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WO2025028566A1 - Procédé de production d'adn double brin - Google Patents

Procédé de production d'adn double brin Download PDF

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Publication number
WO2025028566A1
WO2025028566A1 PCT/JP2024/027365 JP2024027365W WO2025028566A1 WO 2025028566 A1 WO2025028566 A1 WO 2025028566A1 JP 2024027365 W JP2024027365 W JP 2024027365W WO 2025028566 A1 WO2025028566 A1 WO 2025028566A1
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Prior art keywords
stranded dna
polypeptide
double
activity
dna
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Japanese (ja)
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正幸 末次
角　悟
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Moderna Enzymatics Co Ltd
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Moderna Enzymatics Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to a method for obtaining linear or circular double-stranded DNA by accumulating multiple single-stranded DNA fragments at extremely low concentrations in a cell-free system at regions having complementary base sequences, and a method for amplifying the obtained DNA.
  • the synthesis of artificial genes is achieved by annealing the homologous regions of multiple chemically produced oligo-DNAs (single-stranded DNA) to generate double-stranded DNA.
  • Multiple linear single-stranded DNA fragments are assembled (assembled) by binding their complementary regions together to produce linear or circular double-stranded DNA, making it possible to obtain longer double-stranded DNA that is difficult to synthesize chemically.
  • Non-Patent Document 1 discloses a method in which 28 types of single-stranded DNA fragments (each 57-60 bases long) with complementary strands are prepared at approximately 140 nM each, for a total of 4.0 ⁇ M, and then accumulated by slow thermal annealing at 96°C to 30°C for 2 hours to obtain a linear double-stranded DNA of 836 base pairs.
  • Non-Patent Document 2 discloses a method in which 2.2 ⁇ M of each of nine overlapping single-stranded DNA fragments (mainly 58 bases long each) 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.
  • LCR ligase chain reaction
  • Non-Patent Document 3 discloses that 56 types of single-stranded DNA fragments (each 40 bases long) with complementary strands were prepared at 22 nM each, for a total of 2.5 ⁇ M, 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) with complementary strands were prepared at 7.5 nM each, for a total of 1 ⁇ M, and the fragments were linked using the PCA method, followed by repeated PCR reactions to obtain double-stranded DNA.
  • PCA polymerase cycling assembly
  • Non-Patent Document 4 discloses that a total of 15 ⁇ M of 48 types of single-stranded DNA fragments (each 50 bases long) with complementary strands were prepared (approximately 0.3 ⁇ M each), and after annealing and linking under stringent conditions using the circular assembly amplification method, error removal was performed using an exonuclease and an endonuclease to obtain circular double-stranded DNA, which was then cut into linear form and amplified by a PCR reaction.
  • Patent Document 1 discloses a method for efficiently linking multiple nucleic acid fragments, each at a concentration of about 1 nM, isothermally using RecA and, if necessary, a linear double-stranded DNA-specific exonuclease.
  • Patent Document 2 discloses a method for producing double-stranded DNA by duplex asymmetric PCR (DA-PCR), comprising the steps of: (1) 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 to prepare a reaction mixture; (2) performing PCR using the reaction mixture of step (1); (3) adding a primer set capable of amplifying the full length of the double-stranded DNA fragment to the reaction mixture of step (2); and (4) performing PCR using the reaction mixture of step (3).
  • DA-PCR duplex asymmetric PCR
  • Patent document 3 discloses a method for assembling a DNA molecule having a desired sequence, in which seven overlapping pools of oligo DNA (14 pieces x 8 mer) with 4 bp overlaps are linked using DNA ligase to form seven pools of 60 mer DNA, and then these seven pools are placed into one pool, and the 240 bp product is assembled by polymerase chain assembly reaction.
  • Non-Patent Document 5 a method for obtaining double-stranded DNA by accumulating a set of low-concentration single-stranded DNA fragments such as oligo pools.
  • a set of barcoded beads is used, and each bead recognizes the single-stranded DNA fragment required for accumulating a specific gene, and accumulation proceeds in separate water droplets, allowing multiple types of double-stranded DNA to be obtained simultaneously.
  • this method focuses on obtaining multiple types of double-stranded DNA such as a library, and also involves many steps.
  • the main objective of the present invention is to provide a method for producing linear or circular double-stranded DNA by assembling multiple single-stranded DNA fragments with high precision.
  • the inventors have found that accumulation efficiency of single-stranded DNA fragments can be increased by carrying out accumulation of single-stranded DNA fragments in the presence of a cationic salt or a cationic substance. They have also found that when accumulating low concentrations of single-stranded DNA fragments, the accumulation of single-stranded DNA can be promoted and double-stranded DNA can be obtained by further adding a polypeptide having specific properties, thus completing the present invention.
  • a method for producing double-stranded DNA comprising the steps of: (1) preparing a set of two or more 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; (2) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments and a cationic salt or a cationic substance; (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;
  • a method for producing double-stranded DNA comprising: [2] The reaction solution further comprises an accumulation-promoting polypeptide, the accumulation-promoting polypeptide being: (i) the accumulation-promoting polypeptide
  • the accumulation-promoting polypeptide 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, histone-like DNA binding proteins, Chromosome replication initiator protein, A polypeptide having adenylate kinase activity, a polypeptide having nucleoside diphosphate kinase activity, and the ⁇
  • SCR supercoiling reaction
  • a method comprising: [12] The method according to any one of [1] to [11] above, further comprising subjecting the double-stranded DNA to a double-stranded DNA amplification reaction.
  • 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 circular double-stranded DNA is subjected to a double-stranded DNA amplification reaction by a replication cycle reaction (RCR) method.
  • RCR replication cycle reaction
  • the method includes 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 a PCR method.
  • the method according to any one of [1] to [11].
  • [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, MutL, and an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and single-strand-specific exonuclease to act on the double-stranded DNA prior to the double-stranded DNA amplification reaction.
  • a method for producing a double-stranded DNA library comprising the steps of: (1) preparing a set of two or more 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; (1') preparing two or more types of library-constituting single-stranded DNA fragments, each of which has a region corresponding to a part of one 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 of (1), the base sequences of the non-complementary regions being 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 constituent single-stranded DNA fragments, a
  • the accumulation-promoting polypeptide comprises: (i) the accumulation-promoting polypeptide is a polypeptide that forms an aggregate when the accumulation-promoting polypeptide and any DNA fragment are heated in a solution at a temperature higher than 40° C.; and (ii) The method, wherein the accumulation-promoting polypeptide, any DNA fragment, and arginine form a reduced amount of aggregates when the accumulation-promoting polypeptide, any DNA fragment, and arginine are heated in a solution at a temperature higher than 40°C, compared to when the accumulation-promoting polypeptide does not contain arginine.
  • multiple single-stranded DNA fragments can be assembled with high precision to produce linear or circular double-stranded DNA.
  • FIG. 1A A schematic diagram of accumulation in Example 1 is shown in FIG. 1A, and the staining results are shown in FIG. 1B.
  • a schematic diagram of the accumulation in Example 3-1 is shown in FIG. 2A, and the staining results are shown in FIG. 2B.
  • a schematic diagram of the accumulation in Example 3-2 is shown in FIG. 3A, and the staining results are shown in FIG. 3B.
  • a schematic diagram of accumulation in Example 4 is shown in FIG. 4A, and the staining results are shown in FIG. 4B.
  • the results of Example 5-1 are shown below.
  • the results of Example 5-2 are shown below.
  • the results of Example 6-1 are shown below.
  • the results of Example 6-2 are shown below.
  • the results of Example 7-1 are shown below.
  • the results of Example 7-2 are shown below.
  • a schematic diagram of the accumulation in Example 8-1 is shown in FIG. 11A, and the staining results are shown in FIG. 12B.
  • the results of Example 8-2 are shown below.
  • a schematic diagram of the accumulation in Example 9 is shown in FIG. 13A, and the staining results are shown in FIG. 13B.
  • a schematic diagram of the accumulation in Example 10 is shown in FIG. 14A, and the staining results are shown in FIG. 14B.
  • a schematic diagram of accumulation in Example 11 is shown in FIG. 15A, and the staining results are shown in FIG. 15B.
  • a schematic diagram of accumulation in Example 12 is shown in FIG. 16A, and the staining results are shown in FIG. 16B.
  • 1 shows the staining results in Example 13. 1 shows the results of introducing the amplification product into E.
  • Example 15 shows a design diagram of a random oligo site in Example 16.
  • the staining results of Example 16 are shown. 1 shows a photograph of a plate after culturing transformed Escherichia coli in Example 16. 1 shows the results of further culturing the colored colonies in Example 16.
  • the staining results in Example 17-1 are shown. 1 shows the results of Cy5 detection from tubes in Example 17-2. 1 shows the staining results after accumulation in Example 17-3.
  • Fig. 27A shows the staining results in Example 18.
  • Fig. 27B shows the percentages of colonies that exhibited the colors derived from each gene in Example 18.
  • a schematic diagram of accumulation in Example 19 is shown in FIG. 28A, and the staining results are shown in FIG. 28B. 13 shows the results of further culturing the colored colonies in Example 19.
  • the method for producing double-stranded DNA of this embodiment includes preparing a set of two or more single-stranded DNA fragments (a set including 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 obtained double-stranded DNA may be linear or circular, and its terminal and/or non-terminal regions may be single-stranded. According to the method for producing double-stranded DNA of this embodiment, even double-stranded DNA having 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, can be produced.
  • the set of single-stranded DNA fragments does not have to correspond to all base sequences of the two strands of double-stranded DNA. It may be a set that, when the single-stranded fragments in the set are accumulated, gives double-stranded DNA with gaps rather than continuous double-stranded DNA.
  • the number of single-stranded DNA fragments 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.
  • the single-stranded DNA fragments can be prepared using known methods such as enzymatic methods and chemical synthesis methods. For example, methods by Ansa Biotechnologies, DNA Script, and the like are known as methods for performing DNA synthesis using enzymatic methods.
  • 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
  • the method of chemical synthesis is not particularly limited, and examples thereof include the method described in Hoose et al., Nature Reviews Chemistry (2023) Vol. 7, pp. 144-161, and the like.
  • a method is used in which desired ones of the four types of nucleosides are linked to the synthetic ends one by one on a solid phase such as a chip or beads, and then extended. In this way, multiple types of single-stranded DNA fragments can be chemically synthesized to prepare a set of single-stranded DNA fragments.
  • 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.
  • oligo pools are a collection of two or more, for example, 10 or more, 50 or more, particularly 100 or more, chemically synthesized DNA fragments.
  • Many services are known that provide the design and creation of oligo pools containing large amounts of single-stranded DNA fragments in a single tube, and such services are provided by, for example, Integrated DNA Technologies (IDT), GenScript, Twist Bioscience, and Agilent Technologies. Because oligo pools are created by pool synthesis, the price per base is inexpensive, and in one embodiment, double-stranded DNA can be inexpensively produced in a cell-free system by using such an oligo pool as a set of single-stranded DNA fragments.
  • IDT Integrated DNA Technologies
  • GenScript GenScript
  • Twist Bioscience Twist Bioscience
  • the length of the single-stranded DNA fragment is not particularly limited, but from the viewpoint of the effect on the Tm value and ease of accumulation at temperatures higher than 40° C., it is 10 bases or more, preferably 20 bases or more, more preferably 40 bases or more, and even more preferably 50 bases or more.
  • the upper limit is also not particularly limited, but it can be, for example, 1,000 bases or less, 500 bases or less, etc.
  • the single-stranded DNA fragment is a chemically synthesized single-stranded DNA fragment, it is not particularly limited as long as it can be chemically synthesized, and it 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 in length.
  • two or more types of single-stranded DNA fragments contained in the set may be amplified by a method including non-exponential amplification using a DNA polymerase.
  • a non-exponential amplification reaction can be performed by repeatedly adding a primer to the single-stranded DNA fragments bound to the microarray, synthesizing oligonucleotides with a DNA polymerase, and 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.).
  • two or more types of single-stranded DNA fragments contained in the set may be amplified by a method including amplifying the two or more types of double-stranded DNA 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. This allows for more accurate accumulation by increasing the concentration of each fragment even when the concentration of single-stranded DNA fragments is very low.
  • PCR method and thermal dissociation techniques are known to those skilled in the art, and the PCR method can be carried out by designing primers and repeating cycles of thermal denaturation, annealing, and extension reaction. Although it depends on the type of single-stranded DNA, thermal dissociation can usually be carried out at 95 to 100°C.
  • the method for producing double-stranded DNA in 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.
  • cationic substance refers to a substance that is positively charged when dissolved in water, other than a cationic salt.
  • the cationic salt or cationic substance is not particularly limited as long as it provides cations in the accumulation reaction liquid, but is preferably one or more types selected from the group consisting of divalent cationic salts, monovalent cationic salts, and polyamines.
  • the cationic salt or cationic substance is selected from the group consisting of magnesium salts such as magnesium acetate [Mg(OAc) 2 ], magnesium chloride [MgCl 2 ], magnesium sulfate [MgSO 4 ], calcium salts such as calcium chloride, calcium sulfate, calcium carbonate, potassium salts such as potassium glutamate [KGlu], potassium aspartate, potassium chloride, potassium acetate [KOAc], sodium glutamate, sodium aspartate, sodium chloride, sodium acetate, ammonium salts such as ammonium chloride, ammonium acetate, polyamines such as putrescine, spermidine, spermine, cationic surfactants such as CTAB (cetyltrimethylammonium bromide), and more preferably, the cationic salt is selected from magnesium salt, potassium salt, sodium salt, and ammonium salt, and the cationic substance is selected from polyamines and cationic surfactants. Particularly preferably, the cationic salt is selected from
  • the concentration of the cationic salt or cationic substance is not particularly limited as long as the accumulation reaction proceeds, and can be changed 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 in the accumulation reaction liquid preferably at a concentration of 0.1 mM to 50 mM, more preferably 0.5 mM to 20 mM, and particularly preferably 1 mM to 20 mM.
  • a monovalent cationic salt it can be added in the accumulation reaction liquid preferably at a concentration of 5 mM to 200 mM, more preferably 10 mM to 150 mM, and particularly preferably 30 mM to 100 mM.
  • a cationic substance e.g., polyamine
  • 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 ⁇ M, for example, the lower limit can be 1 fM or more, 10 fM or more, or 100 pM or more, and the upper limit can be 75 ⁇ M or less, 50 ⁇ M or less, or 25 ⁇ M or less.
  • the concentration of each single-stranded DNA fragment can be preferably 1 fM to 75 ⁇ M, 10 fM to 50 ⁇ M, 0.1 pM to 25 ⁇ M, preferably 1 pM to 1 ⁇ M, more preferably 10 pM to 50 nM, and/or
  • the total concentration of single-stranded DNA fragments in the enrichment reaction solution can be set to 0.1 pM to 10 mM, preferably 1 pM to 100 ⁇ M, and more preferably 10 pM to 5 ⁇ M.
  • 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.
  • 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.
  • the concentration of each single-stranded DNA fragment in the reaction solution for accumulation 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 types of single-stranded DNA fragments in the enrichment 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 ⁇ M or less, 1 ⁇ M 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, which makes it possible to efficiently produce double-stranded DNA by accumulating the single-stranded DNA fragments even when the amount of each single-stranded DNA fragment is small and/or when the total concentration of the single-stranded DNA fragments is low
  • 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 any DNA fragment are heated in 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 in which, when the accumulation-promoting polypeptide, any 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, the amount of aggregates formed is reduced compared to when arginine is not contained.
  • the heating temperature in (i) and (ii) above can be, for example, 100°C or less, 90°C or less, 80°C or less, or 75°C or less, and in one embodiment, can be preferably 45°C or more, more preferably 50°C or more, 55°C or more, 60°C or more, 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 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.
  • solutions in (i) and (ii) above can be prepared with reference to the Examples.
  • a solution in which the accumulation-promoting polypeptide and any DNA fragment have been removed from the accumulation reaction solution described below, or a buffer solution can be used.
  • the buffer solution there are no particular limitations on the buffer solution, so 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 solution is Tris-HCl or Tris-OAc.
  • the concentration of the buffer solution can be appropriately selected by a person skilled in the art and is not particularly limited, but in the case of Tris-HCl or Tris-OAc, a concentration of, for example, 10 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 20 mM to 30 mM can be selected.
  • the buffer solution may contain a component selected from the group consisting of a cationic salt or a cationic substance, a substance having a polymer crowding effect, and a small amount of a surfactant.
  • a component selected from the group consisting of a cationic salt or a cationic substance, a substance having a polymer crowding effect, and a small amount of a surfactant for the types and amounts of these components to be added, please refer to the description of the accumulation reaction solution described below.
  • Aggregates refer to a larger assembly formed by the association of dispersed accumulation-promoting polypeptide and any DNA fragment, and the formation of aggregates can be easily confirmed by centrifugation, as in the examples described below.
  • an accumulation-promoting polypeptide and any DNA fragment are heated in a solution at a temperature higher than 40°C and then centrifuged, aggregates are formed if, compared to a solution not heated, the amount of precipitation is greater; and/or more accumulation-promoting polypeptide is detected in the precipitate fraction after centrifugation.
  • any DNA fragment, and arginine are heated in a solution at 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 not containing arginine, the amount of aggregates formed is reduced.
  • Arginine may be added to the solution as arginine itself or as a salt of arginine. It can be added conveniently in the form of a salt, such as arginine hydrochloride, acetate, sulfate, lactate, phosphate, pyroglutamate, aspartate, maleate, and other known salts. Of these, it is preferable to use arginine hydrochloride from the standpoint of economy and ease of handling.
  • an accumulation-promoting polypeptide causes single-stranded DNA fragments in the accumulation reaction solution to form aggregates with the polypeptide, promoting accumulation into double-stranded DNA. Furthermore, by confirming that any polypeptide has the properties (i) and (ii) prior to the accumulation reaction, an accumulation-promoting polypeptide can be selected.
  • 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.
  • EC creatine kinase activity
  • 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 phosphoglycerate 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.
  • 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 F1-ATPase, for example selected from creatine kinase, phosphopyruvate hydratase, pyruvate kinase, and the epsilon subunit of F1-ATPase, for example selected from creatine
  • the accumulation-promoting polypeptide is 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 ATP-type (T4 phage-derived) DNA ligase activity, a polypeptide having E.
  • coli-derived DNA helicase activity and a polypeptide having RecA family recombinase activity, and is particularly preferably a polypeptide 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, for example, selected from phosphopyruvate kinase, pyruvate kinase, 3-phosphoglycerate kinase, and ATP-type (T4 phage-derived) DNA ligase.
  • the accumulation-promoting polypeptide is not a polypeptide having RecA family recombinase activity.
  • a polypeptide having a specific enzymatic activity may be a wild-type polypeptide, or a variant that retains the specific enzymatic activity by introducing a mutation into the wild-type polypeptide in which 1 to 30 amino acids are deleted, added, or substituted.
  • the concentration of the polypeptide in the accumulation 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.
  • the accumulation reaction solution may contain a set of single-stranded DNA corresponding to multiple types of double-stranded DNA. This makes it possible, for example, to accumulate and synthesize multiple types of genes at once from a set of single-stranded DNA 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, when 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.
  • 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.
  • a buffer solution there are no particular limitations on the buffer solution, so 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, Tricine-HCl, and the like.
  • a preferred buffer solution is Tris-HCl or Tris-OAc.
  • the concentration of the buffer solution can be appropriately selected by a person skilled in the art and is not particularly limited, but in the case of Tris-HCl or Tris-OAc, a concentration of, for example, 10 mM to 100 mM, preferably 10 mM to 50 mM, and more preferably 20 mM to 30 mM can be selected.
  • the accumulation reaction solution in this embodiment may further contain a substance having a polymer crowding effect.
  • the polymer crowding effect enhances the interaction between DNA molecules and can promote the linking 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).
  • PEG particularly PEG 8000, can be preferably used.
  • the concentration of the substance is not particularly limited as long as the substance has a DNA fragment linking promoting effect.
  • the concentration of PEG 8000 contained in the reaction solution in which the linking reaction is performed in this invention is preferably 2-20% by mass, more preferably 2-10% by mass, and even more preferably 4-6% by mass.
  • the accumulation reaction solution in which the accumulation reaction of this embodiment is carried out may further contain a small amount of a surfactant.
  • a surfactant a non-ionic surfactant is preferable, and for example, Tween 20 (0.02 to 0.5%, preferably 0.05 to 0.2%), Triton X-100 (0.05 to 1.0%, preferably 0.1 to 0.5%), NP-40 (0.02 to 1.0%, preferably 0.05 to 0.2%), Brij (trademark) i35 (0.02 to 0.5%, preferably 0.05 to 0.2%), dodecyl- ⁇ -D-maltoside (approximately 0.1 to 0.5%), octyl- ⁇ -D-glucoside (approximately 0.5 to 1.0%), etc. can be used.
  • Tween 20 can be preferably used. This prevents DNA fragments from adsorbing to the inner walls of the tube when the reaction is carried out in the tube, ensuring that they are involved in the accumulation reaction.
  • the accumulation reaction solution 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, at 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, 5 minutes to 60 minutes, etc. Whether the desired double-stranded DNA has been obtained can be confirmed by a known method, for example, gel electrophoresis.
  • the method of this embodiment may further include subjecting the double-stranded DNA obtained in the accumulation reaction to a double-stranded DNA amplification reaction.
  • the method of the amplification reaction 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.
  • Double-stranded DNA can be prepared appropriately depending on the cell used, and simply, circular double-stranded DNA having a replication origin can be prepared and introduced into E. coli for amplification.
  • HDA Helicase-dependent amplification
  • RPA Recombinase polymerase amplification
  • RCA Rolling circle amplification
  • 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.
  • the double-stranded DNA may be pretreated depending on the amplification method; for example, non-circular DNA may be circularized using a known method, and then the circular DNA may be amplified.
  • the method of this embodiment may also include carrying out a supercoiling and repair reaction (SCR) on the double-stranded DNA obtained in the accumulation reaction.
  • SCR supercoiling and repair 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 strong, and being easily separated by electrogel electrophoresis according to size.
  • 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 a circular double-stranded DNA, it is circularized by a known method to obtain a circular double-stranded DNA. Then, a mixture for SCR 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 circular double-stranded DNA in the mixture is supercoiled, and the supercoiling reaction can be carried out.
  • 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, for example 30 minutes or more
  • the circular double-stranded DNA in the mixture is supercoiled, and the supercoiling reaction can be carried out.
  • There is no particular upper limit to the incubation time but it can be, for example, 24 hours, 10 hours, 5 hours, 2 hours, 1 hour, etc.
  • each enzyme such as an enzyme having DNA polymerase I activity, an enzyme having DNA ligase activity, or an enzyme or group of enzymes having DNA gyrase activity
  • the description of the enzymes related to the RCR method described below can be referred to.
  • the enzyme having exonuclease III activity there is no particular limitation on its biological origin, so long as it is an enzyme that has the activity of degrading nucleotides in the 3' to 5' direction from linear or nicked double-stranded DNA, similar to exonuclease III of E. coli.
  • ExoIII derived from E. coli can be suitably used.
  • coli may be contained in the reaction solution in a range of 1 to 500 mU/ ⁇ L, preferably 2 to 400 mU/ ⁇ L, 3 to 300 mU/ ⁇ L, 5 to 200 mU/ ⁇ L, or 10 to 100 mU, but is not limited thereto.
  • 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.
  • 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.
  • the reaction solution contains DNA ligase derived from Escherichia coli, it may also contain NAD (nicotinamide adenine dinucleotide), which is a cofactor for the DNA ligase.
  • NAD nicotinamide adenine dinucleotide
  • a supercoiling reaction can be carried out prior to subjecting the double-stranded DNA to a double-stranded DNA amplification reaction.
  • the double-stranded DNA obtained in the accumulation reaction may be amplified using the Replication Cycle Reaction method (RCR method; see WO2016/080424, WO2017/199991, WO2018/159669).
  • the DNA amplified by the RCR method is a circular DNA containing a replication origin 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 origin sequence that can bind to an enzyme having DnaA activity, such a sequence can be linked or inserted to obtain a circular DNA to be amplified by the RCR method.
  • a replication origin sequence e.g., oriC
  • the replication origin sequence capable of binding to an enzyme having DnaA activity is, for example, a known replication origin sequence present in bacteria such as Escherichia coli and Bacillus subtilis, which can be obtained from a public database such as NCBI.
  • a replication origin sequence can be obtained by cloning a DNA fragment capable of binding to an enzyme having DnaA activity and analyzing its base sequence.
  • the replication origin sequence a sequence in which a mutation has been introduced to replace, delete, or insert one or more bases of a known replication origin sequence, and a modified sequence capable of binding to an enzyme having DnaA activity can also be used.
  • the replication origin sequence is preferably oriC and its modified sequence, and more preferably oriC derived from Escherichia coli and its modified sequence.
  • the replication initiation sequence may have a mutant double-stranded break region (DUE) that has a higher AT content compared to the wild type.
  • DUE double-stranded break region
  • the circular DNA after accumulation may contain a replication origin sequence capable of binding to an enzyme having DnaA activity and a strong gyrase-binding sequence (SGS).
  • SGS is a binding sequence to which DNA gyrase binds and introduces negative supercoils into 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 including this.
  • Mu-SGS SGS derived from bacteriophage Mu
  • the RCR method includes the following steps: (a) a first group of enzymes that catalyze the replication of circular DNA; preparing a reaction mixture containing a reaction solution containing a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and the circular DNA to be amplified; (b) incubating the reaction mixture prepared in step (a) at a constant temperature comprised between 15°C and 80°C or under temperature cycling comprising repeated incubation at two temperatures below 80°C.
  • the first group of enzymes that catalyze the replication of circular DNA for example, the group of enzymes described in Kaguni JM & Kornberg A. Cell. 1984, 38:183-90 can be used.
  • the first enzyme group include one or more enzymes or enzyme groups selected from the group consisting of an enzyme having DnaA activity, one or more types of nucleoid proteins, an enzyme or enzyme group having DNA gyrase activity, a single-stranded DNA 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 an enzyme or enzyme group having DNA polymerase III * activity, or all combinations of the enzymes or enzyme groups.
  • SSB single-stranded DNA binding protein
  • the first enzyme group includes an enzyme having DnaA activity, a single-stranded DNA 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 an enzyme or enzyme group having DNA polymerase III * activity.
  • SSB single-stranded DNA binding protein
  • DnaB-type helicase activity 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 an enzyme or enzyme group having DNA polymerase III * activity.
  • an initiator protein of E. coli As an enzyme having DnaA activity, there is no particular restriction on its biological origin, so long as it has the same initiator activity as DnaA, an initiator protein of E. coli.
  • DnaA derived from E. coli can be preferably used.
  • DnaA derived from E. coli may be contained in the reaction solution as a monomer in the range of 1 nM to 10 ⁇ M, preferably in the range of 1 nM to 5 ⁇ M, 1 nM to 3 ⁇ M, 1 nM to 1.5 ⁇ M, 1 nM to 1.0 ⁇ M, 1 nM to 500 nM, 10 nM to 200 nM, or 20 nM to 150 nM, but is not limited thereto.
  • 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 having the same activity as the nucleoid proteins of E. coli.
  • IHF derived from E. coli i.e., a complex (heterodimer or homodimer) of IhfA and/or IhfB, or HU derived from E. coli, i.e., a complex of hupA and hupB, can be preferably used.
  • coli may be contained in the reaction solution as a hetero/homo dimer 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 the range of 1 nM to 50 nM, preferably 5 nM to 50 nM, or 5 nM to 25 nM, but is not limited thereto.
  • the enzyme or group of enzymes having DNA gyrase activity there is no particular limitation on their biological origin, so long as they have an activity similar to that of E. coli DNA gyrase.
  • 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 as a heterotetramer in the reaction solution in a range of 20 nM to 500 nM, preferably in a range of 20 nM to 400 nM, 20 nM to 300 nM, or 20 nM to 200 nM, but is not limited thereto.
  • SSB single-stranded DNA binding protein
  • coli may be contained in the reaction solution as a homotetramer in a range of 20 nM to 1000 nM, preferably in a range of 20 nM to 500 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 500 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 50 nM to 150 nM, 100 nM to 500 nM, or 100 nM to 400 nM, but is not limited thereto.
  • DnaB-type helicase activity there is no particular limitation on its biological origin, so long as it has activity similar to that of DnaB from E. coli.
  • DnaB derived from E. coli can be preferably 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.
  • DnaC derived from Escherichia coli can be preferably used.
  • DnaC derived from Escherichia 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.
  • DnaG derived from E. coli can be preferably used.
  • DnaG derived from E. coli may be contained in the reaction solution as a monomer in the range of 20 nM to 1000 nM, preferably in the range of 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.
  • DnaN derived from E. coli can be preferably used.
  • DnaN derived from E. coli may be contained in the reaction solution as a homodimer in a range of 10 nM to 1000 nM, preferably in a range of 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.
  • the enzyme or enzyme group having DNA polymerase III * activity is not particularly limited in terms of its biological origin, as long as it has the same activity as the DNA polymerase III * complex of E. coli.
  • an enzyme group containing any of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE derived from E. coli preferably an enzyme group containing a complex of DnaX, HolA, HolB, and DnaE derived from E. coli, more preferably an enzyme group containing a complex of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE derived from E. coli, can be suitably used.
  • the DNA polymerase III* complex derived from E. coli may be contained in the reaction solution as a heteromultimer in a range of 2 nM to 50 nM, preferably in a range of 2 nM to 40 nM, 2 nM to 30 nM, or 2 nM to 20 nM, but is not limited thereto.
  • the two sister circular DNAs that form a catenane refer to two circular DNAs that are synthesized by a DNA replication reaction and are in a linked state.
  • the second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes can be, for example, one or more enzymes selected from the group consisting of enzymes with DNA polymerase I activity, enzymes with DNA ligase activity, and enzymes with RNase H activity, or a combination of such enzymes. In one embodiment, it preferably includes an enzyme with DNA polymerase I activity and an enzyme with DNA ligase activity.
  • E. coli-derived DNA polymerase I As an enzyme having DNA polymerase I activity, there is no particular limitation on its biological origin, so long as it has activity similar to that of E. coli DNA polymerase I.
  • 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.
  • E. coli-derived DNA ligase As an enzyme having DNA ligase activity, there is no particular limitation on its biological origin, so long as it has activity similar to that of E. coli DNA ligase.
  • E. coli-derived DNA ligase or T4 phage DNA ligase can be suitably used.
  • E. coli-derived DNA ligase may be contained in the reaction solution as a monomer in the 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.
  • RNase H derived from Escherichia coli can be suitably used.
  • RNase H derived from Escherichia coli may be contained in the reaction solution as a monomer in the 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.
  • the third enzyme group that catalyzes the separation reaction of the two sister circular DNAs for example, the enzyme group described in Peng H & Marians KJ. PNAS. 1993, 90: 8571-8575 can be used.
  • the third enzyme group can be 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.
  • it is preferable that the third enzyme group includes an enzyme having topoisomerase IV activity and/or an enzyme having topoisomerase III activity.
  • RecQ-type helicase activity there is no particular limitation on its biological origin, so long as it has activity similar to that of E. coli RecQ.
  • RecQ derived from E. coli can be suitably used.
  • RecQ derived from E. coli may be contained in the reaction solution as a monomer in the range of 20 nM to 500 nM, preferably in the range of 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.
  • topoisomerase IV activity there is no particular limitation on its biological origin, so long as it has activity similar to that of Escherichia coli topoisomerase IV.
  • 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 as a heterotetramer in the reaction solution in a range of 0.1 nM to 50 nM, preferably in a range of 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.
  • the reaction solution may contain an additional enzyme.
  • an additional enzyme e.g., 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.
  • 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 may be appropriately carried out using techniques available to those skilled in the art.
  • enzymes other than those derived from E. coli are used as the first, second, and third enzyme groups, they can be used in a concentration range that corresponds in terms of enzyme activity units to the concentration range specified for the enzymes derived from E. coli.
  • the reaction solution may contain a buffer, ATP, GTP, CTP, UTP, dNTP, a magnesium ion source, and an alkali metal ion source.
  • 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.
  • a preferred buffer solution is Tris-HCl or Tris-OAc.
  • the concentration of the buffer solution can be appropriately selected by those skilled in the art and is not particularly limited, but in the case of Tris-HCl or Tris-OAc, a concentration of 10 mM to 100 mM, 10 mM to 50 mM, or 20 mM can be selected, for example.
  • ATP means adenosine triphosphate.
  • 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.
  • GTP, CTP, and UTP refer to guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, respectively.
  • concentrations of GTP, CTP, and UTP contained in the reaction solution at the start of the reaction may each be independently in the range of, for example, 0.1 mM to 3.0 mM, and preferably in the range of 0.5 mM to 3.0 mM, or 0.5 mM to 2.0 mM.
  • dNTP is a general term for deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP).
  • 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, and preferably in the range of 0.05 mM to 1 mM, or 0.1 mM to 1 mM.
  • the magnesium ion source is a substance that provides magnesium ions (Mg 2+ ) in the reaction solution. Examples include 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 range of 5 to 50 mM in the reaction solution.
  • the alkali metal ion source is a substance that provides alkali metal ions in the reaction solution.
  • alkali metal ions include sodium ions (Na + ) and potassium ions (K + ).
  • 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.
  • the reaction solution further contains inhibitors of non-specific adsorption of proteins (bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.), inhibitors of non-specific adsorption of nucleic acids (tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), glycogen, heparin, oligo-DNA, poly(I-C) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyinosine-polydeoxycytidine), poly(A) (polyadenine), and poly(dA) (polydeoxyadenine), etc.), linear DNA-specific exonuclease (RecBCD, ⁇ exonuclease, etc.), and ⁇ -terminal ...
  • proteins bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.
  • nucleic acids t
  • the reaction solution may contain nuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, Plasmid-Safe (registered trademark) ATP-Dependent DNase (epicentre), etc.), RecG-type helicase (E. coli-derived RecG, etc.), ammonium salts (ammonium sulfate, ammonium chloride, ammonium acetate, etc.), and reducing agents (DTT, ⁇ -mercaptoethanol, glutathione, etc.), and if the reaction solution contains E. coli-derived DNA ligase, it may also contain its cofactor NAD (nicotinamide adenine dinucleotide).
  • NAD nicotinamide adenine dinucleotide
  • 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 in the enrichment reaction.
  • the double-stranded DNA with sequence errors may be double-stranded DNA in which at least one of the strands forming a double-stranded structure has a sequence error and normal base pairs are not formed in the sequence error portion. For example, only one of the strands forming the double-stranded structure may have a sequence error, or both strands forming the double-stranded structure may have sequence errors.
  • double-stranded DNA 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, an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and single-strand-specific exonuclease (e.g., 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.
  • an enzyme selected from the group consisting of MutH, UvrD, and a combination of UvrD and single-strand-specific exonuclease e.g., 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.
  • the above enzymes may be added and allowed to act before the double-stranded DNA amplification reaction, and then the amplification reaction may be further carried out in the presence of the above enzymes.
  • the above enzymes that act on double-stranded DNA with sequence errors are also referred to as mismatch repair-related enzymes.
  • the amplification reaction of double-stranded DNA in the presence of a mismatch repair-related enzyme group, and allowing a mismatch repair-related enzyme group to act on double-stranded DNA prior to a double-stranded DNA amplification reaction refer to sequence error recognition by MutS of the mismatch repair-related enzyme group and interaction with MutS by MutL, or sequence error recognition by MutS of the mismatch repair-related enzyme group and hydrolysis by a single-strand-specific exonuclease, and when the mismatch repair-related enzyme group includes additional enzymes, this refers to the individual enzymes exerting the activities described below in conjunction with these.
  • the mismatch repair-related enzyme group can be allowed to act in a 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.
  • a 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.
  • the composition of the reaction solution is not particularly limited as long as it allows the action of the mismatch repair-related enzyme group to proceed.
  • the reaction solution can include ATP.
  • MutS is not particularly limited in its biological origin as long as it is a protein that recognizes and binds to mismatched base pairs in DNA, and known proteins such as MutS and its family proteins derived from various bacteria such as Escherichia coli and heat-resistant bacteria can be used. MutS with mutations introduced into the amino acid sequence of known MutS or with modified amino acids 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 and heat-resistant bacteria or those with mutations introduced or modified amino acids can be used, and MutS derived from Escherichia coli can be preferably used, and in particular, wild-type MutS derived from Escherichia coli can be used.
  • MutS may be contained in the range of 10 nM to 500 nM when acting on sequence errors, preferably in the range of 10 nM to 300 nM, 30 nM to 500 nM, 30 nM to 300 nM, 30 nM to 200 nM, 50 nM to 300 nM, and more preferably in the range of 100 nM to 300 nM, but is not limited thereto.
  • MutL is a protein that interacts with MutS that recognizes a mismatched base pair to form a complex.
  • MutL has endonuclease activity, but MutL derived from Escherichia coli does not have endonuclease activity.
  • 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 in the amino acid sequence of known MutLs or modified amino acids can also be used as long as they have the same action as MutL of Escherichia coli, which interacts with MutS that recognizes a mismatched base pair.
  • MutL derived from Escherichia coli can be preferably used. MutL may be contained in the range of 30nM to 1000nM when acting on sequence errors, preferably 30nM to 500nM, 30nM to 300nM, 30nM to 200nM, 30nM to 500nM, 50nM to 400nM, 50nM to 300nM, 50nM to 200nM, 50nM to 150nM, 100nM to 500nM, 100nM to 400nM, more preferably 100nM to 300nM, but is not limited thereto.
  • MutH is a protein that is activated by MutS and MutL in prokaryotes such as E. coli and has the activity of nicking unmethylated DNA (cutting the double strand when both strands of double-stranded DNA are unmethylated), and known proteins such as MutH derived from E. coli can be used. MutHs in which mutations have been introduced into the amino acid sequence of known MutHs or in which the amino acids have been modified can also be used as long as they have the above activity. In one embodiment, MutH derived from E. 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, 50 nM to 200 nM, and more preferably 100 nM to 200 nM, but is not limited thereto.
  • UvrD is a protein derived from Escherichia coli that has helicase activity to cleave (unwind) double-stranded DNA.
  • known UvrDs with mutations in the amino acid sequence or modified amino acids can be used as long as they have helicase activity.
  • wild-type UvrD derived from Escherichia coli can be used.
  • UvrD may be contained in a range of 1 nM to 100 nM when acting on sequence errors, 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, 10 nM to 20 nM, and more preferably 15 nM, but is not limited thereto.
  • a single-strand-specific exonuclease is an enzyme that sequentially hydrolyzes nucleotides from the 3' or 5' end of linear single-stranded DNA.
  • enzymes that sequentially hydrolyze from the 3' end 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), oligoribonuclease (ORN), and the like.
  • enzymes that sequentially hydrolyze from the 5' end include exonuclease VII, ⁇ exonuclease, exonuclease VIII, T5 exonuclease, T7 exonuclease, and RecJ exonuclease.
  • 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.
  • the mismatch repair-related enzymes can be brought into contact 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.
  • 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 so long as it allows the action of the mismatch repair-related enzymes to proceed. For example, a solution in which a magnesium ion source, ATP, etc.
  • the reaction solution can contain ATP.
  • the present embodiment also relates to a method for producing a double-stranded DNA library, comprising: (1) preparing a set of two or more single-stranded DNA fragments, at least a portion of each of the single-stranded DNA fragments 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; (1') preparing two or more types of library-constituting single-stranded DNA fragments, each of which has a region corresponding to a part of one 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 of (1), the base sequences of the non-complementary regions being 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 constituent single-stranded DNA
  • the single-stranded DNA fragments accumulated in this library production method further include two or more types of library-constituting single-stranded DNA fragments in addition to the set of single-stranded DNA fragments in the above-mentioned double-stranded DNA fragment production method.
  • the library-constituting single-stranded 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 fragments is preferably in a region that is 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 the same sequences as each other except for the library region.
  • the library region may be designed not to have a complementary sequence, or may be designed to have a complementary sequence. If it has a complementary sequence, the complementary sequence portion may also be a library region. By accumulating such a set of single-stranded DNA fragments, a library of double-stranded DNA can be easily obtained.
  • the complementary strand of the random sequence portion can be replicated by a known method, for example, as shown in Example 16 described below.
  • steps (2) and (3) can be carried out in the same manner as 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.
  • the present embodiment also relates to a method for producing double-stranded DNA, comprising: (a) heating a solution containing any polypeptide and any plurality of single-stranded DNA fragments to a temperature higher than 40° C., whereby the polypeptide and at least one of the single-stranded DNA fragments in the solution form aggregates, and selecting the polypeptide that has 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, each single-stranded DNA fragment representing a portion of either strand of the double-stranded DNA; (c) preparing an accumulation reaction solution containing the set of single-stranded DNA fragments, a cationic salt or a cationic substance, and the polypeptide selected in (a); (d) incubating the reaction solution at an incubation temperature higher than 40° C., thereby assembling the set of single-strand
  • a solution containing any polypeptide and any number of single-stranded DNA fragments is heated to a temperature higher than 40° C., and a polypeptide that forms an aggregate is selected, whereby a polypeptide that exhibits an effect as an accumulation-promoting polypeptide can be selected from any polypeptide. 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 exhibits an effect as an accumulation-promoting polypeptide can be further selected.
  • the heating temperature at this time varies depending on the Tm value of the single-stranded DNA fragments used, but is preferably 45° C. or higher, for example, about 75° C. For other selection methods, the description regarding the accumulation-promoting polypeptide can be referred to.
  • steps (b) to (d) can be carried out in the same manner as the method for producing double-stranded DNA of this embodiment.
  • the present embodiment also provides 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 group of enzymes having DNA gyrase activity, a cationic salt or substance, and a polypeptide having creatine kinase activity, - a polypeptide having phosphopyruvate hydratase activity, - a polypeptide having pyruvate kinase activity, -3-A polypeptide having 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, - histone-like DNA binding proteins, - chromosome replication initiator protein, - a polypeptid
  • single-stranded DNA fragments can be accumulated from a set (e.g., an oligo pool) of two or more types of 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 of the strands of the double-stranded DNA, to obtain double-stranded DNA.
  • Some of the single-stranded DNA fragments to be accumulated may be library-constituting single-stranded DNA fragments.
  • the double-stranded DNA can be circularized, and then the above-mentioned supercoiling reaction can be carried out 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 a supercoiled circular double-stranded DNA.
  • the description of the method for producing circular DNA of this embodiment can be referred to.
  • Example 1 Accumulation-promoting effect of specific polypeptides on oligo DNA fragments 24 types of single-stranded DNA fragments (each 200 bases long) having 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.
  • oligo DNA sequences of 200 bases were designed so that double-stranded DNA without single-stranded gaps would be generated by hybridization between the oligo DNAs.
  • Tables 1 to 3 show the sequences of each of the designed oligo DNAs (IDT, Ultramer PAGE purified grade).
  • CK creatine kinase
  • ENO phosphopyruvate hydratase
  • BSA bovine serum albumin
  • BAP bacterial alkaline phosphatase
  • ⁇ 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.
  • the oligo DNA was linearized by heat treatment at 75°C for 5 minutes.
  • the reaction solution (4.0 ⁇ L) was analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.
  • FIG. 1A A schematic diagram of accumulation and linearization is shown in Figure 1A, and the staining results are shown in Figure 1B.
  • Figure 1B A schematic diagram of accumulation and linearization is shown in Figure 1A, and the staining results are shown in Figure 1B.
  • CK was added as the polypeptide
  • a 2.4 kb band was detected at 120 nM and 1,200 nM
  • ENO was added
  • a 2.4 kb band was detected at 120 nM, 1,200 nM, and 12,000 nM.
  • BSA or BAP was added, the 2.4 kb band was not observed at any concentration, and no significant accumulation-promoting effect was observed. It was demonstrated that CK and ENO have a promoting effect on oligo DNA accumulation at a specific range of concentrations.
  • Example 2 Various polypeptides having the effect of promoting accumulation of oligo-DNA fragments From the results of Example 1, it was found that specific polypeptides exhibit the effect of promoting accumulation of oligo-DNA. Other polypeptides having the accumulation-promoting effect were investigated.
  • polypeptides shown in Tables 4 and 5 were added as polypeptides, and the accumulation-promoting effect was confirmed using the same materials and methods as in Example 1, except that the assembly buffer described below was used.
  • CK, ENO, PK, 3-PGK, HU, myokinase, NDK, AtpC, BSA, BAP, SSB, or lysozyme was added, heat treatment (accumulation reaction) was performed at 75°C for 5 minutes.
  • T4 DNA Lig, EcUvrD, RecA, EcLigA, or T4 PNK was added, heat treatment (accumulation reaction) was performed at 65°C for 2 minutes.
  • nucleoside diphosphate kinase was prepared by purifying from an Escherichia coli expression strain of NDK through a process including ammonium sulfate precipitation and affinity column chromatography.
  • ⁇ subunit of F1-ATPase was prepared by the method described in Kato et al. J. of Biological Chemistry (2007) 282(52):37618-37623.
  • Example 3 Effect of Mg(OAc) 2 on promoting accumulation of oligo-DNA (Example 3-1)
  • the 23 types of 200-base oligo DNA used in Example 1 were added to an assembly buffer containing 1200 nM CK and different concentrations of Mg(OAc) 2 so that each had a concentration of 0.87 nM (total 20 nM, 1.3 ng/ ⁇ L), and the oligo DNA was assembled and linearized at the same reaction temperature as in Example 1.
  • the results were then analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.
  • composition of assembly buffer 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.
  • FIG. 2A A schematic diagram of accumulation is shown in Figure 2A, and the results are shown in Figure 2B.
  • Figure 2B As shown in Figure 2B, when a buffer not containing Mg(OAc) 2 was used, the 2.4 kb accumulation product was not observed. On the other hand, when Mg(OAc) 2 was added, the 2.4 kb accumulation product was detected in greater amounts as the concentration increased.
  • Example 3-2 For an artificial gene of about 800 base pairs including the gfp gene, 16 oligo DNAs were designed by dividing the gene into about 95 to 97 bases in the same manner as in Example 1. Table 6 shows the design of IDT's oPools oligo pools (IDT oPools).
  • the pUP-2 fragment (2.3 kb) was prepared by PCR amplification using the pUP fragment (SEQ ID NO: 41) containing oriC, ampicillin resistance gene, pUCori, ⁇ 70 promoter and rrnBT1 terminator as a template and primers 1 and 2 (SEQ ID NO: 42 and 43) containing dU (deoxyuridine).
  • the pUP-2 fragment (2.3 kb) and the 16 types of oligo DNA pools (IDT oPools) shown in Table 6 were added to an assembly buffer containing 10 mU/ ⁇ L Thermolabile USER II Enzyme (NEB), different concentrations of Mg(OAc) 2 , and different concentrations of CK so that the concentration of each was 10 pM.
  • Composition of assembly buffer 20 mM Tris-HCl (pH 8.0), 0.05% Tween 20.
  • Mg(OAc) 2 was added at concentrations of 0 mM, 5 mM, 10 mM or 20 mM.
  • CK was added at concentrations of 0 nM, 120 nM, or 1,200 nM.
  • the DNA was heat-treated at 75°C for 5 minutes and slowly cooled (0.1°C/sec) to accumulate and cyclize the oligo DNA and single-stranded overhangs.
  • 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 consisting of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE derived from E. coli
  • DnaB is DnaB derived from E. coli
  • DnaC is DnaX derived from E. coli.
  • naC and DnaA are DnaA derived from E.
  • 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 is a complex of ParC and ParE derived from E. coli
  • Topo III is topoisomerase III derived from E. coli
  • RecQ is RecQ derived from E. coli.
  • SSB was prepared by purifying an E. coli expression strain of SSB through a process including ammonium sulfate precipitation and ion exchange column chromatography.
  • IHF was prepared by purifying from an Escherichia coli strain co-expressing IhfA and IhfB through a process including ammonium sulfate precipitation and affinity column chromatography.
  • DnaG was prepared by purification from an Escherichia coli expression strain of DnaG through a process including ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography.
  • DnaN was prepared by purification from an Escherichia coli expression strain of DnaN through a process including ammonium sulfate precipitation and anion exchange column chromatography.
  • Pol III* was prepared by purifying from an Escherichia coli strain co-expressing DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ and HolE through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.
  • DnaB and DnaC were purified and prepared from an Escherichia coli strain co-expressing DnaB and DnaC through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.
  • DnaA was prepared by purification from an Escherichia coli expression strain of DnaA through a process including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography.
  • GyrA and GyrB were prepared by purification from a mixture of an E. coli expression strain for GyrA and an E.
  • Topo IV was prepared by purifying a mixture of an E. coli expression strain for ParC and an E. coli expression strain for ParE through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.
  • Topo III was prepared by purification from an E. coli expression strain of Topo III through a process including ammonium sulfate precipitation and affinity column chromatography. RecQ was purified and prepared from an E.
  • coli expression strain of RecQ 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 Escherichia 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.
  • FIG. 3A A schematic diagram of accumulation is shown in Figure 3A, and the staining results are shown in Figure 3B.
  • Figure 3B when the concentration of Mg(OAc) 2 was 0 mM or 5 mM, almost no pUP-GFP band was observed even when CK was included.
  • CK was included.
  • 10 mM and 20 mM Mg(OAc) 2 pUP-GFP was detected.
  • 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.
  • the 23 types of 200-base oligo DNA used in Example 1 were added to an assembly buffer containing 1,200 nM CK and various cationic salts so that the concentration of each was 0.87 nM (total 20 nM, 1.3 ng/ ⁇ L), and the oligo DNA was linearized. After that, it was analyzed by 1.5% agarose gel electrophoresis and SYBR Green staining.
  • FIG. 4A A schematic diagram of accumulation is shown in Figure 4A, and the staining results are shown in Figure 4B.
  • Figure 4B in addition to Mg(OAc) 2 , the effect of which was confirmed in Example 3, NaCl, KOAc and spermidine also showed an accumulation-promoting effect.
  • the effect of 20 mM Mg(OAc) 2 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 oligo-DNA accumulation.
  • Example 5 Properties of polypeptides exhibiting accumulation-promoting effect In Examples 1 and 2, it was found that specific polypeptides have an accumulation-promoting effect on oligo-DNA. Therefore, in order to clarify the properties of polypeptides having an accumulation-promoting effect (APP: Assembly Promoting Peptides ), binding with single-stranded DNA was examined by gel shift analysis, and aggregation of the polypeptide was confirmed by SDS-PAGE.
  • APP Assembly Promoting Peptides
  • Example 5-1 One type of 100-base oligo DNA (SEQ ID NO: 44) fluorescently labeled with Cy5 was added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) to a concentration of 20 nM. 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 solution (5.0 ⁇ L) was subjected to 6% polyacrylamide gel electrophoresis to detect Cy5.
  • 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 solution (5.0 ⁇ L) was subjected
  • Example 5-2 5.0 ⁇ L 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 ⁇ L of 5xSB was added to 5.0 ⁇ L of the reaction solution before centrifugation or 5.0 ⁇ L of the supernatant, and 6.3 ⁇ L of 1xSB was added to the precipitate, and heat treatment was performed at 95° C. for 5 minutes.
  • the composition of SB SDS-PAGE sample preparation buffer
  • the composition of SB SDS-PAGE sample preparation buffer
  • the composition of SB is 25 mM Tris-HCl (pH 8.8), 5% glycerol, 2% SDS, 20 mM EDTA, 1% ⁇ -mercaptoethanol, 0.05% BPB).
  • 6.0 ⁇ L of the solution was electrophoresed by SDS-PAGE, and polypeptides were detected by CBB staining.
  • Example 6 Reaction temperature at which accumulation-promoting effect is observed It was considered that the heat-dependent aggregation of polypeptides in Example 5 exhibits an accumulation-promoting effect of oligo-DNA. Therefore, the temperature at which an accumulation-promoting effect is observed was examined.
  • Example 6-1 The 23 types of 200-base oligo DNA used in Example 1 were added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) so that each concentration was 0.87 nM (total 20 nM, 1.3 ng/ ⁇ L), and the oligo DNAs were assembled and linearized by heat treatment for 1 hour at 40° C., 45° C., or 50° C. in the presence (1200 nM) or absence (0 nM) of CK. Analysis was performed by 1.5% agarose gel electrophoresis and SYBR Green staining.
  • 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 the polypeptide was detected by CBB staining.
  • Example 6 show that the aggregation of the polypeptide corresponds to the accumulation-promoting effect. It is believed that the accumulation-promoting effect of the oligo-DNA is obtained by the reaction at a temperature at which the polypeptide aggregates.
  • Example 7 Inhibition of accumulation reaction and aggregation of polypeptide by addition of arginine From Example 6, it was considered that the accumulation promotion effect of the polypeptide corresponds to the temperature at which the polypeptide aggregates. Therefore, in order to further clarify the relationship between the heat-dependent aggregation of the polypeptide and the accumulation promotion effect, it was examined whether the polypeptide-dependent accumulation of oligo DNA is inhibited by using arginine hydrochloride as an aggregation inhibitor.
  • 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) 2 , 0.05% Tween 20) so that the concentration of each oligo DNA was 0.87 nM (total 20 nM, 1.3 ng/ ⁇ L). 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.
  • assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20
  • CK or ENO was added at a concentration of 0 nM or 1,200 nM
  • arginine hydrochloride (
  • 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 the polypeptide was detected by CBB staining. The results are shown in Figure 10.
  • Figure 10 “t” represents the fraction before centrifugation, “s” represents the supernatant fraction after centrifugation, and “p” represents the precipitate fraction after centrifugation.
  • Example 8 Accumulation from low concentration oligo DNA and amplification of the accumulated product It was examined whether low concentration oligo DNA could be accumulated using APP and amplified by PCR reaction.
  • the pUP-2 fragment (2.3 kb) and 16 types of IDT oPools used in Example 3-2 were subjected to an assembly reaction and an 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 in the assembly reaction was added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) at a concentration of 1 pM, 10 pM, 100 pM, or 1000 pM (total DNA concentration was 17 times that of each DNA concentration).
  • CK was used at the concentrations shown in Figure 11B and Figure 12.
  • FIG. 11A A schematic diagram of accumulation is shown in Figure 11A, and the staining results are shown in Figures 11B and 12.
  • Figure 11B for oligo DNA concentrations of 10 pM, a band of the amplified product pUP-GFP could be detected when 120 nM or 1,200 nM CK was added.
  • Figure 11B for oligo DNA concentrations of 10 pM, a band of the amplified product pUP-GFP could be detected when 120 nM or 1,200 nM CK was added.
  • a band of pUP-GFP could be detected when 120 nM CK was added.
  • 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.
  • oligo DNA sequences For the artificial gene 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).
  • the pUP-3 fragment (2.3 kb) was prepared by PCR amplification using the pUP fragment (sequence number 53) containing oriC, the ampicillin resistance gene, and pUCori as a template and primers 3 and 4, both of which contain dU.
  • 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 added to the assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 50 mM KOAc) at a concentration of 1 nM each.
  • CK was added at concentrations of 0 nM, 232 nM, 464 nM, or 696 nM.
  • E. coli DH5 ⁇ strain 0.01 ⁇ L of the PCR amplification product was used to transform E. coli DH5 ⁇ strain by chemical method.
  • the transformed E. coli was plated on an LB plate containing 50 ⁇ g/mL carbencillin and cultured overnight at 37°C. Plasmids were extracted from the resulting colonies and sequenced using the Sanger method.
  • FIG. 13A A schematic diagram of accumulation is shown in Figure 13A, and the staining results are shown in Figure 13B.
  • 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 of pUP-cfrUR was confirmed as an amplification product.
  • Table 14 shows the results of plasmid sequence analysis of the amplified product obtained when CK was added at a concentration of 464 nM, which was then introduced into E. coli. The correct sequence was obtained for 6 of the 8 clones.
  • sequences with a high AT ratio which are considered difficult to synthesize, can also be constructed using a method using APP.
  • Example 10 Synthesis of an artificial gene with high GC content (GC 70%)
  • GC 70% a region of 825 bases (IJ101 frg8) of 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 to be a difficult sequence to synthesize, could be constructed by a method using APP.
  • the pUP-3 fragment (2.3 kb) used in Example 9 and the 20 types of IDT oPools in Table 15 were subjected to the 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 added to the assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) at a concentration of 1 pM each.
  • CK was added at a concentration of 0 nM, 120 nM, or 1,200 nM.
  • the resulting PCR amplification product was used to transform the cells in the same manner as in Example 9, and plasmids were extracted from the resulting colonies and subjected to sequence analysis by the Sanger method.
  • FIG. 14A 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 for pUP-IJ101-frg5 was confirmed as an amplification product.
  • Table 16 shows the results of plasmid sequence analysis of the amplified product obtained when CK was added at a concentration of 120 nM, which was then introduced into E. coli. The correct sequence was obtained for 3 of the 7 clones.
  • sequences with a high GC ratio which are considered difficult to synthesize, can also be constructed using a method using APP.
  • oligo DNA sequences 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, desalted grade).
  • the pUP-3 fragment (2.3 kb) used in Example 9 and the 42 types of IDT oPools in Tables 17 to 19 were subjected to the 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 added to the assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) at a concentration of 100 pM each. ENO was added at concentrations of 0 nM, 36 nM, 120 nM, 360 nM, or 1,200 nM.
  • FIG. 15A A schematic diagram of 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 pUP-Luc band was confirmed as an amplification product.
  • the 24 types of 200-base oligo DNAs of Example 1 were added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 ) at a total concentration of 0.02 nM, 0.2 nM, or 2 nM (the concentration of each oligo DNA was almost the same).
  • CK was added at a concentration of 0 nM, 23.2 nM, 69.6 nM, or 232 nM.
  • the oligo DNA was accumulated and circularized by heat treatment at 75°C for 5 minutes and slow cooling (0.1°C/sec).
  • a PCR amplification reaction similar to that in Example 3-2 (but without adding Tus) was carried out, and a portion (1.0 ⁇ L) was analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.
  • FIG. 16A A schematic diagram of accumulation is shown in Figure 16A, and the staining results are shown in Figure 16B.
  • Figure 16B when oligo DNA at a total concentration of 0.2 nM or 2 nM was used, it was possible to amplify pOligoCP without adding CK.
  • pOligoCP When oligo DNA at a total concentration of 0.02 nM was used, pOligoCP could be constructed by adding 232 nM CK. It was demonstrated that the oriC plasmid can be constructed by accumulation using only oligo DNA, without using the oriC vector.
  • oligo DNA sequences For artificial genes of approximately 800 bases each, 64 types of oligo DNA sequences of 85 to 97 bases were designed according to Example 1 for the four genes encoding the pigment proteins GFP, efoRed, amilCP, and fwYellow (Liljeruhm et al. Journal of Biological Engineering (2016) 12:8).
  • the designed oligo DNA sequences (IDT, oPool, desalted grade) are shown in Tables 20 to 23.
  • the pUPKm3 fragment (3.2 kb) was prepared by PCR amplification using the pUPKm fragment (SEQ ID NO: 182) containing oriC, kanamycin resistance gene, pUCori, ⁇ 70 promoter, and rrnBT1 terminator as a template and primers 1 and 2 (see Example 3-1) containing dU.
  • the pUP-2 fragment (2.3 kb) and 16 types of oligo DNA used in Example 3-2 were added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) containing 100 mU/ ⁇ L Thermolabile USER II Enzyme (NEB) so that each DNA had a concentration of 15 nM.
  • Example 15 Preparation of artificial genes by PCR amplification of assembled products It was examined whether a synthetic gene could be obtained by PCR amplification after oligo-DNA assembly using APP.
  • the pUP-2 fragment (2.3 kb) and 16 types of oligo DNA used in Example 3-2 were subjected to an assembly reaction using the same method as in Example 3-2.
  • Each DNA was added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) containing 10 mU/ ⁇ L Thermolabile USER II Enzyme (NEB) so that the concentration of each DNA was 10 pM.
  • CK was used at a concentration of 0 nM, 36 nM, or 120 nM.
  • 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 ⁇ L of the reaction mixture and the diluted SCR mix were added to a PCR tube and incubated at 37°C for 30 min using a ProFlex PCR system (Applied Biosystems) in a 10 ⁇ L reaction volume.
  • This nick repair product was amplified by PCR using primers 5 and 6. 2.0 ⁇ L of the amplified product was analyzed by 1.0% agarose gel electrophoresis and ethidium bromide staining.
  • Example 16 Preparation of site saturation library-1 (Example 16-1)
  • a site-saturated library could be created by oligo-DNA accumulation using APP using random oligo-DNA.
  • the random oligo-DNA site was intentionally designed to have a single-stranded gap.
  • amilCP mutant (amilCP-N6)
  • 16 types of oligo DNA sequences of 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 was a single-stranded gap.
  • Figure 20 shows the design diagram 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.
  • 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 an assembly reaction and an 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 added at a concentration of 1 nM to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) containing 10 mU/ ⁇ L Thermolabile USER II Enzyme (NEB). CK was added at a concentration of 0 nM (-) or 1,200 nM (+).
  • Example 16-2 The colonies that exhibited 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.
  • Table 29 shows the results of sequencing 27 of the colored colonies.
  • Example 16-3 The PCR product obtained in Example 16-1 was subjected to NGS analysis to perform UMI counting. Library preparation was performed using NEBNext Ultra II FS DNA Library Prep Kit (NEB). Indexing during library preparation was performed by PCR, and the following primers were used in combination. NGS analysis was performed using Illumina's iSeq 100.
  • Example 16 demonstrated that this accumulation method can be used to create a site-saturated library.
  • 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 examined.
  • Example 17-1 An assembly reaction was carried out for 2 minutes at 75°C in the same manner as in Example 3-2 for one type of 100-base oligo DNA (SEQ ID NO: 205) labeled with Cy5 fluorescence.
  • the DNA was added at a concentration of 20 nM to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 ) containing 0% or 0.05% Tween 20.
  • Example 17-2 The solution was removed from the tube in which the accumulation reaction was carried out in Example 17-1, and Cy5 detection was carried out.
  • Typhoon FLA9500 manufactured by GE Healthcare was used. The results are shown in FIG. 25.
  • 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) 2 ) so that the concentration of each oligo DNA was 0.87 nM (total 20 nM, 1.3 ng/ ⁇ L). 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. It was added at a concentration of 0.05%. The ligation and linearization reaction of the oligo DNA was carried out by heat treatment at 75°C for 5 minutes.
  • Examples 17-1 to 17-3 suggest that Tween 20 promotes the accumulation of oligo-DNA by inhibiting its adsorption to the tube wall.
  • the APP-dependent accumulation effect was obtained even without the addition of Tween 20.
  • 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, and an investigation was conducted into whether the accuracy of the genes synthesized by this accumulation method could be improved by combining them with a mismatch repair enzyme group.
  • the OriC7.0 fragment (3.0 kb) was used instead of the pUPKm fragment (3.2 kb) in Example 13. As in Example 13, enrichment reactions and PCR amplification reactions were performed for 1 nM OriC7.0 fragment (3.0 kb) and 64 types of IDT oPools with DNA concentrations of 250 pM.
  • PCR amplification reactions were also performed without the addition of mismatch repair-related enzymes. Each was analyzed by 1.2% agarose gel electrophoresis and SYBR Green staining.
  • E. coli DH5 ⁇ strain was transformed using the same method as in Example 13, except that 0.01 ⁇ L of the PCR amplification product was used during transformation.
  • the transformed E. coli was plated and cultured, and the number of colonies that exhibited the color derived from each gene was counted.
  • the amplification product was 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 three times independently for three samples, and the average values and standard deviations are shown in Figure 27B.
  • the OriC7.0 fragment was prepared by PCR amplification using primers 1 and 2 (see Example 3-1) containing dU 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 (sequence number 206), the pUCori portion was replaced with p15A, and a sequence incorporating Mu-SGS (sequence number 207) was used as a template.
  • oriC5.6AT (SEQ ID NO: 206) is shown in Table 33.
  • the bolded part is the DUE part
  • the boxed part in the bolded part is the mutated part in the mutant DUE, which has been mutated to DUE with a high AT content.
  • Example 19 Preparation of site saturation library-2 (Example 19-1)
  • a site-saturation library could be created using random oligo DNA.
  • the random oligo DNA site was designed so that no single-stranded gaps would be formed.
  • amilCP mutant (amilCP-N6)
  • 14 types of oligo DNA sequences ranging from 60 bases to 103 bases were designed for the regions other than the random oligo 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.
  • the OriC7.0-2 fragment (3.0 kb) and the 14 types of IDT oPools shown in Table 35 were subjected to accumulation 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 concentrations were 10 pM for the OriC7.0-2 fragment and 30 pM for each of the oligo DNAs of the IDT oPools, which were added to an assembly buffer (20 mM Tris-HCl (pH 8.0), 20 mM Mg(OAc) 2 , 0.05% Tween 20) containing 20 mU/ ⁇ L Thermolabile USER II Enzyme (NEB). CK was added at a concentration of 120 nM.
  • the OriC7.0-2 fragment (3.0 kb) was prepared by PCR amplification using the following primers containing dU, using the OriC7.0 fragment as a template, which has oriC5.6AT (sequence number 206) and Mu-SGS (sequence number 207) instead of oriC in the pUPKm fragment (3.2 kb) used in Example 13 and has p15A as the plasmid replication origin.
  • Example 19-2 Next, 0.003 ⁇ L of the PCR amplification product was used to transform E. coli DH5 ⁇ strain by chemical method.
  • the transformed E. coli was plated on an LB plate containing 50 ⁇ g/mL kanamycin, and cultured at 30° C. for two days, and then at 4° C. for three days. The number of colonies exhibiting the color derived from each gene was counted. Furthermore, when the colored colonies were inoculated onto a new plate and cultured, colonies of about 10 colors were visually confirmed (FIG. 29. In FIG.
  • Example 19-3 The PCR product obtained in Example 19-1 was subjected to NGS analysis to perform UMI counting. 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

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Abstract

La présente invention concerne un procédé de production d'un ADN double brin linéaire ou circulaire par assemblage d'une pluralité de fragments d'ADN simple brin. La présente invention concerne, par exemple, un procédé de production d'un ADN double brin, le procédé impliquant : (1) préparer un ensemble de deux ou plusieurs fragments d'ADN simple brin, au moins une partie de chaque fragment d'ADN simple brin présentant une séquence complémentaire à au moins un des autres fragments d'ADN simple brin de l'ensemble, et chaque fragment d'ADN simple brin correspondant à une partie d'un brin de l'ADN double brin ; (2) préparer un liquide de réaction pour l'assemblage comprenant l'ensemble de fragments d'ADN simple brin et un sel cationique ou une substance cationique ; et (3) incuber le liquide de réaction à une température d'incubation supérieure à 40 °C, assemblant ainsi l'ensemble de fragments d'ADN simple brin en un ADN double brin.
PCT/JP2024/027365 2023-07-31 2024-07-31 Procédé de production d'adn double brin Pending WO2025028566A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000501615A (ja) * 1995-12-15 2000-02-15 アマーシャム・ライフ・サイエンス・インコーポレーテッド 酵素増幅中に生じる変異配列の検出および除去のためのミスマッチ修復系を用いる方法
US20100081575A1 (en) * 2008-09-22 2010-04-01 Robert Anthony Williamson Methods for creating diversity in libraries and libraries, display vectors and methods, and displayed molecules
JP2015536689A (ja) * 2012-12-13 2015-12-24 シンセティック ゲノミクス、インク． Ｐｅｇを介した核酸分子のアセンブリ
WO2019009361A1 (fr) * 2017-07-05 2019-01-10 国立研究開発法人科学技術振興機構 Méthode de production d'adn et kit d'assemblage de fragment d'adn

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JP2000501615A (ja) * 1995-12-15 2000-02-15 アマーシャム・ライフ・サイエンス・インコーポレーテッド 酵素増幅中に生じる変異配列の検出および除去のためのミスマッチ修復系を用いる方法
US20100081575A1 (en) * 2008-09-22 2010-04-01 Robert Anthony Williamson Methods for creating diversity in libraries and libraries, display vectors and methods, and displayed molecules
JP2015536689A (ja) * 2012-12-13 2015-12-24 シンセティック ゲノミクス、インク． Ｐｅｇを介した核酸分子のアセンブリ
WO2019009361A1 (fr) * 2017-07-05 2019-01-10 国立研究開発法人科学技術振興機構 Méthode de production d'adn et kit d'assemblage de fragment d'adn

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