing no arginine. [3] The method of [2] above, wherein the concentration of each single-stranded DNA fragment in the reaction solution is 1 nM or less, and / or the concentration of all single-stranded DNA fragments is 1 μM or less. [4] The method according to [2] or [3] above, wherein the accumulation-promoting polypeptide is selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having RecA family recombinase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA-binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and the epsilon subunit of F1-ATPase.[5] The method according to [4], wherein the accumulation-promoting polypeptide is selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having ATP-type DNA ligase activity, a polypeptide having Escherichia coli-derived DNA helicase activity, and a polypeptide having RecA family recombinase activity. [6] The method according to any of [1] to [5], wherein the cationic salt or cationic substance is selected from the group consisting of divalent cationic salts, monovalent cationic salts, and polyamines. [7] The method according to any of [1] to [5], wherein the cationic salt or cationic substance is selected from the group consisting of magnesium salts, calcium salts, sodium salts, potassium salts, ammonium salts, putrescine, spermidine, and spermine. [8] The method according to any of [1] to [7], wherein at least one of the single-stranded DNA fragments is obtained by chemical synthesis and / or the set of single-stranded DNA fragments is an oligopool. [9] The method according to any one of [1] to [7], comprising, prior to (2), non-exponentially amplifying two or more types of single-stranded DNA fragments contained in the set using a DNA polymerase, or amplifying two or more types of single-stranded DNA fragments contained in the set by PCR to obtain two or more types of double-stranded DNA, and thermally dissociating the two or more types of double-stranded DNA to obtain two or more types of single-stranded DNA.

[10] The method according to any one of [1] to [9], wherein the reaction solution contains a set of single-stranded DNAs corresponding to the multiple types of double-stranded DNAs, and multiple types of double-stranded DNAs are produced.

[11] The method for producing double-stranded DNA according to any one of [1] to

[10] , wherein the produced double-stranded DNA is supercoiled double-stranded DNA, and the method further comprises: (4) if the double-stranded DNA obtained in (3) is not circular double-stranded DNA, circularizing the obtained double-stranded DNA to obtain circular double-stranded DNA; (5) forming a mixture for supercoiling reaction (SCR) containing the circular double-stranded DNA and the following: an enzyme having exonuclease III activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, and an enzyme or enzymes having DNA gyrase activity; and (6) incubating the SCR mixture, thereby supercoiling the circular double-stranded DNA in the mixture.

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

[11] , further comprising subjecting the double-stranded DNA to a double-stranded DNA amplification reaction.

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

[11] , wherein the double-stranded DNA is circular and has a replication initiation sequence capable of binding to an enzyme having DnaA activity, and the method comprises subjecting the circular double-stranded DNA to a double-stranded DNA amplification reaction by the replication cycle reaction (RCR) method, or the method comprises inserting a replication initiation sequence capable of binding to an enzyme having DnaA activity into the double-stranded DNA to obtain a circular double-stranded DNA having the replication initiation sequence, and subjecting the circular double-stranded DNA to a double-stranded DNA amplification reaction by the RCR method.

[14] The method of

[12] or

[13] , wherein the double-stranded DNA is circular and the double-stranded DNA amplification reaction is carried out in the presence of MutS and MutL, or in the presence of enzymes selected from the group consisting of MutS and MutL, MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease.

[15] The method according to any one of

[12] to

[14] , comprising allowing MutS and MutL to act on the double-stranded DNA, or allowing MutS and MutL and an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease to act on the double-stranded DNA prior to the double-stranded DNA amplification reaction.

[16] A method for producing a double-stranded DNA library, comprising: (1) preparing a set of two or more types of single-stranded DNA fragments, wherein at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of either strand of the double-stranded DNA; (1') preparing two or more types of library-constituting single-stranded DNA fragments, wherein each library-constituting single-stranded DNA fragment has a region corresponding to a portion of either strand of the double-stranded DNA and a region non-complementary to any single-stranded DNA fragment in the set of single-stranded DNA fragments in (1), and the base sequences of the non-complementary regions are different from each other; (2) preparing an enrichment reaction solution containing the set of single-stranded DNA fragments, the two or more types of library-constituting single-stranded DNA fragments, a cationic salt or cationic substance, and an enrichment-promoting polypeptide; and (3) Incubating the reaction solution at an incubation temperature higher than 40°C, thereby accumulating the set of single-stranded DNA fragments and two or more of the library-constituting single-stranded DNA fragments into a double-stranded DNA library, wherein the accumulation-promoting polypeptide is: (i) a polypeptide that forms aggregates when the accumulation-promoting polypeptide and any DNA fragment are heated in solution at a temperature higher than 40°C, and (ii) a polypeptide that forms a reduced amount of aggregates when the accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in solution at a temperature higher than 40°C, compared to when arginine is not contained.

[17] A method for producing double-stranded DNA, comprising: (a) heating a solution containing a given polypeptide and a given number of single-stranded DNA fragments to a temperature higher than 40°C, wherein the polypeptide and at least one of the single-stranded DNA fragments in the solution form aggregates, and selecting the polypeptide that formed the aggregate; (b) providing a set of single-stranded DNA fragments, wherein at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of one of the strands of the double-stranded DNA; (c) preparing a reaction solution containing the set of single-stranded DNA fragments, a cationic salt or cationic substance, and the polypeptide selected in (a); and (d) incubating the reaction solution at an incubation temperature higher than 40°C, thereby assembling the set of single-stranded DNA fragments into double-stranded DNA.

[18] A kit for producing circular double-stranded DNA, comprising: an enzyme having exonuclease III activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, an enzyme or enzymes having DNA gyrase activity, a cationic salt or a cationic substance, and an accumulation-promoting polypeptide selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having RecA family recombinase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA-binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and an ε subunit of F1-ATPase.

[0017] According to the present invention, a linear or circular double-stranded DNA can be produced by assembling a plurality of single-stranded DNA fragments with high precision.

[0018] A schematic diagram of accumulation in Example 1 is shown in Figure 1A, and the staining results are shown in Figure 1B. A schematic diagram of accumulation in Example 3-1 is shown in Figure 2A, and the staining results are shown in Figure 2B. A schematic diagram of accumulation in Example 3-2 is shown in Figure 3A, and the staining results are shown in Figure 3B. A schematic diagram of accumulation in Example 4 is shown in Figure 4A, and the staining results are shown in Figure 4B. Results of Example 5-1 are shown. Results of Example 5-2 are shown. Results of Example 6-1 are shown. Results of Example 6-2 are shown. Results of Example 7-1 are shown. Results of Example 7-2 are shown. A schematic diagram of accumulation in Example 8-1 is shown in Figure 11A, and the staining results are shown in Figure 12B. Results of Example 8-2 are shown. A schematic diagram of accumulation in Example 9 is shown in Figure 13A, and the staining results are shown in Figure 13B. A schematic diagram of accumulation in Example 10 is shown in Figure 14A, and the staining results are shown in Figure 14B. A schematic diagram of accumulation in Example 11 is shown in Figure 15A, and the staining results are shown in Figure 15B. FIG. 16A shows a schematic diagram of enrichment in Example 12, and FIG. 16B shows the staining results. FIG. 16B shows the staining results in Example 13. FIG. 13 shows the results of introducing an amplification product into E. coli in Example 13. FIG. 15 shows the staining results in Example 15. FIG. 16B shows a design diagram of a random oligo site in Example 16. FIG. 16C shows the staining results in Example 16. FIG. 16D shows a photograph of a plate after culturing transformed E. coli in Example 16. FIG. 16E shows the results of further culturing colored colonies in Example 16. FIG. 17A shows the staining results in Example 17-1. FIG. 17B shows the results of Cy5 detection from a tube in Example 17-2. FIG. 17C shows the staining results after enrichment in Example 17-3. FIG. 27A shows the staining results in Example 18. FIG. 27B shows the percentage results of counting colonies that exhibited the color derived from each gene in Example 18. FIG. 28A shows a schematic diagram of enrichment in Example 19, and FIG. 28B shows the staining results. FIG. 17B shows the results of further culturing colored colonies in Example 19.

[0019] 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.

[0020] The method for producing double-stranded DNA of this embodiment includes preparing a set of two or more single-stranded DNA fragments (a set containing two or more single-stranded DNA fragments), in which at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of one of the strands of the double-stranded DNA. "A single-stranded DNA fragment corresponds to a portion of one of the strands of a double-stranded DNA" means that the single-stranded DNA fragment has the same sequence as a DNA sequence constituting a portion of one of the strands of the double-stranded DNA. The resulting double-stranded DNA may be linear or cyclic, and its terminal and / or non-terminal regions may be single-stranded. The method for producing double-stranded DNA of this embodiment can produce double-stranded DNA even if it has a sequence that is considered difficult to synthesize, such as an AT content of more than 50%, 60% or more, or 70% or more, or a GC content of more than 50%, 60% or more, or 70% or more.

[0021] The set of single-stranded DNA fragments does not have to correspond to all base sequences of the two strands of double-stranded DNA. When the single-stranded fragments included in the set are assembled, they may be a set that results in a double-stranded DNA having gaps rather than a continuous double-stranded DNA. The number of single-stranded DNA fragments included in the set is not particularly limited as long as it is two or more types, and may include, for example, at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 types or more, with the upper limit being 10,000, 5,000, 3,000, 2,000, 1,000, 800, 600, 500, 400, or 300 types or less of single-stranded DNA fragments.

[0022] Single-stranded DNA fragments can be prepared using known techniques, such as enzymatic methods and chemical synthesis. For example, methods such as those used by Ansa Biotechnologies and DNA Script are known as methods for enzymatic DNA synthesis. In one aspect, from the viewpoint of cost, at least one of the single-stranded DNA fragments in the set (e.g., 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% of the multiple single-stranded DNA fragments in the set) is preferably a single-stranded DNA fragment artificially obtained by chemical synthesis. The chemical synthesis method is not particularly limited, and examples include the method described in Hoose et al., Nature Reviews Chemistry (2023) Vol. 7, pp. 144-161. A typical example is a method in which desired nucleosides from among four types of nucleosides are linked one by one to the synthetic termini on a solid phase such as a chip or beads, followed by elongation. In this way, a set of single-stranded DNA fragments can be prepared by chemically synthesizing multiple types of single-stranded DNA fragments.

[0023] In one embodiment, the set of single-stranded DNA fragments is an oligo pool, which is a collection of two or more, for example, 10 or more, 50 or more, particularly 100 or more, chemically synthesized DNA fragments. Numerous services are known that provide the design and creation of oligo pools containing large amounts of single-stranded DNA fragments in a single tube, for example, provided by Integrated DNA Technologies (IDT), GenScript, Twist Bioscience, Agilent Technologies, and others. Because oligo pools are created by pool synthesis, the price per base is inexpensive. In one embodiment, double-stranded DNA can be inexpensively produced in a cell-free system by using such oligo pools as a set of single-stranded DNA fragments.

[0024] The length of the single-stranded DNA fragment is not particularly limited. ｍFrom the viewpoint of the influence on the chromatin value and facilitating accumulation at temperatures higher than 40° C., the length is 10 bases or more, preferably 20 bases or more, more preferably 40 bases or more, and even more preferably 50 bases or more. There is no particular upper limit to the length, and it can be, for example, 1,000 bases or less, 500 bases or less, etc. When the single-stranded DNA fragment is a chemically synthesized single-stranded DNA fragment, there is no particular limit as long as it can be chemically synthesized, and the length can be, for example, 350 bases or less, 300 bases or less, 200 bases or less, 150 bases or less, or 100 bases or less.

[0025] In one embodiment, prior to preparation of the enrichment reaction solution, two or more single-stranded DNA fragments contained in the set (e.g., oligo pool, etc.) may be amplified non-exponentially using a DNA polymerase. For example, a non-exponential amplification reaction can be performed by repeatedly adding primers to the single-stranded DNA fragments bound to the microarray, synthesizing oligonucleotides with a DNA polymerase, and then removing the oligonucleotides with a nicking endonuclease (Hughes and Ellington, Cold Spring Harb Perspect Biol. 2017, 9(1):a023812, Quan et al. (2011) Nat Biotechnol. 29: 449-452).

[0026] In one embodiment, prior to preparation of the enrichment reaction solution, two or more single-stranded DNA fragments contained in the set (e.g., oligo pool, etc.) may be amplified by a method including: amplifying the two or more double-stranded DNA fragments by PCR to obtain two or more double-stranded DNA fragments; and thermally dissociating the two or more double-stranded DNA fragments to obtain two or more single-stranded DNA fragments. This allows for more accurate enrichment even when the concentration of single-stranded DNA fragments is very low, by increasing the concentration of each fragment. PCR and thermal dissociation techniques are well known to those skilled in the art. PCR can be performed by designing primers and repeating cycles of thermal denaturation, annealing, and extension. Thermal dissociation can typically be performed at 95 to 100°C, depending on the type of single-stranded DNA.

[0027] The method for producing double-stranded DNA of this embodiment includes preparing an accumulation reaction solution containing the set of single-stranded DNA fragments prepared above and a cationic salt or a cationic substance. In this specification, the term "cationic substance" refers to a substance that becomes positively charged when dissolved in water, other than a cationic salt.

[0028] The cationic salt or cationic substance is not particularly limited as long as it provides cations in the accumulation reaction solution, but is preferably one or more selected from the group consisting of divalent cationic salts, monovalent cationic salts, and polyamines. More preferably, the cationic salt or cationic substance is, for example, magnesium acetate [Mg(OAc)]. ２ ], magnesium chloride [MgCl ２ ], magnesium sulfate [MgSO ４

[0023] , calcium salts such as calcium chloride, calcium sulfate, and calcium carbonate, potassium salts such as potassium glutamate [KGlu], potassium aspartate, potassium chloride, and potassium acetate [KOAc], sodium salts such as sodium glutamate, sodium aspartate, sodium chloride, and sodium acetate, ammonium salts such as ammonium chloride and ammonium acetate, polyamines such as putrescine, spermidine, and spermine, and cationic surfactants such as CTAB (cetyltrimethylammonium bromide). More preferably, the cationic salt is selected from magnesium salts, potassium salts, sodium salts, and ammonium salts, and the cationic substance is selected from polyamines and cationic surfactants. Particularly preferably, the cationic salt or cationic substance is selected from magnesium salts, potassium salts, sodium salts, ammonium salts, and polyamines.

[0029] The concentration of the cationic salt or cationic substance is not particularly limited as long as the accumulation reaction proceeds, and can be varied depending on the cationic salt or cationic substance used. For example, it can be added at a concentration of 0.1 mM to 200 mM. For example, in the case of a divalent cationic salt, it can be added to the accumulation reaction solution at a concentration of preferably 0.1 mM to 50 mM, more preferably 0.5 mM to 20 mM, and particularly preferably 1 mM to 20 mM. For example, in the case of a monovalent cationic salt, it can be added to the accumulation reaction solution at a concentration of preferably 5 mM to 200 mM, more preferably 10 mM to 150 mM, and particularly preferably 30 mM to 100 mM. In the case of a cationic substance (e.g., a polyamine), it can be added to the accumulation reaction solution at a concentration of preferably 0.2 mM to 100 mM, more preferably 0.3 mM to 50 mM, and particularly preferably 0.4 mM to 10 mM, for example, 0.5 mM to 5 mM.

[0030] The concentration of the single-stranded DNA fragments in the accumulation reaction solution is not particularly limited, and for example, the concentration of each single-stranded DNA fragment can be 0.1 fM to 100 μM, with the lower limit being 1 fM or more, 10 fM or more, or 100 pM or more, and the upper limit being 75 μM or less, 50 μM or less, or 25 μM or less. The concentration of each single-stranded DNA fragment can be preferably 1 fM to 75 μM, 10 fM to 50 μM, or 0.1 pM to 25 μM, preferably 1 pM to 1 μM, and more preferably 10 pM to 50 nM, and / or the concentration of the total single-stranded DNA fragments in the accumulation reaction solution can be 0.1 pM to 10 mM, preferably 1 pM to 100 μM, and more preferably 10 pM to 5 μM.

[0031] The accumulation reaction solution may contain other DNA fragments in addition to the set of single-stranded DNA fragments, for example, double-stranded DNA having an overhang. For example, each DNA can be designed so that the double-stranded DNA obtained by accumulating the set of single-stranded DNA fragments accumulates with this double-stranded DNA having an overhang. The length of the overhang is not particularly limited as long as accumulation proceeds, and can be, for example, 10 bases or more, preferably 20 bases or more, more preferably 40 bases or more, and even more preferably 50 bases or more.

[0032] When the concentration of each single-stranded DNA fragment in the accumulation reaction solution is 15 nM or less, for example, 1 nM or less, preferably less than 1 nM, more particularly 100 pM or less, and / or the number of single-stranded DNA fragments in the accumulation reaction solution is 5 or more, for example, 10 or more, preferably 20 or more, 30 or more, 40 or more, or 50 or more, and / or the concentration of the total single-stranded DNA fragments in the accumulation reaction solution is 1.5 μM or less, 1 μM or less, for example, 100 nM or less, preferably 50 nM or less, 30 nM or less, more particularly 10 nM or less, the accumulation reaction solution preferably contains an accumulation-promoting polypeptide. This allows efficient production of double-stranded DNA by accumulation of the single-stranded DNA fragments, even when the amount of each single-stranded DNA fragment is small and / or the total concentration of single-stranded DNA fragments is low.

[0033] In this embodiment, the accumulation-promoting polypeptide refers to a polypeptide having the following properties (i) and (ii): (i) a polypeptide that forms aggregates when the accumulation-promoting polypeptide and an arbitrary DNA fragment are heated in a solution to a temperature higher than 40°C, for example, 41°C or higher, 43°C or higher, or 45°C or higher; (ii) a polypeptide that forms a reduced amount of aggregates when the accumulation-promoting polypeptide, an arbitrary DNA fragment, and arginine are heated in a solution to a temperature higher than 40°C, for example, 41°C or higher, 43°C or higher, or 45°C or higher, compared to when arginine is not contained.

[0034] The heating temperature in (i) and (ii) above can be, for example, 100° C. or lower, 90° C. or lower, 80° C. or lower, or 75° C. or lower, and in one embodiment, can be preferably 45° C. or higher, more preferably 50° C. or higher, 55° C. or higher, or 60° C. or higher, for example, 45° C. to 100° C., preferably 50° C. to 90° C., 60° C. to 80° C., or for example, about 65° C. to 75° C. The heating time can refer to the accumulation reaction time described below, and can be, for example, 30 seconds to 180 minutes, 1 minute to 120 minutes, 5 minutes to 60 minutes, etc.

[0035] The solutions in (i) and (ii) above can be prepared with reference to the Examples. Conveniently, a solution obtained by removing the accumulation-promoting polypeptide and any DNA fragment from the accumulation reaction solution described below, or a buffer solution can be used. The buffer 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. A preferred buffer is Tris-HCl or Tris-OAc. The buffer concentration 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, preferably 10 mM to 50 mM, and more preferably 20 mM to 30 mM. To more clearly confirm the formation of aggregates even when the amount of DNA fragments is small, the buffer solution may contain a component selected from the group consisting of a cationic salt or cationic substance, a substance having a macromolecular crowding effect, and a small amount of a surfactant. For the types and amounts of these components, see the description of the accumulation reaction solution below.

[0036] Aggregates refer to larger aggregates formed by the association of dispersed accumulation-promoting polypeptides with any DNA fragments, and the formation of aggregates can be easily confirmed by centrifugation, as described in the Examples below. When an accumulation-promoting polypeptide and any DNA fragment are heated in solution to a temperature higher than 40°C and then centrifuged, aggregates have been formed if, compared to a solution without heating, the amount of precipitation is greater; and / or more accumulation-promoting polypeptide is detected in the precipitate fraction after centrifugation. Furthermore, when an accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in solution to a temperature higher than 40°C and then centrifuged, the amount of precipitation is smaller; less accumulation-promoting polypeptide is detected in the precipitate fraction after centrifugation; and / or more accumulation-promoting polypeptide is detected in the supernatant fraction after centrifugation, compared to a solution without arginine, the amount of aggregates formed is reduced.

[0037] Arginine can be added to the solution as arginine itself or as arginine salt.Easily, it can be added in the form of salt, for example, known salts such as arginine hydrochloride, acetate, sulfate, lactate, phosphate, pyroglutamate, aspartate, maleate, etc.Among them, from the viewpoint of economical aspect and easy handling, it is preferable to use arginine hydrochloride.

[0038] Without being bound by theory, it is believed that the inclusion of such a polypeptide causes single-stranded DNA fragments in the accumulation reaction solution to form aggregates with the polypeptide, thereby promoting accumulation into double-stranded DNA. Furthermore, an accumulation-promoting polypeptide can be selected by confirming, prior to the accumulation reaction, that any polypeptide has properties (i) and (ii).

[0039] The accumulation-promoting polypeptide is not particularly limited in type or origin as long as it has the above-mentioned properties, and is preferably a polypeptide that does not damage nucleic acids itself, and more preferably a polypeptide having creatine kinase activity (EC: 2.7.3.2. Hereinafter, even when no EC number is specified, it refers to the enzyme of this EC number unless otherwise specified. The same applies to other enzymes.), a polypeptide having phosphopyruvate hydratase activity (EC: 4.2.1.11.), a polypeptide having pyruvate kinase activity (EC: 2.7.1.40), a 3- ... Polypeptides having glycerate kinase activity (EC: 2.7.2.3.), polypeptides having DNA ligase activity (EC: 6.5.1.2 or EC: 6.5.1.1), polypeptides having DNA helicase activity (EC: 5.6.2.4), polypeptides having RecA family recombinase activity, polypeptides having polynucleotide kinase activity (EC: 2.7.1.78), histone-like DNA binding proteins, chromosome replication initiator proteins, polypeptides having adenylate kinase activity, and nucleoside diphosphate kinase activity (EC:2.7.4.6) and the epsilon subunit of an F1-ATPase, and even more preferably selected from a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity and the epsilon subunit of an F1-ATPase, for example, selected from creatine kinase, phosphopyruvate hydratase, pyruvate kinase, 3-phosphoglycerate kinase, DNA ligase, DNA helicase, polynucleotide kinase, a histone-like DNA binding protein, a chromosome replication initiator protein, adenylate kinase, nucleoside diphosphate kinase and the epsilon subunit of an F1-ATPase.More preferably, the accumulation-promoting polypeptide is selected from the group consisting of a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having ATP-type (T4 phage-derived) DNA ligase activity, a polypeptide having Escherichia coli-derived DNA helicase activity, and a polypeptide having RecA family recombinase activity. Particularly preferred are polypeptides having creatine kinase activity, a polypeptide having phosphopyruvate kinase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, and a polypeptide having ATP-type (T4 phage-derived) DNA ligase activity, such as phosphopyruvate kinase, pyruvate kinase, 3-phosphoglycerate kinase, and ATP-type (T4 phage-derived) DNA ligase. One or more of these polypeptides can be added to the accumulation reaction solution. In one aspect, the accumulation-promoting polypeptide is not a polypeptide having RecA family recombinase activity.

[0040] When referring to an accumulation-promoting polypeptide, a polypeptide having a specific enzymatic activity may be a wild-type polypeptide, or a variant that retains the specific enzymatic activity, obtained by introducing a mutation into the wild-type polypeptide by deleting, adding, or substituting 1 to 30 amino acids.

[0041] The concentration of the polypeptide in the enrichment reaction solution can be adjusted appropriately depending on the polypeptide used, and can be, for example, 20 nM to 50,000 nM, preferably 100 nM to 2000 nM.

[0042] The accumulation reaction solution may contain a set of single-stranded DNAs corresponding to multiple types of double-stranded DNA. This allows, for example, multiple types of genes to be accumulated and synthesized at once from a set of single-stranded DNAs corresponding to multiple types of genes. The number of single-stranded DNA sets can be two or more, for example, 10 or more, 100 or more, 1,000 or more, for example, 10,000 or more. For example, if accumulation is performed from a set of 10,000 types of single-stranded DNA, 10,000 types of genes can be accumulated and synthesized as double-stranded DNA.

[0043] The accumulation reaction solution for carrying out the accumulation reaction of this embodiment can be prepared, for example, by adding single-stranded DNA fragments, a cationic salt or cationic substance, and, if necessary, an accumulation-promoting polypeptide to a buffer solution. There are no particular limitations on the buffer solution, 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 buffer solutions are Tris-HCl and Tris-OAc. The buffer concentration 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, for example, from 10 mM to 100 mM, preferably from 10 mM to 50 mM, and more preferably from 20 mM to 30 mM.

[0044] The enrichment reaction solution used in the enrichment reaction of this embodiment may further contain a substance with a polymer crowding effect. The polymer crowding effect can enhance interactions between DNA molecules and promote ligation of DNA fragments. Examples of such substances include polyethylene glycol (PEG) 200-20,000, polyvinyl alcohol (PVA) 200-20,000, dextran 40-70, Ficoll 70, and bovine serum albumin (BSA). In one embodiment, PEG, particularly PEG 8000, can be preferably used. In the present invention, when a substance with a polymer crowding effect is added to the reaction solution used in the ligation reaction, the concentration of the substance is not particularly limited as long as it is a concentration that can promote ligation of DNA fragments. For example, when PEG 8000 is used as the substance, the concentration of PEG 8000 added to the reaction solution used in the ligation reaction of this invention is preferably 2-20% by mass, more preferably 2-10% by mass, and even more preferably 4-6% by mass.

[0045] The enrichment reaction solution used in the enrichment reaction of this embodiment may further contain a small amount of surfactant. As the surfactant, a nonionic surfactant is preferred, and examples that can be used include Tween 20 (0.02-0.5%, preferably 0.05-0.2%), Triton X-100 (0.05-1.0%, preferably 0.1-0.5%), NP-40 (0.02-1.0%, preferably 0.05-0.2%), Brij™ i35 (0.02-0.5%, preferably 0.05-0.2%), dodecyl-β-D-maltoside (approximately 0.1-0.5%), and octyl-β-D-glucoside (approximately 0.5-1.0%). In one embodiment, Tween 20 is preferably used. This suppresses adsorption of DNA fragments to the inner wall of the tube when the reaction is carried out in a tube, ensuring their reliable participation in the enrichment reaction.

[0046] When the accumulation reaction solution is incubated at an incubation temperature higher than 40°C, preferably 45°C or higher, more preferably 50°C or higher, 55°C or higher, or 60°C or higher, for example, 45°C to 100°C, preferably 50°C to 90°C, 60°C to 80°C, for example, about 65°C to 75°C, the set of single-stranded DNA fragments accumulates into double-stranded DNA (accumulation reaction). The accumulation reaction time is not particularly limited as long as the set of single-stranded DNA fragments accumulates into double-stranded DNA, and can be, for example, 30 seconds to 180 minutes, 1 minute to 120 minutes, or 5 minutes to 60 minutes. The fact that the desired double-stranded DNA has been obtained can be confirmed by known techniques, such as gel electrophoresis.

[0047] The method of this embodiment may further include subjecting the double-stranded DNA obtained in the enrichment reaction to a double-stranded DNA amplification reaction. The amplification reaction method is not particularly limited, and may be cell-free amplification or intracellular amplification. In the case of cell-free amplification, it may be isothermal amplification or non-isothermal amplification. In one aspect, cell-free amplification is preferred, and in the case of cell-free amplification, isothermal amplification or amplification performed at a temperature of 80°C or less is preferred, and amplification performed at a temperature of 65°C or less is more preferred.

[0048] In the case of intracellular amplification, techniques known in the art can be used, and the type of cell is not particularly limited, for example, Escherichia coli, Bacillus subtilis, yeast, etc. Double-stranded DNA may be prepared appropriately depending on the cell used, and conveniently, circular double-stranded DNA having a replication origin can be prepared and introduced into Escherichia coli for amplification.

[0049] In the case of cell-free amplification, techniques known in the art can also be used. When using isothermal amplification in a cell-free system, various techniques are known (e.g., J. Li and J. Macdonald, Biosensors and Bioelectronics, 2015, vol. 64, pp. 196-211; Gill, P., & Ghaemi, Nucleosides, Nucleotides & Nucleic Acids, 2008, 27(3), 224-243; Zhao, Y. et al., Chemical Reviews, 2015, 115(22), 12491-12545), for example, helicase-dependent amplification (HDA) (Vincent, et al., EMBO Rep., 2004, vol. 5 (8), pp. 795-800), recombinase polymerase (Recombinase Polymerase) amplification (RPA) (Piepenburg, et al, PLoS. Biol., 2006, vol.4 (7), e204), Rolling circle amplification (RCA) (Fire, et al, Proc. Natl. Acad. Sci., 1995, vol.92 (10), p.4641-4645), Ramification amplification (RAM) (Zhang, et al, Mol. Diagn., 2001, vol.6 (2), p.141-150), Multiple displacement amplification (MDA) (Dean, et al, Genome Res., 2001, vol.11 (6), pp. 1095-1099, and Spits, et al., Nat. Protoc., 2006, vol. 1 (4), pp. 1965-1970), Loop-mediated isothermal amplification (LAMP) (Notomi, et al., Nucleic Acids Res., 2000, vol. 28 (12), E63), and the like can be used.When non-isothermal amplification in a cell-free system is used, known techniques such as PCR can be used. Either method can be performed in a conventional manner, and the method to be used can be appropriately selected depending on the shape of the DNA to be amplified (linear or circular), etc. In addition, depending on the amplification method, double-stranded DNA may be pretreated. For example, non-circular DNA can be circularized by a known method, and then the circular DNA can be amplified.

[0050] The method of this embodiment may also include performing a supercoiling and repair reaction (SCR) on the double-stranded DNA obtained in the accumulation reaction. This repairs gaps and nicks, producing intact supercoiled circular DNA. Supercoiled circular DNA has the advantages of being exonuclease resistant, having a compact shape, being physically and chemically robust, and being easily separated by size-based electrophoresis.

[0051] The supercoiling reaction can be carried out as described in the literature (Fujita et al., ACS Synthetic Biol. (2022) 11, 9, 3088-3099). Briefly, first, if the obtained double-stranded DNA is not circular, it is circularized by a known method to obtain circular double-stranded DNA. Then, an SCR mixture is formed containing the circular double-stranded DNA and an enzyme having exonuclease III activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, and an enzyme or enzymes having DNA gyrase activity. The mixture is then incubated at a temperature of, for example, 16 to 45°C, preferably 20 to 40°C, more preferably 24 to 37°C, for an appropriate time, for example, 10 minutes or more, 20 minutes or more, or 30 minutes or more, whereby the circular double-stranded DNA in the mixture is supercoiled, thereby enabling the supercoiling reaction to be carried out. The upper limit of the incubation time is not particularly limited, but can be, for example, 24 hours, 10 hours, 5 hours, 2 hours, or 1 hour.

[0052] For details of each enzyme, such as an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, or an enzyme or enzymes having DNA gyrase activity, please refer to the description of enzymes related to the PCR method described below. The enzyme having exonuclease III activity is not particularly limited in terms of its biological origin, as long as it has the activity of degrading nucleotides from linear or nicked double-stranded DNA in the 3' to 5' direction, similar to E. coli exonuclease III. For example, ExoIII derived from E. coli can be suitably used. E. coli-derived ExoIII may be contained in the reaction solution in a range of 1 to 500 mU / μL, preferably 2 to 400 mU / μL, 3 to 300 mU / μL, 5 to 200 mU / μL, or 10 to 100 mU / μL, but is not limited thereto.

[0053] The SCR mixture may further contain a buffer solution, ATP, dNTP, a magnesium ion source, an alkali metal ion source, an inhibitor of non-specific adsorption of proteins, an inhibitor of non-specific adsorption of nucleic acids, etc. For these components, see the description of the SCR reaction solution below. When the reaction solution contains DNA ligase derived from Escherichia coli, it may also contain its cofactor, nicotinamide adenine dinucleotide (NAD).

[0054] In one embodiment, a supercoiling reaction can be carried out prior to subjecting the double-stranded DNA to a double-stranded DNA amplification reaction.

[0055] In one embodiment, the double-stranded DNA obtained in the accumulation reaction may be amplified using the replication cycle reaction (RCR) method (see WO2016 / 080424, WO2017 / 199991, and WO2018 / 159669). The DNA amplified by the RCR method is circular DNA containing a replication initiation sequence (e.g., oriC) that can bind to an enzyme having DnaA activity. If the double-stranded DNA obtained in the accumulation reaction does not have a replication initiation sequence that can bind to an enzyme having DnaA activity, such a sequence can be ligated or inserted to obtain circular DNA that can be amplified by the RCR method.

[0056] In one embodiment, replication initiation sequences capable of binding to enzymes having DnaA activity (hereinafter sometimes simply referred to as "replication initiation sequences") include, for example, 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. As replication initiation sequences, sequences in which mutations have been introduced into known replication initiation sequences by substituting, deleting, or inserting one or more bases, and which are capable of binding to enzymes having DnaA activity, can also be used. In the present invention, the replication initiation sequence is preferably oriC and its modified sequence, and more preferably oriC derived from E. coli and its modified sequence.

[0057] In one embodiment, the replication initiation sequence may have a mutated double-stranded cleavage region (DUE) with an increased AT content compared to the wild type.

[0058] In one embodiment, when the double-stranded DNA obtained in the accumulation reaction is amplified by PCR, the circular DNA after accumulation may contain a replication initiation sequence capable of binding to an enzyme having DnaA activity and a strong gyrase-binding sequence (SGS). The SGS is a binding sequence to which DNA gyrase binds and introduces negative supercoils into DNA. From the viewpoint of fully achieving the effect of increasing the amplification efficiency of the circular DNA, the SGS is preferably an SGS derived from bacteriophage Mu, more preferably an SGS derived from bacteriophage Mu (Mu-SGS) (Folarin et al., Bioengineering, 2019, vol. 6(2):54.), and even more preferably SEQ ID NO: 207 described below or a sequence containing same.

[0059] The PCR method comprises the following steps: (a) preparing a reaction mixture containing a reaction solution containing a first group of enzymes that catalyze the replication of circular DNA, a second group of enzymes that catalyze the Okazaki fragment ligation reaction to 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 the circular DNA to be amplified; (b) incubating the reaction mixture prepared in step (a) at a constant temperature in the range of 15°C to 80°C, or incubating under temperature cycles that repeat incubation at two temperatures below 80°C.

[0060] 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.

[0061] The enzyme having DnaA activity is not particularly limited in terms of its biological origin, 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 nM to 10 μM, preferably 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 10 nM to 200 nM, or 20 nM to 150 nM, but is not limited thereto.

[0062] 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.

[0063] The biological origin of the enzyme or enzymes having DNA gyrase activity is not particularly limited, as long as they have an 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, or 20 nM to 200 nM, but is not limited thereto.

[0064] 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 its biological origin is not particularly limited. 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 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.

[0065] 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.

[0066] 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.

[0067] 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, or 200 nM to 500 nM, but is not limited thereto.

[0068] 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, or 20 nM to 100 nM, but is not limited thereto.

[0069] 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 suitably used. Preferably, an enzyme group containing a complex of DnaX, HolA, HolB, and DnaE 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 concentration ranging from 2 nM to 50 nM, preferably from 2 nM to 40 nM, from 2 nM to 30 nM, or from 2 nM to 20 nM, but is not limited thereto.

[0070] 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.

[0071] The second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes can include, for example, 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 preferably includes an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity.

[0072] The enzyme having DNA polymerase I activity is not particularly limited in terms of its biological origin, 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, or 20 nM to 80 nM, but is not limited thereto.

[0073] 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.

[0074] 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, 2 nM to 50 nM, or 5 nM to 50 nM, but is not limited thereto.

[0075] 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.

[0076] 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 20 to 80 nM, but is not limited thereto.

[0077] 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-derived 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 20 to 80 nM, but is not limited thereto.

[0078] 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.

[0079] The reaction solution may further contain an enzyme. For example, when the circular DNA to be amplified by PCR has a pair of ter sequences inserted facing outward relative to a replication origin sequence (e.g., oriC) capable of binding to an enzyme having DnaA activity, 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 sequences and inhibiting replication.

[0080] The first, second, and third enzyme groups may be commercially available 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.

[0081] 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.

[0082] The reaction solution may contain a buffer, ATP, GTP, CTP, UTP, dNTP, a magnesium ion source, and an alkali metal ion source.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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 in the range of 0.05 to 1 mM, or 0.1 to 1 mM.

[0087] The magnesium ion source is a magnesium ion (Mg ２＋ ) is a substance that gives Mg(OAc) ２, MgCl ２ , and MgSO ４ 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 reaction solution in the range of 5 to 50 mM.

[0088] The alkali metal ion source is a substance that provides alkali metal ions to the reaction solution. Examples of alkali metal ions include sodium ions (Na ＋ ), 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.

[0089] 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).

[0090] In one embodiment, double-stranded DNA without sequence errors can be selectively amplified by applying a specific combination of enzymes to double-stranded DNA with sequence errors before and / or during amplification of the double-stranded DNA obtained by the enrichment reaction. The double-stranded DNA with sequence errors may be double-stranded DNA in which at least one of the strands forming the double-stranded structure has a sequence error and normal base pairs are not formed in the sequence error portion. For example, the double-stranded DNA may have a sequence error in only one of the strands forming the double-stranded structure, or may have sequence errors in both strands forming the double-stranded structure.

[0091] More specifically, when the double-stranded DNA is circular and the double-stranded DNA amplification reaction is performed in the presence of MutS and MutL, or in addition to MutS and MutL, in the presence of an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease (for example, when performed in the presence of MutS, MutL, and MutH), double-stranded DNA that does not have sequence errors can be selectively amplified during the amplification reaction. Furthermore, prior to the double-stranded DNA amplification reaction, MutS and MutL may be allowed to act on the double-stranded DNA, or in addition to MutS and MutL, an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease may be allowed to act on the double-stranded DNA (e.g., MutS, MutL, UvrD, and a single-strand-specific exonuclease may be allowed to act on the double-stranded DNA), thereby selectively amplifying double-stranded DNA that does not contain sequence errors during the subsequent double-stranded DNA amplification reaction. The above enzymes may be added and allowed to act before the double-stranded DNA amplification reaction, and then the amplification reaction may be carried out in the presence of the above enzymes. In this embodiment, the above enzymes that act on double-stranded DNA with sequence errors are also referred to as mismatch repair-related enzymes. The double-stranded DNA amplification reaction in the presence of mismatch repair-related enzymes, and the act of mismatch repair-related enzymes on double-stranded DNA prior to the double-stranded DNA amplification reaction, refer to sequence error recognition by MutS of the mismatch repair-related enzymes and interaction with MutS by MutL, or sequence error recognition by MutS of the mismatch repair-related enzymes and hydrolysis by single-strand-specific exonuclease. If the mismatch repair-related enzymes include additional enzymes, these enzymes also exert the activities described below. Conveniently, the mismatch repair-related enzymes can be acted on in solution, for example, at 15 to 40°C, 16 to 40°C, 25 to 40°C, preferably 30 to 40°C, for 5 to 120 minutes, preferably 10 to 60 minutes. When the mismatch repair-related enzymes are acted on during the double-stranded DNA amplification reaction, they can be simultaneously carried out under the reaction conditions of the amplification reaction.The composition of the reaction solution is not particularly limited as long as it allows the action of the mismatch repair-related enzymes to proceed. For example, a solution obtained by adding a magnesium ion source, ATP, etc. to a buffer solution such as Tris-HCl buffer, and further adding additional components such as an alkali metal ion source as necessary, to which the mismatch repair-related enzymes are added can be used. In particular, when the mismatch repair-related enzymes include UrvD, the reaction solution can contain ATP.

[0092] The biological origin of MutS is not particularly limited as long as it is a protein that recognizes and binds to mismatched base pairs in DNA. For example, known MutS derived from various bacteria, such as Escherichia coli or thermostable bacteria, and its family proteins can be used. MutS derived from known MutSs, such as MutS derived from various bacteria or MutS derived from thermostable bacteria, can also be used, as long as it has the activity of recognizing and binding to mismatched base pairs in DNA. In one aspect, MutS derived from Escherichia coli or thermostable bacteria, or MutS derived from these MutSs or MutS derived from these MutSs with mutations or modified amino acids, can be used. MutS derived from Escherichia coli is preferably used, and wild-type MutS derived from Escherichia coli can be particularly used. MutS may be contained in a range of 10 nM to 500 nM when acting on sequence errors, preferably 10 nM to 300 nM, 30 nM to 500 nM, 30 nM to 300 nM, 30 nM to 200 nM, or 50 nM to 300 nM, and more preferably 100 nM to 300 nM, but is not limited thereto.

[0093] MutL is a protein that interacts with MutS upon recognizing a mismatched base pair to form a complex. While MutL in some biological species has endonuclease activity, MutL derived from Escherichia coli does not. MutL can be used regardless of whether it has endonuclease activity, and known MutLs, such as MutL derived from Escherichia coli and related species, and its family proteins, can be used. MutLs with mutations introduced into the amino acid sequence of known MutLs or modified amino acids can also be used as long as they have the same effect as Escherichia coli MutL, which interacts with MutS upon recognizing a mismatched base pair. In one aspect, MutL derived from Escherichia coli can be preferably used. MutL may be contained in the range of 30 nM to 1000 nM when acting on sequence errors, preferably 30 nM to 500 nM, 30 nM to 300 nM, 30 nM to 200 nM, 30 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, and more preferably, 100 nM to 300 nM, but is not limited to this range.

[0094] MutH is a protein that is activated by MutS and MutL in prokaryotes such as Escherichia coli and has the activity of nicking unmethylated DNA (when both strands of double-stranded DNA are unmethylated, it cleaves the double strand). For example, a known MutH such as MutH derived from Escherichia coli can be used. MutHs with mutations introduced into the amino acid sequence of known MutHs or modified amino acids can also be used as long as they have the above-mentioned activity. In one aspect, MutH derived from Escherichia coli can be preferably used. MutH may be present in the range of 10 nM to 500 nM when acting on sequence errors, preferably 10 nM to 300 nM, 10 nM to 200 nM, 30 nM to 500 nM, 30 nM to 300 nM, 30 nM to 200 nM, 50 nM to 300 nM, or 50 nM to 200 nM, and more preferably 100 nM to 200 nM, but is not limited thereto.

[0095] UvrD is a protein derived from Escherichia coli that has helicase activity, cleaving (unwinding) double-stranded DNA. In addition to known UvrDs, known UvrDs with mutations introduced into their amino acid sequences or modified amino acids can also be used as long as they retain helicase activity. In one embodiment, wild-type UvrD derived from Escherichia coli can be used. UvrD may be present in a range of 1 nM to 100 nM, preferably 1 nM to 50 nM, 1 nM to 30 nM, 1 nM to 20 nM, 3 nM to 50 nM, 3 nM to 30 nM, 3 nM to 20 nM, 5 nM to 50 nM, 5 nM to 30 nM, 5 nM to 20 nM, or 10 nM to 20 nM, and more preferably 15 nM, when acting on sequence errors. However, the present invention is not limited thereto.

[0096] The single-strand-specific exonuclease is an enzyme that sequentially hydrolyzes nucleotides from the 3'-end or 5'-end of linear single-stranded DNA. The single-strand-specific exonuclease used in this embodiment is not particularly limited in type or biological origin, as long as it has the enzymatic activity of sequentially hydrolyzing linear DNA from the 3'-end or 5'-end.

[0097] When a single-strand-specific exonuclease is used together with UvrD, for example, examples of enzymes that sequentially hydrolyze from the 3' end (3'→5' single-strand-specific exonucleases) include exonuclease VII, exonuclease I (ExoI), exonuclease T (Exo T) (also known as RNase T), exonuclease X, DNA polymerase III epsilon subunit, DNA polymerase I, DNA polymerase II, T7 DNA polymerase, T4 DNA polymerase, Klenow DNA polymerase, Phi29 DNA polymerase, ribonuclease III (RNase D), and oligoribonuclease (ORN). Examples of enzymes that sequentially hydrolyze from the 5' end (5'→3' single-strand-specific exonucleases) that can be used include exonuclease VII, λ exonuclease, exonuclease VIII, T5 exonuclease, T7 exonuclease, and RecJ exonuclease.

[0098] Since ExoVII has both 5'-3' single-strand-specific exonuclease activity and 3'-5' single-strand-specific exonuclease activity, in one embodiment, it is preferable to use ExoVII as the single-strand-specific exonuclease to be used together with UvrD.

[0099] The mismatch repair-related enzymes can be contacted with the double-stranded DNA mixture and allowed to act, conveniently, in a solution at, for example, 15 to 40°C, 16 to 40°C, 25 to 40°C, preferably 30 to 40°C, for 5 to 120 minutes, preferably 10 to 60 minutes. When the mismatch repair-related enzymes are allowed to act during the double-stranded DNA amplification reaction, they can be simultaneously carried out under the reaction conditions of the amplification reaction. The composition of the reaction solution is not particularly limited as long as it allows the action of the mismatch repair-related enzymes to proceed. For example, a solution containing a magnesium ion source, ATP, etc., added to a buffer solution such as Tris-HCl buffer, and further containing additional components such as an alkali metal ion source as needed, to which the mismatch repair-related enzymes are added can be used. In particular, when the mismatch repair-related enzymes include UrvD, the reaction solution can contain ATP.

[0100] This embodiment also relates to a method for producing a double-stranded DNA library, including: (1) preparing a set of two or more types of single-stranded DNA fragments, at least a portion of each single-stranded DNA fragment having a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponding to a portion of either strand of the double-stranded DNA, (1') preparing two or more types of library-constituting single-stranded DNA fragments, each of which has a region corresponding to a portion of either strand of the double-stranded DNA and a region non-complementary to any single-stranded DNA fragment in the set of single-stranded DNA fragments in (1), and the base sequences of the non-complementary regions differ from one another, (2) preparing an enrichment reaction solution containing the set of single-stranded DNA fragments, the two or more types of library-constituting single-stranded DNA fragments, a cationic salt or cationic substance, and an enrichment-promoting polypeptide, and (3) Incubating the reaction solution at an incubation temperature higher than 40°C, thereby accumulating the set of single-stranded DNA fragments and two or more types of library-constituting single-stranded DNA fragments into a double-stranded DNA library.

[0101] The single-stranded DNA fragments accumulated in this library production method include two or more types of library-constituting single-stranded DNA fragments in addition to the set of single-stranded DNA fragments used in the above-described method for producing double-stranded DNA fragments. The library-constituting single-stranded DNA fragments have two or more sequences, preferably random DNA sequences, in part or in whole (library region). The library region in the library-constituting single-stranded DNA fragments is preferably located in a region non-complementary to any of the single-stranded DNA fragments included in the set of single-stranded DNA fragments (1) above. The two or more types of library-constituting single-stranded DNA fragments preferably have identical sequences to each other except for the library region. In the accumulated double-stranded DNA, the library region may be designed to have no complementary sequence, or may be designed to have a complementary sequence. If a complementary sequence is present, the complementary sequence portion may also be included in the library region. By accumulating such a set of single-stranded DNA fragments, a double-stranded DNA library can be easily obtained. The complementary strand of the random sequence portion can be replicated using known techniques, such as those described in Example 16 below.

[0102] The above steps (2) and (3) can be carried out in the same manner as in the method for producing double-stranded DNA of this embodiment. In this manner, a library of double-stranded DNA in which part of the double-stranded DNA has a random sequence can be obtained.

[0103] This embodiment also relates to a method for producing double-stranded DNA, including: (a) heating a solution containing a given polypeptide and a given number of single-stranded DNA fragments to a temperature higher than 40°C, so that the polypeptide and at least one of the single-stranded DNA fragments in the solution form aggregates, and selecting the polypeptide that formed the aggregate; (b) providing a set of single-stranded DNA fragments, at least a portion of each single-stranded DNA fragment having a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponding to a portion of one of the strands of the double-stranded DNA; (c) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments, a cationic salt or cationic substance, and the polypeptide selected in (a); and (d) incubating the reaction solution at an incubation temperature higher than 40°C, so that the set of single-stranded DNA fragments assemble into double-stranded DNA.

[0104] A polypeptide that is effective as an accumulation-promoting polypeptide can be selected from the desired polypeptides by heating a solution containing any polypeptide and any plurality of single-stranded DNA fragments to a temperature higher than 40°C and selecting the polypeptide that forms aggregates. Furthermore, by confirming that the amount of aggregates formed when heated to a temperature higher than 40°C is reduced compared to when arginine is not contained, a polypeptide that is effective as an accumulation-promoting polypeptide can be further selected. The heating temperature in this case is determined based on the T of the single-stranded DNA fragments used. ｍ The temperature varies depending on the value, etc., but is preferably 45° C. or higher, for example, about 75° C. For other selection methods, please refer to the description of the accumulation-promoting polypeptide above.

[0105] The above steps (b) to (d) can be carried out in the same manner as in the method for producing double-stranded DNA of this embodiment.

[0106] The present embodiment also relates to a kit for producing circular double-stranded DNA, comprising: an enzyme having exonuclease III activity; an enzyme having DNA polymerase I activity; an enzyme having DNA ligase activity; an enzyme or enzymes having DNA gyrase activity; a cationic salt or cationic substance; and an accumulation-promoting polypeptide selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having RecA family recombinase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA-binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and an ε subunit of F1-ATPase.

[0107] Using the cationic salt or cationic substance and the accumulation-promoting polypeptide of this kit, single-stranded DNA fragments can be accumulated from a set (e.g., an oligo pool) of two or more single-stranded DNA fragments, where at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set and each single-stranded DNA fragment corresponds to a portion of one strand of the double-stranded DNA. Some of the accumulated single-stranded DNA fragments may be library-constituting single-stranded DNA fragments. If the resulting double-stranded DNA is not circular, the double-stranded DNA can be circularized and then further subjected to the supercoiling reaction described above using the enzyme having exonuclease III activity, the enzyme having DNA polymerase I activity, the enzyme having DNA ligase activity, and the enzyme or enzymes having DNA gyrase activity of this kit to obtain supercoiled circular double-stranded DNA. For details about each enzyme, please refer to the description of the method for producing circular DNA of this embodiment.

[0108] Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0109] Example 1: Accumulation-promoting effect of specific polypeptides on oligo-DNA fragments. Twenty-four types of single-stranded DNA fragments (each 200 bases long) with complementary strands were accumulated in the presence of various accumulation-promoting polypeptides to produce 2.4 kb linear double-stranded DNA. Four types of polypeptides were examined as accumulation-promoting polypeptides.

[0110] For a 2,400-base artificial gene containing the oriC sequence, 24 oligo DNA sequences of 200 bases each were designed so that hybridization between the oligo DNAs would result in double-stranded DNA containing no single-stranded gaps. Tables 1 to 3 show the sequences of each of the designed oligo DNAs (IDT, Ultramer PAGE-purified grade).

[0111]

[0112]

[0113]

[0114] A mixture of 23 types of oligo DNAs (excluding F24) in Tables 1 to 3 was heat-treated at 95°C for 1 minute and then rapidly cooled on ice to prevent misassembly. Next, each mixture was diluted with assembly buffer (20 mM Tris-HCl pH 8.0, 20 mM Mg(OAc)) to a concentration of 0.87 nM (total 20 nM, 1.3 ng / μL). ２ , 0.05% Tween 20). Four polypeptides, creatine kinase (CK), phosphopyruvate hydratase (enolatase, ENO), bovine serum albumin (BSA), and bacterial alkaline phosphatase (BAP), were added under the following conditions. The respective sources are shown in Example 2.

[0115] Control: No polypeptide added (Polypeptide-) CK, ENO, BSA and BAP: added at concentrations of 12 nM, 120 nM, 1,200 nM or 12,000 nM, respectively.

[0116] The oligo DNA was then linearized by heat treatment for 5 minutes at 75° C. The reaction mixture (4.0 μL) was analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.

[0117] A schematic diagram of the linearization and accumulation process is shown in Figure 1A, and the staining results are shown in Figure 1B. When no polypeptide was added, no 2.4 kb accumulation product was observed. When CK was added as a polypeptide, a 2.4 kb band was detected at 120 nM and 1,200 nM, and when ENO was added, a 2.4 kb band was detected at 120 nM, 1,200 nM, and 12,000 nM. When BSA or BAP was added, the 2.4 kb band was not observed at any concentration, and no significant accumulation-promoting effect was observed. CK and ENO were shown to have a promoting effect on oligo DNA accumulation within a specific range of concentrations.

[0118] [Example 2] Various polypeptides that have the effect of promoting accumulation of oligo-DNA fragments From the results of Example 1, it was found that certain polypeptides exhibit the effect of promoting accumulation of oligo-DNA. Other polypeptides that have the effect of promoting accumulation were investigated.

[0119] The accumulation-promoting effect was confirmed using the same materials and procedures as in Example 1, except that the polypeptides shown in Tables 4 and 5 were added and the assembly buffer described below was used. When CK, ENO, PK, 3-PGK, HU, myokinase, NDK, AtpC, BSA, BAP, SSB, or lysozyme was added, heat treatment (accumulation reaction) was carried out at 75°C for 5 minutes. When T4 DNA Lig, EcUvrD, RecA, EcLigA, or T4 PNK was added, heat treatment (accumulation reaction) was carried out at 65°C for 2 minutes.

[0120] Twenty-three types of 200-base oligo DNAs were added to the assembly buffer to a concentration of 0.87 M (total 20 nM, 1.3 ng / μL), and the oligo DNAs were linearized and assembled. Analysis was then performed by 1.5% agarose gel electrophoresis and SYBR Green staining. The concentrations, optimal concentrations, and assembly efficiencies investigated using the 2.4 kb assembly product as an index are shown in Tables 4 and 5.

[0121]

[0122]

[0123] Note 1: In Table 4, DNA helicase was prepared by the method described in Hasebe et al., Life (2018) 8(4), 43. Note 2: In Table 4, histone-like DNA-binding protein (HU) was prepared by purifying it from an E. coli expression strain of HU using a process including ammonium sulfate precipitation and affinity column chromatography. Note 3: In Table 4, chromosomal replication initiator protein was prepared by the method described in Su'etsugu et al., Nucleic Acids Res. (2017) 45(20):11525-11534. Note 4: In Table 5, nucleoside diphosphate kinase (NDK) was prepared by purifying it from an E. coli expression strain of NDK using a process including ammonium sulfate precipitation and affinity column chromatography. Note 5: In Table 5, the ε subunit of F1-ATPase was prepared by the method described in Kato et al. J. of Biological Chemistry (2007) 282(52):37618-37623.

[0124] In Tables 4 and 5, the numbers *1 to *3 attached to the polypeptides indicate the assembly buffers used. *1: 20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 0.05% Tween20 *2: 20mM Tris-HCl (pH 8.0), 20mM Mg (OAc) ２ *3: 20mM Tris-HCl (pH 8.0), 20mM Mg (OAc) ２ , 5% PEG8000

[0125] In Tables 4 and 5, the notation in the "Assembly efficiency" column indicates the assembly efficiency as follows: "-": Almost no 2.4 kb assembly product was detected. "+": A small amount of 2.4 kb assembly product was detected. "++": More 2.4 kb assembly product was detected than +. "+++": More 2.4 kb assembly product was detected than ++. Specific assembly efficiencies are shown in Figure 1.

[0126] As shown in Tables 4 and 5, of the 19 polypeptides tested, five - CK, ENO, PK, 3-PGK, and T4 DNA ligase - showed a high accumulation effect, while EcUvrD and RecA were confirmed to have a lower accumulation effect. On the other hand, only a slight accumulation effect was observed with EcLigA, T4 PNK, HU, DnaA, Tte UvrD, Myokinase, NDK, and AtpC, and no accumulation effect was observed with BSA, BAP, SSB, and lysozyme. This indicates that certain polypeptides have the effect of promoting oligo DNA accumulation.

[0127] [Example 3] Mg(OAc) ２ Example 3-1: Accumulation promotion effect of oligo DNA by 1200 nM CK and different concentrations of Mg(OAc) were added to the 23 types of 200-base oligo DNA used in Example 1 so that each concentration was 0.87 nM (total 20 nM, 1.3 ng / μL). ２ The oligo DNA was added to an assembly buffer containing the above and linearized at the same reaction temperature as in Example 1. Then, the oligo DNA was analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.

[0128] Assembly buffer composition: 20 mM Tris-HCl (pH 8.0), 0.05% Tween 20. Mg(OAc) ２ was added at concentrations of 0 mM, 0.5 mM, 2 mM, 5 mM, 10 mM or 20 mM.

[0129] A schematic diagram of the accumulation is shown in Figure 2A, and the results are shown in Figure 2B. As shown in Figure 2B, Mg(OAc) ２ When a buffer containing no Mg(OAc) was used, the 2.4 kb accumulation product was not observed.２ When the concentration of ATP was increased, more 2.4 kb accumulation products were detected.

[0130] (Example 3-2) For an artificial gene of approximately 800 base pairs containing the gfp gene, 16 oligo DNAs were designed, divided into approximately 95 to 97 base segments, as in Example 1. Table 6 shows the design of IDT's oPools oligo pools (IDT oPools).

[0131]

[0132] The pUP-2 fragment (2.3 kb) was prepared by PCR amplification using a pUP fragment (SEQ ID NO: 41) containing oriC, an ampicillin resistance gene, pUCori, a δ70 promoter, and an rrnBT1 terminator as a template and primers 1 and 2 (SEQ ID NOs: 42 and 43) containing dU (deoxyuridine).

[0133]

[0134]

[0135] The pUP-2 fragment (2.3 kb) and the 16 types of oligo DNA pools (IDT oPools) shown in Table 6 were diluted to a concentration of 10 pM with 10 mU / μL Thermolabile USER II Enzyme (NEB), different concentrations of Mg(OAc), and ２ and added to assembly buffer containing different concentrations of CK.

[0136] Assembly buffer composition: 20 mM Tris-HCl (pH 8.0), 0.05% Tween 20. Mg(OAc) ２ was added at a concentration of 0 mM, 5 mM, 10 mM, or 20 mM. CK was added at a concentration of 0 nM, 120 nM, or 1,200 nM.

[0137] After uracil removal reaction at 37°C for 30 minutes, the mixture was heat treated at 75°C for 5 minutes and slowly cooled (0.1°C / sec) to carry out integrated circularization of the oligo DNA and single-stranded overhangs.

[0138] One microliter of the resulting reaction mixture was added to the PCR reaction mixture shown in Table 9 (enzymes were diluted to 50% of the amounts shown in Table 9) (total volume: 10 μL), and PCR amplification was carried out at 30°C for 16 hours. Finalization treatment was then carried out (the reaction mixture was diluted 5-fold with reaction buffer, followed by incubation at 33°C for 30 minutes) to supercoil the replication intermediates. A 1.0 μL aliquot was analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.

[0139]

[0140] 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.

[0141] 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.

[0142] A schematic diagram of the accumulation is shown in Figure 3A, and the staining results are shown in Figure 3B. As shown in Figure 3B, Mg(OAc) ２ When the concentration of Mg(OAc) was 0 mM or 5 mM, almost no band of pUP-GFP was observed even when CK was contained. ２ In its presence, pUP-GFP was detected.

[0143] From the results of Examples 3-1 and 3-2, it is clear that the Mg(OAc) added to this system ２ is essential for the accumulation of oligo DNA, and it was thought that the accumulation-promoting effect could be obtained by adding 10 mM or more.

[0144] Example 4 Accumulation-Promoting Effect of Cationic Salts or Cationic Substances Other Than Mg The accumulation-promoting effect of cationic salts and cationic substances other than Mg was examined.

[0145] The 23 types of 200-base oligo DNAs used in Example 1 were added to an assembly buffer containing 1,200 nM CK and various cationic salts to a concentration of 0.87 nM each (total 20 nM, 1.3 ng / μL), and the oligo DNAs were assembled and linearized. The results were then analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.

[0146] Composition of assembly buffer: 20 mM Tris-HCl (pH 8.0), 0.05% Tween 20. Furthermore, the following cationic salts were added: Mg(OAc) ２ (Sigma): 20 mM NaCl (Fuji Film): 50 mM or 100 mM KOAc (Sigma): 50 mM or 100 mM Spermidine (Sp) (MP Biomedicals): 0.6 mM or 2 mM

[0147] A schematic diagram of the accumulation is shown in Figure 4A, and the staining results are shown in Figure 4B. As shown in Figure 4B, the effect of Mg(OAc) confirmed in Example 3 was ２In addition, NaCl, KOAc, and spermidine also showed an accumulation-promoting effect. ２ The effect was the highest, followed by spermidine, KOAc, and NaCl in that order. It was shown that cationic salts and cationic substances other than Mg also promote the assembly of oligo-DNA.

[0148] [Example 5] Properties of polypeptides that exhibit an assembly-promoting effect In Examples 1 and 2, it was found that specific polypeptides have the effect of promoting the assembly of oligo-DNA. Therefore, in order to clarify the properties of polypeptides that have an assembly-promoting effect (APP: Assembly Promoting Peptides), binding to single-stranded DNA was examined by gel shift analysis, and aggregation of the polypeptide was confirmed by SDS-PAGE.

[0149] (Example 5-1) One type of 100-base oligo DNA (SEQ ID NO: 44) fluorescently labeled with Cy5 was diluted to a concentration of 20 nM with assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 0.05% Tween 20). CK, ENO, BSA, and BAP were each added at a concentration of 1,200 nM. The mixture was then heat-treated at 75°C for 5 minutes, and the resulting reaction mixture (5.0 µL) was subjected to 6% polyacrylamide gel electrophoresis to detect Cy5.

[0150]

[0151] The results are shown in Figure 5. "-" indicates the electrophoresis results of samples left on ice for 5 minutes or more, and "+" indicates the electrophoresis results of samples incubated at 75°C for 5 minutes. CK and ENO were observed to bind to single-stranded DNA after incubation, although only slightly. The results showing that aggregated polypeptides bind to DNA are consistent with previous reports (Litberg et al., Biophys J. (2020) Jan 7;118(1):162-171). On the other hand, BSA and BAP, which did not exhibit an accumulation-promoting effect in Examples 1 and 2, did not show any binding to single-stranded DNA.

[0152] Example 5-2: 5.0 μL of the reaction solution obtained in Example 5-1 was centrifuged at 15,000 rpm for 15 minutes at 4°C, and the supernatant and precipitate were separated. 1.3 μL of 5xSB was added to 5.0 μL of the reaction solution before centrifugation or 5.0 μL of the supernatant, and 6.3 μL of 1xSB was added to the precipitate, followed by heat treatment at 95°C for 5 minutes. The composition of SB (SDS-PAGE sample preparation buffer) was 25 mM Tris-HCl (pH 8.8), 5% glycerol, 2% SDS, 20 mM EDTA, 1% β-mercaptoethanol, and 0.05% BPB. 6.0 μL of the resulting solution was electrophoresed by SDS-PAGE, and polypeptides were detected by CBB staining.

[0153] The staining results are shown in Figure 6. "t" represents the fraction before centrifugation (total), "s" represents the supernatant fraction after centrifugation, and "p" represents the precipitate fraction after centrifugation. Furthermore, "-" represents a sample that was left on ice for 5 minutes or more, and "+" represents a sample that was incubated at 75°C for 5 minutes.

[0154] As shown in Figure 6, for CK and ENO, without incubation, the respective polypeptides were detected in the supernatant fraction, but with incubation, the respective polypeptides were detected in the precipitate fraction. On the other hand, for BSA and BAP, the respective polypeptides were detected in the supernatant fraction, regardless of whether incubation was performed. From these results, it was found that polypeptides with accumulation-promoting effects aggregate in a heat-dependent manner and have the property of binding to single-stranded DNA.

[0155] [Example 6] Reaction temperature at which accumulation-promoting effect is observed It was thought that heat-dependent aggregation of polypeptides would exhibit the effect of promoting oligo-DNA accumulation in Example 5. Therefore, the temperature at which the accumulation-promoting effect is observed was investigated.

[0156] (Example 6-1) The 23 types of 200-base oligo DNA used in Example 1 were each diluted with assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) to a concentration of 0.87 nM (total 20 nM, 1.3 ng / μL). ２The oligo DNA was linearized by heat treatment at 40°C, 45°C, or 50°C for 1 hour in the presence (1200 nM) or absence (0 nM) of CK, in addition to 0.05% Tween 20. Analysis was performed by 1.5% agarose gel electrophoresis and SYBR Green staining.

[0157] The staining results are shown in Figure 7. At 40°C, the 2.4 kb band of the accumulation product could not be confirmed even with the addition of CK. On the other hand, at 45°C and 50°C, the 2.4 kb band was detected.

[0158] Example 6-2 The reaction solution of Example 6-1 containing CK was centrifuged in the same manner as in Example 5-2, electrophoresed by SDS-PAGE, and polypeptides were detected by CBB staining.

[0159] The results are shown in Figure 8. In Figure 8, "t" represents the fraction before centrifugation, "s" represents the supernatant fraction after centrifugation, and "p" represents the precipitate fraction after centrifugation. As shown in Figure 8, at 40°C, CK was detected only in the supernatant fraction, whereas at 45°C, half of the CK was detected in the precipitate fraction, and at 50°C, almost all of the CK was detected in the precipitate fraction.

[0160] The results of Example 6 showed that the aggregation of the polypeptide corresponds to the accumulation-promoting effect. It was thought that the accumulation-promoting effect of the oligo-DNA was obtained by the reaction at a temperature at which the polypeptide aggregates.

[0161] Example 7: Suppression of accumulation reaction and polypeptide aggregation by addition of arginine From Example 6, it was thought that the accumulation-promoting effect of the polypeptide corresponds to the temperature at which the polypeptide aggregates. Therefore, to further clarify the relationship between the heat-dependent aggregation of the polypeptide and the accumulation-promoting effect, we investigated whether polypeptide-dependent accumulation of oligo-DNA is suppressed using arginine hydrochloride as an aggregation inhibitor.

[0162] (Example 7-1) The 23 types of 200-base oligo DNA used in Example 1 were assembled and linearized, and analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining. Each oligo DNA was added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) to a concentration of 0.87 nM (total 20 nM, 1.3 ng / μL). ２ , 0.05% Tween 20). CK or ENO was added at a concentration of 0 nM or 1,200 nM, and arginine hydrochloride (Fuji Film) was added at a concentration of 200 mM.

[0163] The results are shown in Figure 9. As shown in Figure 9, when arginine hydrochloride (Arg-HCl) was not contained, a 2.4 kb band was detected. On the other hand, when arginine hydrochloride was contained, the 2.4 kb band was not detected, indicating that the accumulation-promoting effect was inhibited.

[0164] (Example 7-2) The reaction solution of Example 7-1 containing CK or ENO was centrifuged in the same manner as in Example 5, electrophoresed by SDS-PAGE, and polypeptides were detected by CBB staining. The results are shown in Figure 10. In Figure 10, "t" represents the fraction before centrifugation, "s" represents the supernatant fraction after centrifugation, and "p" represents the precipitate fraction after centrifugation.

[0165] 10, when arginine hydrochloride was not contained, CK or ENO was detected in the precipitate fraction but not in the supernatant fraction, as in Example 5. On the other hand, when arginine hydrochloride was contained, CK or ENO was detected not only in the precipitate fraction but also in the supernatant fraction. From these results, it was considered that the polypeptide that forms fewer aggregates when arginine is contained compared to when arginine is not contained has an oligo-DNA accumulation effect.

[0166] Example 8 Accumulation from Low Concentration Oligo DNA and Amplification of Accumulation It was examined whether low concentration oligo DNA was accumulated using APP and whether amplification could be achieved by PCR reaction.

[0167] The pUP-2 fragment (2.3 kb) and 16 types of IDT oPools used in Example 3-2 were subjected to assembly reactions and PCR amplification reactions in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. Each DNA used in the assembly reaction was diluted to a concentration of 1 pM, 10 pM, 100 pM, or 1000 pM in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 0.05% Tween 20) (total DNA concentration was 17 times the concentration of each DNA). CK was used at the concentrations shown in FIG. 11B and FIG. 12.

[0168] A schematic diagram of the accumulation is shown in Figure 11A, and the staining results are shown in Figures 11B and 12. As shown in Figure 11B, for each 10 pM oligo DNA concentration, a band of the amplified product pUP-GFP could be detected when 120 nM or 1,200 nM CK was added. For each 1 pM oligo DNA concentration, a band of pUP-GFP could be detected when 120 nM CK was added. As shown in Figure 12, when the oligo DNA concentration was high, a band of pUP-GFP could be detected even when the CK concentration was low or zero.

[0169] It was shown that the addition of APP enabled the accumulation and amplification of oligo DNA at a very low concentration of 1 pM.

[0170] [Example 9] Synthesis of an artificial gene with a high AT content (GC 31%) We investigated whether the 487 bases of the upstream region of the cfr gene (cfrUR) of Bacillus bacteria, which has a high AT content (GC 31%) and is a difficult-to-synthesize sequence, could be constructed by a method using APP.

[0171] For the artificial gene of cfrUR (487 bases), eight types of oligo DNA sequences of 119 to 140 bases were designed according to Example 1. Table 11 shows the designed oligo DNA sequences (IDT, oPool, desalted grade).

[0172]

[0173] The pUP-3 fragment (2.3 kb) was prepared by PCR amplification using the pUP fragment (SEQ ID NO: 53) having oriC, ampicillin resistance gene and pUCori as a template with primers 3 and 4 containing dU.

[0174]

[0175]

[0176] The pUP-3 fragment (2.3 kb) and eight types of IDT oPools were subjected to assembly reaction and PCR amplification reaction in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. The DNA was diluted to a concentration of 1 nM each in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 50 mM KOAc). CK was added at concentrations of 0 nM, 232 nM, 464 nM, or 696 nM.

[0177] Next, 0.01 μL of the PCR amplification product was used to transform Escherichia coli DH5α by chemical transformation. The transformed Escherichia coli was plated on an LB plate containing 50 μg / mL carbencillin and cultured overnight at 37°C. Plasmids were extracted from the resulting colonies and sequenced by the Sanger method.

[0178] A schematic diagram of the accumulation is shown in Figure 13A, and the staining results are shown in Figure 13B. As shown in Figure 13B, when CK was not included, almost no amplification of pUP-cfrUR was observed. On the other hand, when 232 nM, 464 nM, or 696 nM CK was added, a band corresponding to pUP-cfrUR was confirmed as an amplification product.

[0179] Table 14 shows the results of plasmid sequencing analysis of the amplified product obtained when CK was added at a concentration of 464 nM and introduced into E. coli. Correct sequences were obtained for 6 of the 8 clones.

[0180]

[0181] It was shown that sequences with a high AT ratio, which are considered difficult to synthesize, can also be constructed using a method using APP.

[0182] Example 10 Synthesis of an Artificial Gene with a High GC Content (GC 70%) We investigated whether a region of 825 bases (IJ101 frg8) of the plasmid pIJ101 (Kieser et al., Mol Gen Genet. (1982) 185, 223-238) derived from Streptomyces bacteria, which has a high GC content (GC 70%) and is considered difficult to synthesize, could be constructed by a method using APP.

[0183] For the artificial gene IJ101 frg8 (825 bases), 20 types of oligo DNA sequences of 76 to 91 bases were designed according to Example 1. Table 15 shows the designed oligo DNA sequences (IDT, oPool, desalted grade).

[0184]

[0185] The pUP-3 fragment (2.3 kb) used in Example 9 and the 20 types of IDT oPools in Table 15 were subjected to assembly reaction and PCR amplification reaction in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. The DNA was diluted to a concentration of 1 pM each in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 0.05% Tween 20). CK was added at concentrations of 0 nM, 120 nM, or 1,200 nM.

[0186] Next, the resulting PCR amplification product was used to transform the cells in the same manner as in Example 9, and the plasmids were extracted from the resulting colonies and subjected to sequence analysis by the Sanger method.

[0187] A schematic diagram of the accumulation is shown in Figure 14A, and the results are shown in Figure 14B. As shown in Figure 14B, almost no amplification of pUP-IJ101-frg5 was observed in the absence of CK or in the presence of 1,200 nM CK. On the other hand, when 120 nM CK was added, a band corresponding to pUP-IJ101-frg5 was confirmed as an amplification product.

[0188] The amplification products obtained when CK was added at a concentration of 120 nM were introduced into E. coli and the resulting plasmids were sequenced. The correct sequences were obtained from 3 of the 7 clones.

[0189]

[0190] It was shown that sequences with a high GC ratio, which are considered difficult to synthesize, can also be constructed using a method using APP.

[0191] [Example 11] Synthesis of an artificial gene using 42 oligo DNAs In order to examine whether a large number of oligo DNAs can be assembled using an APP-based technique, an artificial gene (Luc) of 2000 bases containing the luciferase gene was constructed.

[0192] For the 2000-base Luc, 42 types of oligo DNA sequences of 91 to 100 bases were designed according to Example 1. Tables 17 to 19 show the designed oligo DNA sequences (IDT, oPool, desalting grade).

[0193]

[0194]

[0195]

[0196] The pUP-3 fragment (2.3 kb) used in Example 9 and the 42 types of IDT oPools listed in Tables 17 to 19 were subjected to assembly reactions and PCR amplification reactions in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. The DNA was diluted to a concentration of 100 pM each in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ , 0.05% Tween 20). ENO was added at concentrations of 0 nM, 36 nM, 120 nM, 360 nM, or 1,200 nM.

[0197] A schematic diagram of the accumulation is shown in Figure 15A, and the staining results are shown in Figure 15B. As shown in Figure 15B, when 120 nM or 360 nM ENO was added, a band of pUP-Luc was confirmed as the amplification product.

[0198] [Example 12] Gene synthesis using only oligo DNA It was examined whether the 2400-base plasmid pOligoCP could be constructed using only oligo DNA without using the oriC vector.

[0199] The 24 types of 200-base oligo DNAs of Example 1 were mixed at a total concentration of 0.02 nM, 0.2 nM, or 2 nM (the concentration of each oligo DNA was almost the same) in an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) ２ CK was added at concentrations of 0 nM, 23.2 nM, 69.6 nM, or 232 nM.

[0200] Next, the oligo DNA was assembled and circularized by heat treatment at 75°C for 5 minutes and slow cooling (0.1°C / sec). Using 1 µL of the resulting reaction solution, a PCR amplification reaction similar to that in Example 3-2 (except that Tus was not added) was carried out, and a portion (1.0 µL) was analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.

[0201] A schematic diagram of the accumulation is shown in Figure 16A, and the staining results are shown in Figure 16B. As shown in Figure 16B, when oligo DNAs were used at a total concentration of 0.2 nM or 2 nM, pOligoCP could be amplified without adding CK. When oligo DNAs were used at a total concentration of 0.02 nM, pOligoCP could be constructed by adding 232 nM CK. This demonstrated that the oriC plasmid could be constructed by accumulation using only oligo DNAs, without using the oriC vector.

[0202] [Example 13] Simultaneous synthesis of multiple artificial genes An investigation was conducted into whether multiple genes could be simultaneously synthesized by carrying out an enrichment reaction and an amplification reaction in a single test tube using a single oligo pool.

[0203] For artificial genes of approximately 800 bases each, four genes encoding the chromoproteins GFP, efoRed, amilCP, and fwYellow (Liljeruhm et al. Journal of Biological Engineering (2018) 12:8), 64 types of oligo DNA sequences of 85 to 97 bases were designed according to Example 1. Tables 20 to 23 show the designed oligo DNA sequences (IDT, oPool, desalting grade).

[0204]

[0205]

[0206]

[0207]

[0208] The pUPKm3 fragment (3.2 kb) was prepared by PCR amplification using the pUPKm fragment (SEQ ID NO: 182) having oriC, a kanamycin resistance gene, pUCori, the σ70 promoter, and the rrnBT1 terminator as a template and primers 1 and 2 containing dU (see Example 3-1).

[0209]

[0210] Enrichment reaction and PCR amplification reaction were carried out in the same manner as in Example 3-2 for 1 nM pUPKm fragment (3.2 kb) and 64 types of IDT oPools with each DNA at a concentration of 250 pM, and the results were analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.

[0211] The DNA was lysed in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 10 mU / μL Thermolabile USER II Enzyme (NEB) and 1,200 nM CK. ２ , 0.05% Tween 20). Then, 0.01 μL of the PCR amplification product was used to transform E. coli DH5α strain by chemical method. The transformed E. coli was plated on an LB plate containing 50 μg / mL kanamycin and cultured at 30°C for two days. The number of colonies exhibiting color due to each gene was counted.

[0212] The staining results are shown in Figure 17. Four bands thought to contain pUPKm-CP were detected by the enrichment reaction and PCR reaction.

[0213] The results of introducing the amplified products into E. coli are shown in Figure 18 along with a schematic diagram. Colonies with different colors were obtained, with GFP at 33.3%, eforRed at 11.8%, amilCP at 9.3%, and fwYellow at 12.7%, demonstrating successful simultaneous synthesis of four genes. White colonies are believed to contain an error in one of the four genes.

[0214] It was demonstrated that multiple artificial genes can be synthesized simultaneously by performing a single enrichment and amplification reaction using a single oligo pool.

[0215] [Example 14] Introduction of the assembled product into Escherichia coli It was examined whether circular DNA constructed from APP and oligo DNA could be obtained by directly transforming Escherichia coli without in vitro DNA amplification.

[0216] The pUP-2 fragment (2.3 kb) and 16 types of oligo DNA used in Example 3-2 were mixed in an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 100 mU / μL Thermolabile USER II Enzyme (NEB) so that each DNA had a concentration of 15 nM. ２ , 0.05% Tween 20).

[0217] Next, a uracil removal reaction was carried out at 37°C for 30 minutes, followed by heat treatment at 75°C for 5 minutes and slow cooling (0.1°C / sec) to assemble and circularize the oligo DNA and single-stranded overhangs. Five microliters of the resulting reaction mixture was used to transform Escherichia coli DH5α by chemical transformation. The transformed E. coli was plated on an LB plate containing 50 μg / mL carbencillin and cultured overnight at 37°C.

[0218] The number of colonies that exhibited fluorescence due to the gfp gene was counted, and the ratio of these to the total number of colonies was calculated as GFP positive. The results are shown in Table 25.

[0219]

[0220] As shown in Table 25, when CK was not added, 19 colonies were obtained, of which 89.5% showed GFP fluorescence. On the other hand, when CK was added, a significant increase in the number of colonies was observed, and when CK was added at a concentration of 14 μM, the number of colonies increased 17.4 times compared to when CK was not added. Furthermore, when CK was added, 83.7% or more of the colonies showed GFP fluorescence.

[0221] These findings demonstrate that circular DNA constructed by accumulating oligo DNA can be obtained without an in vitro amplification reaction, and that the number of circular DNAs obtained by APP can be increased.

[0222] [Example 15] Creation of artificial genes by PCR amplification of assembled products We investigated whether synthetic genes could be obtained by PCR amplification after oligo-DNA assembly using APP.

[0223] The pUP-2 fragment (2.3 kb) and 16 types of oligo DNA used in Example 3-2 were subjected to assembly reaction using the same method as in Example 3-2. Each DNA was adjusted to a concentration of 10 pM using an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 10 mU / μL Thermolabile USER II Enzyme (NEB). ２ , 0.05% Tween 20). CK was used at concentrations of 0 nM, 36 nM, or 120 nM.

[0224] To repair nicks at the DNA integration site, 1 μL of the resulting reaction mixture was subjected to Supercoiling and Repair Reaction (SCR) according to the literature (Fujita et al., ACS Synthetic Biol. (2022) 11, 9, 3088-3099). Briefly, a 10× SCR mix containing 1 mg / mL BSA (Roche), 140 nM LigA, 170 nM PolI, 200 mU / μL ExoIII (Takara Bio), and 250 nM GyrAB (prepared as described in Example 3-2) was prepared. The SCR mix was diluted 10-fold using dilution buffer (dilution buffer: 10% glycerol, 20 mM Tris-HCl (pH 7.5), 0.1 mg / mL BSA, 8 mM DTT, 10 mM Mg(OAc) ２ , and 125 mM KOAc). 1 μL of the reaction mixture and the diluted SCR mix were added to a PCR tube, and the reaction was incubated at 37°C for 30 minutes using a ProFlex PCR system (Applied Biosystems) in a 10 μL reaction volume. The nick repair product was amplified by PCR using primers 5 and 6. 2.0 μL of the amplified product was analyzed by 1.0% agarose gel electrophoresis and ethidium bromide staining.

[0225]

[0226] The results are shown in Figure 19. When CK was added at concentrations of 36 nM and 120 nM, a band of approximately 1.1 kb was detected as a PCR amplification product. This demonstrates that a synthetic gene can be constructed from oligo DNA assembled using APP, even when a PCR-mediated amplification system is used.

[0227] Example 16: Preparation of Site-Saturated Library-1 (Example 16-1) We investigated whether a site-saturated library could be prepared by assembling oligo-DNA using APP using random oligo-DNA. Using two consecutive random oligo-DNAs (N=6) at two amino acid sequences that form the chromophore of amilCP, we evaluated whether mutants exhibiting various colors could be obtained (Liljeruhm et al. Journal of Biological Engineering (2018) 12:8). The random oligo-DNA sites were intentionally designed to allow for single-stranded gaps.

[0228] For the amilCP mutant (amilCP-N6), 16 types of oligo DNA sequences ranging from 85 to 95 bases were designed so that the regions other than the random oligo were similar to Example 1, and the random oligo site would have a single-stranded gap. Figure 20 shows the design of the random oligo site. Table 27 shows the designed oligo DNA sequences (IDT, oPool, desalted grade). The oligo DNA "amilCP-N6_05r" contains a random sequence.

[0229]

[0230] The pUPKm fragment (3.2 kb) used in Example 13 and 16 types of oligo DNA containing random oligo sites shown in Table 27 were subjected to assembly reaction and PCR amplification reaction in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. Each DNA was diluted to a concentration of 1 nM in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 10 mU / μL Thermolabile USER II Enzyme (NEB). ２ , 0.05% Tween 20). CK was added at a concentration of 0 nM (-) or 1,200 nM (+).

[0231] The staining results are shown in Figure 21. Even when CK was not added, a band of pUPKm-amilCP N6 was observed as an amplification product, but when CK was added, a larger amount of pUPKm-amilCP N6 was amplified.

[0232] Next, 0.01 μL of the PCR amplification product was used to transform E. coli DH5α strain by chemical transformation. The transformed E. coli was plated on an LB plate containing 50 μg / mL kanamycin and cultured at 30°C for two days. Figure 22 shows a photograph and a schematic diagram of the plate after culture. Colonies exhibiting the color derived from each gene were also counted.

[0233] The colony count showed that 6.4% of the 801 colonies obtained were colored. Furthermore, when the colored colonies were inoculated onto a new plate and cultured, approximately 10 different colored colonies were visually confirmed (Figure 23). In Figure 23, 1, 12, 30 = dark blue; 2, 3, 10, 28 = dark purple; 4, 14, 31 = light blue; 5, 8, 24, 26, 32 = light purple; 6, 7, 25, 27 = purple; 9 = white purple; 13, 21, 22 = pink; 15 = light brown purple; 11 = gray; 23 = light yellow; 29 = dark yellow). It was believed that the enrichment method using APP and random oligo DNA could be used to form a library.

[0234] Example 16-2 The colonies that developed color in Example 16-1 were amplified by colony PCR using the primers shown in Table 28, and the products were subjected to sequence analysis by the Sanger method.

[0235]

[0236] Table 29 shows the results of sequencing 27 of the colored colonies.

[0237]

[0238] As shown in Table 29, 19 variants were obtained, 18 of which were novel. The remaining 93.6% were white colonies, and their sequences were combinations of amino acids not involved in color development, or sequences containing stop codons or frameshift mutations due to deletions.

[0239] (Example 16-3) The PCR products obtained in Example 16-1 were subjected to NGS analysis to perform UMI counting. Library preparation was performed using the NEBNext Ultra II FS DNA Library Prep Kit (NEB). Indexing during library preparation was performed by PCR, using the following primer combinations. NGS analysis was performed using an Illumina iSeq 100.

[0240]

[0241] As a result, as shown in Table 31, 1,357 types of UMIs were detected in the absence of CK, while 1,812 types of UMIs were detected in the presence of CK, indicating that the addition of APP increases the number of accumulated DNA molecules.

[0242]

[0243] The results of Example 16 demonstrated that a site-saturated library can be produced by this enrichment method.

[0244] Example 17 Effect of Tween 20 and Its Influence on Accumulation To suppress the adsorption of oligo-DNA to the tube wall, the effect of Tween 20 and its influence on accumulation were investigated.

[0245] (Example 17-1) For one type of 100-base oligo DNA (SEQ ID NO: 205) fluorescently labeled with Cy5, an assembly reaction was carried out at 75°C for 2 minutes in the same manner as in Example 3-2. The DNA was added at a concentration of 20 nM in an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 0% or 0.05% Tween 20. ２ ) was added.

[0246]

[0247] 5.0 μL of the reaction solution was subjected to electrophoresis on a 6% polyacrylamide gel, and Cy5 was detected. The results are shown in Figure 24. As shown in Figure 24, the addition of Tween 20 resulted in a stronger detection of the oligo-DNA band. It was thought that without the addition of Tween 20, the oligo-DNA was lost during the procedures leading up to electrophoresis.

[0248] (Example 17-2) The solution was removed from the tube in which the enrichment reaction was carried out in Example 17-1, and Cy5 detection was carried out. For detection, a Typhoon FLA9500 (manufactured by GE Healthcare) was used. The results are shown in Figure 25.

[0249] 25, when Tween 20 was not added, Cy5 was strongly detected from the tube, whereas when Tween 20 was added, Cy5 was hardly detected from the tube. It was thought that oligo-DNA was easily adsorbed to the tube, and that this adsorption was suppressed by adding Tween 20.

[0250] (Example 17-3) The 23 types of 200-base oligo DNA used in Example 1 were assembled and linearized, and analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining. Each oligo DNA was added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) to a concentration of 0.87 nM (total 20 nM, 1.3 ng / μL). ２ CK was added at a concentration of 0 nM or 2,300 nM, and Tween 20 was added at a concentration of 0% or 200 mM. Ligation and linearization of the oligo DNA was carried out by heat treatment at 75°C for 5 minutes.

[0251] The results are shown in Figure 26. As shown in Figure 26, the addition of Tween 20 in the absence of CK resulted in an upshift of the band. In the presence of CK, the addition of Tween 20 promoted the accumulation effect. On the other hand, a CK-dependent accumulation effect was observed even in the absence of Tween 20.

[0252] From the results of Examples 17-1 to 17-3, it was considered that Tween 20 acts to promote the accumulation of oligo-DNA by suppressing the adsorption of oligo-DNA to the tube wall. On the other hand, the APP-dependent accumulation effect was obtained even without the addition of Tween 20.

[0253] Example 18 Simultaneous Synthesis of Four Genes in Combination with a Mismatch Repair Enzyme Group As in Example 13, multiple genes were simultaneously accumulated in a single test tube using a single oligo pool and amplified by PCR. We investigated whether the accuracy of the genes synthesized by this accumulation method could be improved by combining them with a mismatch repair enzyme group.

[0254] The OriC7.0 fragment (3.0 kb) was used instead of the pUPKm fragment (3.2 kb) in Example 13. Enrichment reactions and PCR amplification reactions were carried out in the same manner as in Example 13 for 1 nM of the OriC7.0 fragment (3.0 kb) and 64 types of IDT oPools, each containing a DNA concentration of 250 pM.

[0255] When carrying out the PCR amplification reaction, 150 nM MutS and 150 nM MutL (Mut_SL), or 150 nM MutS, 150 nM MutL, and 25 nM MutH (Mut_SLH) were added as mismatch repair-related enzymes. MutS, MutL, and MutH are all derived from Escherichia coli, and were each mass-expressed in Escherichia coli as an N-terminal histidine tag fusion using a known sequence, followed by purification and preparation using a process including affinity column chromatography. The molar concentrations are expressed as values ​​for the monomer.

[0256] As a control, PCR amplification reactions were also performed without adding the mismatch repair-related enzymes. Each reaction was analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.

[0257] Thereafter, Escherichia coli DH5α strain was transformed in the same manner as in Example 13, except that 0.01 μL of the PCR amplification product was used during transformation. The transformed Escherichia coli was plated and cultured, and then the number of colonies exhibiting the color derived from each gene was counted.

[0258] The staining results are shown in Figure 27A. Bands thought to contain four types of OriC7.0-CP were detected by the enrichment reaction and PCR reaction. Three independent PCR amplification reactions were performed on three samples.

[0259] The amplification products were introduced into E. coli, and the percentage of each colony that grew on an LB plate is shown in Figure 27B. Note that PCR amplification reactions were performed independently three times for three samples, and the average values ​​and standard deviations are shown in Figure 27B.

[0260] As mentioned in Example 13, white colonies are thought to contain errors in one of the four genes. As shown in Figure 27B, the addition of MutS and MutL reduced the white colonies from 22.1% (None) to 9.7%, and the addition of MutH further reduced it to 5.0%. Although the oligo pool contains a certain number of sequence errors, in this enrichment reaction, the sequence errors were eliminated by combining it with a group of mismatch repair enzymes during amplification by PCR, thereby improving the accuracy of the artificial gene.

[0261] The OriC7.0 fragment was prepared by PCR amplification using primers 1 and 2 containing dU (see Example 3-1) in the same manner as the pUPKm fragment, except that in the pUPKm fragment (3.2 kb) used in Example 13, the oriC portion was replaced with oriC5.6AT (SEQ ID NO: 206), the pUCori portion was replaced with p15A, and a sequence in which Mu-SGS (SEQ ID NO: 207) was incorporated was used as a template.

[0262] oriC5.6AT (SEQ ID NO: 206) is shown in Table 33. In Table 33, the bolded part is the DUE part, and the boxed part in the bolded part is the mutated part in the mutant DUE, which has been mutated to a DUE with a high AT content.

[0263]

[0264]

[0265] [Example 19] Preparation of Site-Saturation Library-2 (Example 19-1) As in Example 16, we investigated whether a site-saturation library could be prepared using random oligo-DNA. We evaluated whether mutants exhibiting various colors could be obtained by using random oligo-DNA (N=6) at two amino acid sequences that form the chromophore of amilCP (Liljeruhm et al. Journal of Biological Engineering (2018) 12:8). Unlike Example 16, the random oligo-DNA sites were designed to prevent single-strand gaps.

[0266] For the amilCP mutant (amilCP-N6), 14 types of oligo DNA sequences ranging from 60 to 103 bases were designed for the regions other than the random oligos according to Example 1. Figure 28A shows a design diagram of the random oligo site. Table 35 shows the designed oligo DNA sequences (IDT, oPools oligo pool (IDT oPools), desalted grade). The oligo DNAs "amilCP-N6_04f" and "amilCP-N6_05r" contain random sequences.

[0267]

[0268] The OriC7.0-2 fragment (3.0 kb) and the 14 types of IDT oPools shown in Table 35 were subjected to assembly reactions and PCR amplification reactions in the same manner as in Example 3-2, and analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining. The DNA concentrations were adjusted to 10 pM for the OriC7.0-2 fragment and 30 pM for each of the IDT oPools oligo DNAs in assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc)) containing 20 mU / μL Thermolabile USER II Enzyme (NEB). ２ , 0.05% Tween 20). CK was added at a concentration of 120 nM.

[0269] The OriC7.0-2 fragment (3.0 kb) was prepared by PCR amplification using the OriC7.0 fragment as a template, which had oriC5.6AT (SEQ ID NO: 206) and Mu-SGS (SEQ ID NO: 207) instead of oriC in the pUPKm fragment (3.2 kb) used in Example 13 and had p15A as the plasmid replication origin, with the following primers containing dU:

[0270]

[0271] The staining results are shown in Figure 28B. A band thought to be OriC7.0-amilCP-N6 was detected as an amplification product from the enrichment reaction and PCR reaction. Two independent PCR amplification reactions were performed on one enrichment reaction sample.

[0272] (Example 19-2) Next, 0.003 μL of the PCR amplification product was used to transform E. coli DH5α strain by chemical transformation. The transformed E. coli was plated on an LB plate containing 50 μg / mL kanamycin and cultured at 30°C for two days, followed by three days at 4°C. Colonies exhibiting the color associated with each gene were counted. Furthermore, colored colonies were inoculated onto a new plate and cultured. Approximately 10 colored colonies were visually confirmed (Figure 29; in Figure 29: 1 = light blue, 2 = dark blue, 3 = dark purple, 4 = purple, 5 = light purple, 6 = whitish purple, 7 = light brownish purple, 8 = dark pink, 9 = pink, 10 = red, 11 = dark yellow, 12 = light yellow). It was believed that a library had been formed. The colored colonies were amplified by colony PCR using the primers listed in the table below, and the resulting products were sequenced by the Sanger method.

[0273]

[0274] As a result, 29.4% (#1) and 29.0% (#2) of the total colonies obtained were colored. The results of sequencing 12 of the colored colonies are shown in Table 35. As shown in Table 35, 12 variants were obtained, 11 of which were novel.

[0275]

[0276] (Example 19-3) NGS analysis was performed on the PCR products obtained in Example 19-1 to perform UMI counting. The NEBNext Ultra II FS DNA Library Prep Kit (NEB) was used for library preparation. Indexing during library preparation was performed by PCR, and the following primers were used in combination. Illumina's iSeq 100 was used for NGS analysis. #1: Primers H702-R and H501-F #2: Primers H703-R and H501-F

[0277]

[0278] As a result, 3,676 types (#1) or 3,618 types (#2) of UMIs were detected in each sample, an increase in the number of UMIs detected compared to Example 16. Even when the random oligo DNA design method was changed, a site-saturation library could be created.

Claims

1. A method for producing double-stranded DNA comprising: (1) preparing a set of two or more types of single-stranded DNA fragments, at least a portion of each of which has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of one of the strands of the double-stranded DNA; (2) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments and a cationic salt or a cationic substance; and (3) incubating the reaction solution at an incubation temperature higher than 40°C, thereby accumulating the set of single-stranded DNA fragments into double-stranded DNA.

2. The method according to claim 1, wherein the reaction solution further comprises an accumulation-promoting polypeptide, the accumulation-promoting polypeptide being: (i) a polypeptide that forms aggregates when the accumulation-promoting polypeptide and any DNA fragment are heated in solution to a temperature higher than 40°C, and (ii) a polypeptide that reduces the amount of aggregates formed when the accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in solution to a temperature higher than 40°C, compared to when the accumulation-promoting polypeptide does not contain arginine.

3. The method of claim 2, wherein the concentration of each single-stranded DNA fragment in the reaction solution is 1 nM or less and / or the concentration of the total single-stranded DNA fragments is 1 μM or less.

4. The method of claim 2, wherein the accumulation-promoting polypeptide is selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having RecA family recombinase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and the ε subunit of F1-ATPase.

5. The method according to claim 4, wherein the accumulation-promoting polypeptide is selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having ATP-type DNA ligase activity, a polypeptide having Escherichia coli-derived DNA helicase activity, and a polypeptide having RecA family recombinase activity.

6. The method of claim 1 or 2, wherein the cationic salt or cationic substance is selected from the group consisting of divalent cationic salts, monovalent cationic salts, and polyamines.

7. The method of claim 1 or 2, wherein the cationic salt or cationic substance is selected from the group consisting of magnesium salts, calcium salts, sodium salts, potassium salts, ammonium salts, putrescine, spermidine, and spermine.

8. The method according to claim 1 or 2, wherein at least one of the single-stranded DNA fragments is obtained by chemical synthesis and / or the set of single-stranded DNA fragments is an oligo pool.

9. The method according to claim 1 or 2, comprising, prior to (2), non-exponentially amplifying two or more types of single-stranded DNA fragments contained in the set using a DNA polymerase, or amplifying two or more types of single-stranded DNA fragments contained in the set by a PCR method to obtain two or more types of double-stranded DNA, and thermally dissociating the two or more types of double-stranded DNA to obtain two or more types of single-stranded DNA.

10. The method according to claim 1 or 2, wherein the reaction solution contains a set of single-stranded DNAs corresponding to multiple types of double-stranded DNAs, and multiple types of double-stranded DNAs are produced.

11. A method according to claim 1 or 2, wherein the double-stranded DNA produced is a supercoiled double-stranded DNA, the method further comprising: (4) if the double-stranded DNA obtained in (3) is not a circular double-stranded DNA, circularizing the obtained double-stranded DNA to obtain a circular double-stranded DNA; (5) forming a mixture for supercoiling reaction (SCR) containing the circular double-stranded DNA and the following: an enzyme having exonuclease III activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, and an enzyme or enzymes having DNA gyrase activity; and (6) incubating the mixture for SCR, thereby supercoiling the circular double-stranded DNA in the mixture.

12. The method of claim 1 or 2, further comprising subjecting the double-stranded DNA to a double-stranded DNA amplification reaction.

13. The method according to claim 1 or 2, wherein the double-stranded DNA is a circular double-stranded DNA having a replication origin sequence capable of binding to an enzyme having DnaA activity, and the method comprises subjecting the circular double-stranded DNA to a double-stranded DNA amplification reaction by a replication cycle reaction (RCR) method, or the method comprises inserting a replication origin sequence capable of binding to an enzyme having DnaA activity into the double-stranded DNA to obtain a circular double-stranded DNA having the replication origin sequence, and subjecting the circular double-stranded DNA to a double-stranded DNA amplification reaction by the RCR method.

14. The method of claim 12 or 13, wherein the double-stranded DNA is circular and the double-stranded DNA amplification reaction is carried out in the presence of MutS and MutL, or in the presence of MutS and MutL and an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease.

15. The method according to claim 12 or 13, comprising acting MutS and MutL on the double-stranded DNA prior to the double-stranded DNA amplification reaction, or acting MutS and MutL and an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and a single-strand-specific exonuclease on the double-stranded DNA.

16. A method for producing a double-stranded DNA library, comprising: (1) preparing a set of two or more types of single-stranded DNA fragments, at least a portion of each of which has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of either strand of the double-stranded DNA; (1') preparing two or more types of library-constituting single-stranded DNA fragments, each of which has a region corresponding to a portion of either strand of the double-stranded DNA and a region non-complementary to any of the single-stranded DNA fragments contained in the set of single-stranded DNA fragments in (1), and the base sequences of the non-complementary regions are different from each other; (2) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments, the two or more types of library-constituting single-stranded DNA fragments, a cationic salt or cationic substance, and an accumulation-promoting polypeptide; and (3) incubating the reaction solution at an incubation temperature of higher than 40°C, thereby accumulating the set of single-stranded DNA fragments and two or more of the single-stranded DNA fragments constituting the library into a double-stranded DNA library, wherein the accumulation-promoting polypeptide is: (i) a polypeptide that forms aggregates when the accumulation-promoting polypeptide and any DNA fragment are heated in a solution at a temperature of higher than 40°C, and (ii) a polypeptide that forms a reduced amount of aggregates when the accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in a solution at a temperature of higher than 40°C, compared to when arginine is not contained.

17. A method for producing double-stranded DNA, comprising: (a) heating a solution containing any polypeptide and any number of single-stranded DNA fragments to a temperature higher than 40°C, wherein the polypeptide and at least one of the single-stranded DNA fragments in the solution form an aggregate, and selecting the polypeptide that formed the aggregate; (b) preparing a set of single-stranded DNA fragments, wherein at least a portion of each single-stranded DNA fragment has a sequence complementary to at least one other single-stranded DNA fragment in the set, and each single-stranded DNA fragment corresponds to a portion of one of the strands of the double-stranded DNA; (c) preparing a reaction solution containing the set of single-stranded DNA fragments, a cationic salt or cationic substance, and the polypeptide selected in (a); and (d) incubating the reaction solution at an incubation temperature higher than 40°C, thereby accumulating the set of single-stranded DNA fragments into double-stranded DNA.

18. A kit for producing circular double-stranded DNA, comprising: an enzyme having exonuclease III activity, an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, an enzyme or enzymes having DNA gyrase activity, a cationic salt or cationic substance, and an accumulation-promoting polypeptide selected from the group consisting of: a polypeptide having creatine kinase activity, a polypeptide having phosphopyruvate hydratase activity, a polypeptide having pyruvate kinase activity, a polypeptide having 3-phosphoglycerate kinase activity, a polypeptide having DNA ligase activity, a polypeptide having DNA helicase activity, a polypeptide having RecA family recombinase activity, a polypeptide having polynucleotide kinase activity, a histone-like DNA binding protein, a chromosome replication initiator protein, a polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and an ε subunit of F1-ATPase.

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