DNA species selection method and isolation method in cell-free system

Abstract

The present invention provides: a method for easily isolating a plurality of gene species from the gene species in a cell-free system; and a method for easily isolating and selecting a specific gene species in a cell-free system using indexes derived from individual gene species. The present invention is a DNA amplification method comprising: preparing a mixture of DNA that is circular DNA having a replication origin sequence capable of binding to an enzyme having a DnaA activity; preparing a reaction gel solution containing the mixture, a first enzyme group which catalyze the replication of the circular DNA, a second enzyme group which synthesize two sister circular DNA molecules that catalyze an Okazaki fragment linking reaction to form a catenane, a third enzyme group which catalyze the reaction for separating the two sister circular DNA molecules, and a gelatinizing agent; gelatinizing the reaction gel solution to produce an amplification gel plate containing the circular DNA; and subjecting the DNA to a circular DNA amplification reaction at a temperature of 80°C or lower using the gel plate to produce amplified DNA.

Description

Methods for selecting and isolating DNA species in a cell-free system

[0001] The present invention relates to a method for selecting and isolating DNA species in a cell-free system, and in particular to a method for simultaneously amplifying and selecting and isolating multiple DNA species with very low molecular weights in a cell-free system.This application claims priority to Japanese Patent Application No. 2023-090459, filed on May 31, 2023, the contents of which are incorporated herein by reference.

[0002] Attempts have been made to create pools of heterogeneous genes with different sequences, such as DNA libraries, and then select useful genes from those pools. The selection of genes from such pools of heterogeneous genes is useful in a variety of situations, such as screening for target genes such as therapeutic genes, screening for disease risks associated with mutations, and elucidating interactions between DNA or proteins encoded by DNA, and may be applicable to a variety of fields related to genome characterization, gene expression studies, medical diagnosis, population genetics, etc.

[0003] Furthermore, unlike DNA isolated using cells, plasmids constructed and amplified in a cell-free system contain plasmids with mutations that arose during amplification, non-specific amplification products, etc. This can be problematic when using the plasmid in subsequent experiments, and may require isolation and selection of individual amplification products.

[0004] As a method for isolating and selecting such heterologous genes, research is being conducted on creating colonies of individual genes (so-called DNA colonies) from a pool of heterologous genes. For example, Non-Patent Document 1 and Patent Document 1 (Example 15) disclose the creation of colony-like "Polony (PCR+Colony)" made of monoclonal DNA by PCR on a polyacrylamide film on a glass slide.

[0005] Non-Patent Document 2 discloses various improvements to DNA nanocolonies using polyacrylamide gel and PCR amplification, and also discloses the creation of RNA nanocolonies using agarose gel and Qβ replicase. Non-Patent Document 3 discloses a method for amplifying single molecules of template DNA using loop-mediated isothermal amplification (LAMP) in a microchamber containing polyacrylamide gel, primers, and polymerase. Non-Patent Document 4 provides a review of single-molecule DNA amplification and analysis methods using microfluidics. Non-Patent Document 5 discloses the amplification of cell-derived DNA in gel droplets using Phi29 DNA polymerase.

[0006] On the other hand, the PCR method is known as a method for amplifying DNA in a cell-free system (Patent Documents 2 to 4). As a method for isolating and selecting genes using the PCR method, a method has been attempted in which a pool of heterologous genes is subjected to limiting dilution followed by amplification by the PCR method, but limiting dilution does not always result in the isolation of a single molecule, and this is probabilistic, and a large amount of sample is required for dilution.

[0007] International Publication No. WO 2000 / 053812 International Publication No. WO 2016 / 080424 International Publication No. WO 2017 / 199991 International Publication No. WO 2018 / 159669

[0008] Mitra and Church, Nucleic Acids Research, 1999, vol.27, No.24, e34.Chetverina and Chetverin, Biochemistry (Moscow), 2008, Vol.73, No.13, p.1361-1387.Lam et al., Biomed Microdevices, 2008, Vol.10, p.539-546.Zhang and Xing, Chemical Reviews, 2010, Vol.110, No.8, p.4910-4947.Michikawa et al., Anal Biochem., 2008, Vol.383, No.2, p.151-158.

[0009] The conventional single-molecule DNA amplification methods described above have had problems such as difficulty in simple implementation due to the need for temperature cycling, the need to prepare minute compartments, and the need to prepare large amounts of sample. Another problem is that single DNA molecules are not always isolated. Furthermore, the phenotype (functional expression) of the target gene, as in blue-white selection using E. coli, has not been confirmed for colonies created in a cell-free system to select the target gene.

[0010] Against this background, the main object of the present invention is to provide a method for easily isolating individual gene species from multiple gene species in a cell-free system, and a method for easily isolating and selecting specific gene species in a cell-free system using indicators derived from the individual gene species.

[0011] The present inventors have discovered that by performing PCR on a gel scaffold, a heterogeneous mixture of DNA at very low concentrations can be amplified individually, and then isolated and selected as colonies of DNA with a single sequence. Furthermore, they have discovered that functions such as protein expression linked to DNA information can also be confirmed in the colonies in the gel, thereby completing the present invention.

[0012] That is, the methods etc. according to the present invention are the following [1] to

[22] : [1] A method for amplifying DNA, comprising: preparing a mixture of DNA, wherein the DNA is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing the mixture, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to prepare an amplification gel plate containing DNA; and performing a circular DNA amplification reaction on the DNA using the gel plate at a temperature of 80°C or less to obtain amplified DNA. [2] A method for amplifying DNA, comprising: preparing a mixture of DNA, the DNA being circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to prepare an amplification gel plate; plating the mixture on the gel plate; and using the gel plate to perform a circular DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA. [3] The method according to [1] or [2], wherein the gelling agent is selected from the group consisting of agarose, polyacrylamide, agar, carrageenan, alginic acid, alginate salts, gellan gum, pectin, collagen, gelatin, gluten, polyvinyl alcohol, polyethylene glycol, polyacrylamide, agaropectin, polyacrylic acid, polyvinylpyrrolidone, and combinations of one or more thereof. [4] The method according to [3], wherein the gelling agent is agarose.[5] A method for amplifying DNA, comprising: preparing a DNA mixture; preparing a reaction gel solution containing the mixture, agarose, and one or more DNA amplification enzymes; gelling the reaction gel solution to create an amplification gel plate containing DNA; and performing a DNA amplification reaction on the DNA using the gel plate at a temperature of 80°C or less to obtain amplified DNA. [6] A method for amplifying DNA, comprising: preparing a DNA mixture; preparing a reaction gel solution containing agarose and one or more DNA amplification enzymes; gelling the reaction gel solution to create an amplification gel plate; plating the mixture on the gel plate; and performing a DNA amplification reaction on the DNA using the gel plate at a temperature of 80°C or less to obtain amplified DNA. [7] Any of the methods [1] to [4] above, wherein the mixture contains two or more types of DNA, and the amplified DNA contains two or more types of DNA colonies. [8] The method of [7] above, further comprising detecting at least one of the two or more types of DNA colonies. [9] The method of [7] or [8] above, further comprising isolating at least one of the two or more types of DNA colonies and performing a DNA amplification reaction on the isolated DNA colonies to obtain amplified DNA derived from the colonies.

[10] The method of [5] or [6] above, wherein the mixture contains two or more types of DNA and the amplified DNA contains two or more types of DNA colonies.

[11] The method of

[10] above, further comprising detecting at least one of the two or more types of DNA colonies.

[12] The method of

[10] or

[11] above, further comprising isolating at least one of the two or more types of DNA colonies and performing a DNA amplification reaction on the isolated DNA colonies to obtain amplified DNA derived from the colonies.

[13] Any of the methods of [1] to [4] and [7] to [9] above, wherein the gel plate further contains RNA polymerase, and further comprising performing a transcription reaction in the gel plate using the amplified DNA as a template to obtain RNA.

[14] The method of

[13] , wherein the gel plate further comprises ribosomes, tRNA, and amino acids and / or amino acid derivatives, and further comprising carrying out a translation reaction in the gel plate using the RNA as a template to obtain a protein.

[15] Any of the methods of [5], [6], and

[10] to

[12] , wherein the gel plate further comprises RNA polymerase, and further comprising carrying out a transcription reaction in the gel plate using the amplified DNA as a template to obtain RNA.

[16] The method of

[15] , wherein the gel plate further comprises ribosomes, tRNA, and amino acids and / or amino acid derivatives, and further comprising carrying out a translation reaction in the gel plate using the RNA as a template to obtain a protein.

[17] A method for detecting a circular DNA, comprising: preparing a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction solution containing the circular DNA, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, an RNA polymerase, a ribosome, tRNA, and an amino acid and / or an amino acid derivative; translating the circular DNA using the reaction solution to obtain a translation product; and detecting the translation product.

[18] A composition for translating circular DNA, comprising: a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; a first group of enzymes that catalyze the replication of the circular DNA, 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; an RNA polymerase, a ribosome, and a tRNA.

[19] A method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, wherein the DNAs encode proteins; creating a gel plate containing the mixture, a gelling agent, one or more DNA amplification enzymes, an RNA polymerase, a ribosome, and a tRNA; and translating the DNA using the gel plate to obtain colonies of translation products.

[20] A method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, the DNA encoding a protein; creating a gel plate containing a gelling agent, one or more DNA amplification enzymes, an RNA polymerase, a ribosome, and a tRNA; plating the mixture on the gel plate; and translating the two or more types of DNA using the gel plate to obtain colonies of translation products.

[21] A gel plate for amplifying circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and agarose, wherein the circular DNA has a replication initiation sequence that can bind to an enzyme having DnaA activity.

[22] A gel plate for translating circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA; 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; agarose; an RNA polymerase, a ribosome, and a tRNA; wherein the circular DNA has a replication initiation sequence that can bind to an enzyme having DnaA activity.

[0013] According to the production method of the present invention, individual gene species can be easily isolated from multiple gene species in a cell-free system, and specific gene species can be easily isolated and selected in a cell-free system using indicators derived from the individual gene species.

[0014] FIG. 1 shows an overview of Example 1. FIG. 1 is a schematic diagram of the method for preparing an RCR plate and the DNA amplification method using the RCR plate. FIG. 2 shows the results of Example 1, photographing RCR plates after amplifying DNA with different numbers of molecules using a fluorescence microscope. FIG. 3 shows DNA colonies prepared in Example 2 by spreading DNA on an RCR plate prepared without DNA. FIG. 4 shows the results of Example 3. FIG. 4(A) shows DNA colonies prepared by premixing 2.3 kb to 23 kb circular DNA with a gel (2% agarose). FIG. 4(B) shows DNA colonies prepared by applying 93 kb circular DNA to an RCR plate (2% agarose). FIG. 4(C) shows DNA colonies prepared by premixing 2.3 kb circular DNA with a gel (3% agarose). FIG. 5 is a schematic diagram of the experiment in Example 4. FIG. 6 is a graph showing the frequency of UMI sequences in each DNA colony obtained by NGS analysis in Example 4. Figure 7 shows the results of Example 5. Figures 7(A) and (B) are red fluorescent microscopic images (A) and green fluorescent microscopic images (B), respectively, of circular DNA after reaction using a PURE-RCR plate. Figure 7(C) is a superimposed image of Figures 7(A) and 7(B), and Figure 7(D) is an illustration of the photograph in 7(C). Figure 8 is a schematic diagram of the DNA size amplified when the two types of plasmid DNA used in Example 5 were subjected to PCR using the same primers. Figure 9 shows the results of electrophoresis of the PCR amplified product after colony picking, from the results of Example 5. Figure 10 shows the results of Example 6. Figures 10(A) and (B) are red fluorescent microscopic images (A) and green fluorescent microscopic images (B), respectively, of circular DNA after reaction using a PURE-RCR plate. Figure 10(C) is a superimposed image of Figures 10(A) and 10(B).

[0015] Hereinafter, embodiments for carrying out the present invention (hereinafter also referred to as the present embodiment) will be specifically described, but the present invention is not limited to these.

[0016] In one aspect, this embodiment relates to a method for amplifying DNA, comprising: preparing a mixture of DNA, the DNA being circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing the mixture, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to prepare an amplification gel plate containing DNA; and using the gel plate to perform a circular DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA.

[0017] This embodiment also relates to a method for amplifying DNA, in one aspect, comprising: preparing a mixture of DNA, the DNA being circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to prepare an amplification gel plate; plating the mixture on the gel plate; and performing a circular DNA amplification reaction using the gel plate at a temperature of 80°C or less to obtain amplified DNA.

[0018] In one aspect, the DNA used in the DNA amplification method of this embodiment is circular DNA. As long as the DNA has a circular structure, it may be either single-stranded or double-stranded, but double-stranded is preferred. There are no particular limitations on its origin, and examples include natural circular DNA such as the circular chromosome of a microorganism, circular DNA obtained by circularizing non-circular DNA, circular DNA obtained by linking multiple non-circular DNAs, and artificially synthesized circular DNA. Examples of such non-circular DNA include natural circular DNA cleaved by enzyme treatment or the like, natural non-circular DNA, and artificially synthesized non-circular DNA.

[0019] When the circular DNA contains a replication initiation sequence capable of binding to an enzyme having DnaA activity (hereinafter, sometimes simply referred to as a "replication initiation sequence"), examples of the replication initiation sequence capable of binding to an enzyme having DnaA activity include known replication initiation sequences present in bacteria such as Escherichia coli and Bacillus subtilis, which can be obtained from public databases such as NCBI. Alternatively, a replication initiation sequence can be obtained by cloning a DNA fragment capable of binding to an enzyme having DnaA activity and analyzing its nucleotide sequence. The replication initiation sequence used in the present invention may also be a sequence in which a mutation has been introduced into a known replication initiation sequence by substituting, deleting, or inserting one or more bases, and which is capable of binding to an enzyme having DnaA activity. The replication initiation sequence used in the present invention is preferably oriC and a modified sequence thereof, more preferably oriC derived from E. coli and a modified sequence thereof.

[0020] In this embodiment, the circular DNA containing a replication initiation sequence may have, in addition to the replication initiation sequence, a pair of ter sequences inserted outward relative to the replication initiation sequence. Alternatively, the circular DNA may have a base sequence recognized by a DNA multimer separation enzyme. This suppresses the production of DNA multimers when the circular DNA is amplified by PCR (Patent Documents 2 to 4) or the like, enabling the amplification product of the circular DNA to be efficiently obtained. Alternatively, the circular DNA may have both a replication initiation sequence and a base sequence recognized by a DNA multimer separation enzyme. (Patent Document 4)

[0021] With respect to ter sequences, "inserting outward relative to the replication initiation sequence" means inserting the ter sequence in a direction that allows replication outward from the replication initiation sequence, such as oriC, while terminating replication inward toward the replication initiation sequence, due to the action of a combination of proteins that bind to the ter sequence and inhibit replication. Therefore, with respect to ter sequences, "a pair of ter sequences inserted outward relative to the replication initiation sequence" means that one is inserted on the 5' side of the replication initiation sequence and the other is inserted on the 3' side of the replication initiation sequence. Examples of ter sequences inserted on the 5' side of the replication initiation sequence include sequences shown in Patent Document 4 and International Publication No. 2023 / 140325. Examples of ter sequences inserted on the 3' side of the replication initiation sequence include sequences containing a complementary sequence to the nucleotide sequence inserted on the 5' side of the replication initiation sequence. The ter sequences may be present in any position, as long as a pair of ter sequences is inserted outward relative to the replication initiation sequence.

[0022] For example, replication termination systems using a ter sequence and a protein that has the activity of binding to a ter sequence on DNA and inhibiting replication include the Tus-ter system in Escherichia coli (Hiasa and Marians, The Journal of Biological Chemistry, 1994, vol. 269(43), pp. 26959-26968; Neylon, et al., Microbiology and Molecular Biology Reviews, 2005, vol. 69(3), pp. 501-526.) and the RTP-ter system in Bacillus bacteria (Vivian, et al., Journal of Molecular Biology, 2007, vol. 370, pp. 481-491.). As the ter sequence, a ter sequence that can be used in these systems or a modified sequence thereof can be used. A modified ter sequence refers to a base sequence in which a mutation has been introduced to substitute, delete, or insert one or more bases in the base sequence before modification while retaining the function of the ter sequence. When the Tus-ter system is used, the Tus protein is used during plasmid replication, and when the RTP-ter system is used, the RTP protein is used during plasmid replication.

[0023] In this embodiment, the ter sequence of the circular DNA containing a replication initiation sequence may be wild-type or mutant. In one aspect, an example of a wild-type ter sequence is 5'-GN[A / G][T / A]GTTGTAAC[T / G]A-3' (SEQ ID NO: 1), and an example of a mutant ter sequence is a base sequence containing a modified sequence in which one or several bases in the base sequence of SEQ ID NO: 1 are substituted. In one aspect, the ter sequence of the circular DNA containing a replication initiation sequence of this embodiment is preferably a mutant ter sequence, such as 5'-GN[A / G][T / A]GTTGTAcC[T / G]A-3' (SEQ ID NO: 2) or 5'-GTATGTTGTAcCTA-3' (SEQ ID NO: 3) (the lowercase bases in SEQ ID NOs: 2 and 3 are mutation sites from the wild type).

[0024] In one aspect of this embodiment, a DNA mixture is prepared to contain multiple identical or different circular DNAs. In one aspect of this embodiment, the DNA mixture may be a mixture containing DNA. For example, multiple types of circular DNAs with different partial sequences of the circular DNA can be prepared. More specifically, multiple types of fragments with randomized partial sequences of the circular DNA can be prepared and inserted into the circular DNA to obtain a circular DNA library, and the obtained circular DNA library can be prepared as a DNA mixture. In one aspect, preferably, the mixture contains multiple different types of circular DNA.

[0025] The size of the circular DNA is not particularly limited and can be, for example, 1 kb (1,000 bases) or more, 5 kb or more, 8 kb or more, 10 kb or more, 50 kb or more, 100 kb or more, 200 kb or more, 500 kb or more, or 1,000 kb or more. The length can be 1 Mb (1,000,000 bases) or less, 100 kb or less, 50 kb or less, 30 kb or less, 20 kb or less, or 10 kb or less. The length can also be 1 Mb (1,000,000 bases) or less, 100 kb or less, 50 kb or less, 30 kb or less, 20 kb or less, or 10 kb or less. In one aspect, the circular DNA has a size of 1 kb to 100 kb, 1 kb to 10 kb, 1 kb to 8 kb, or 1 kb to 5 kb. The DNA mixture may contain circular DNAs of different sizes.

[0026] In one aspect of this embodiment, the reaction gel solution comprises a first group of enzymes that catalyze the replication of circular DNA, 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 a gelling agent.

[0027] When the reaction gel solution contains the first enzyme group, the second enzyme group, and the third enzyme group, and the circular DNA contains a replication initiation sequence (e.g., oriC) that can bind to an enzyme having DnaA activity, the DNA can be amplified using the Replication Cycle Reaction method (hereinafter, referred to as the RCR method; see Patent Documents 2 to 4, etc.).

[0028] Examples of the first group of enzymes that catalyze the replication of circular DNA include the enzymes described in Kaguni JM & Kornberg A. Cell, 1984, vol. 38, pp. 183-90. Specifically, examples of the first group of enzymes include one or more enzymes or enzyme groups selected from the group consisting of an enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or enzyme groups having DNA gyrase activity, a single-strand 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 groups having DNA polymerase III* activity, and all combinations of such enzymes or enzyme groups are also exemplified. In one embodiment, the first group of enzymes includes an enzyme having DnaA activity, an enzyme or group of enzymes 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 group of enzymes having DNA polymerase III* activity.

[0029] The biological origin of the enzyme having DnaA activity is not particularly limited, as long as it has initiator activity similar to that of DnaA, an initiator protein of E. coli. For example, DnaA derived from E. coli can be suitably used. DnaA derived from E. coli may be contained in the reaction gel solution as a monomer in a range of 1 nM to 10 μM, preferably 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 200 nM, or 50 nM to 150 nM, but is not limited thereto.

[0030] Nucleoid protein refers to a protein contained in a nucleoid. The one or more nucleoid proteins used in the present invention are not particularly limited in terms of their biological origin, as long as they are enzymes with activity similar to that of E. coli nucleoid proteins. For example, E. coli-derived IHF, i.e., a complex (heterodimer or homodimer) of IhfA and / or IhfB, or E. coli-derived HU, i.e., a complex of hupA and hupB, can be suitably used. E. coli-derived IHF may be contained in the reaction gel solution as a hetero / homodimer in a range of 5 nM to 400 nM, preferably 5 nM to 200 nM, 5 nM to 100 nM, 5 nM to 50 nM, 10 nM to 50 nM, 10 nM to 40 nM, or 10 nM to 30 nM, but is not limited thereto. The concentration of HU derived from E. coli in the reaction gel solution may be in the range of 1 nM to 50 nM, preferably in the range of 5 nM to 50 nM, or 5 nM to 25 nM, but is not limited thereto.

[0031] The biological origin of the enzyme or enzymes having DNA gyrase activity is not particularly limited, as long as they have an activity similar to that of E. coli DNA gyrase. For example, a complex consisting of GyrA and GyrB derived from E. coli can be suitably used. The complex consisting of GyrA and GyrB derived from E. coli may be contained in the reaction gel solution as a heterotetramer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 50 nM to 200 nM, or 100 nM to 200 nM, but is not limited thereto.

[0032] The single-stranded DNA binding protein (SSB) may be an enzyme having the same activity as the single-stranded DNA binding protein of Escherichia coli, and its biological origin is not particularly limited. For example, SSB derived from Escherichia coli can be suitably used. The SSB derived from Escherichia coli may be contained in the reaction gel 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.

[0033] The biological origin of the enzyme having DnaB-type helicase activity is not particularly limited, as long as it has activity similar to that of DnaB from Escherichia coli. For example, DnaB derived from Escherichia coli can be suitably used. DnaB derived from Escherichia coli may be contained in the reaction gel 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.

[0034] The enzyme having DNA helicase loader activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaC. For example, E. coli-derived DnaC can be suitably used. E. coli-derived DnaC may be contained in the reaction gel 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.

[0035] The enzyme having DNA primase activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaG. For example, E. coli-derived DnaG can be suitably used. E. coli-derived DnaG may be contained in the reaction gel solution as a monomer in a range of 20 nM to 1000 nM, preferably 20 nM to 800 nM, 50 nM to 800 nM, 100 nM to 800 nM, 200 nM to 800 nM, 250 nM to 800 nM, 250 nM to 500 nM, or 300 nM to 500 nM, but is not limited thereto.

[0036] The enzyme having DNA clamping activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DnaN. For example, E. coli-derived DnaN can be suitably used. E. coli-derived DnaN may be contained in the reaction gel solution as a homodimer in a range of 10 nM to 1000 nM, preferably 10 nM to 800 nM, 10 nM to 500 nM, 20 nM to 500 nM, 20 nM to 200 nM, 30 nM to 200 nM, or 30 nM to 100 nM, but is not limited thereto.

[0037] The biological origin of the enzyme or enzymes having DNA polymerase III* activity is not particularly limited, as long as they have activity similar to that of the E. coli DNA polymerase III* complex. For example, 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, and 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 E. coli-derived DNA polymerase III* complex may be contained as a heteromultimer in the reaction gel solution in a range of 2 nM to 50 nM, preferably in a range of 2 nM to 40 nM, 2 nM to 30 nM, 2 nM to 20 nM, 5 nM to 40 nM, 5 nM to 30 nM, or 5 nM to 20 nM, but is not limited thereto.

[0038] In the present invention, two sister circular DNAs forming a catenane refer to two circular DNAs that are synthesized by a DNA replication reaction and are in a linked state.

[0039] Examples of the second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes include one or more enzymes selected from the group consisting of enzymes having DNA polymerase I activity, enzymes having DNA ligase activity, and enzymes having RNase H activity, or a combination of such enzymes. In one embodiment, the second group of enzymes preferably includes an enzyme having DNA polymerase I activity and an enzyme having DNA ligase activity.

[0040] The biological origin of the enzyme having DNA polymerase I activity is not particularly limited, as long as it has activity similar to that of E. coli DNA polymerase I. For example, E. coli-derived DNA polymerase I can be suitably used. E. coli-derived DNA polymerase I may be contained in the reaction gel solution as a monomer in a range of 10 nM to 200 nM, preferably 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 40 nM to 150 nM, 40 nM to 100 nM, or 40 nM to 80 nM, but is not limited thereto.

[0041] The enzyme having DNA ligase activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of E. coli DNA ligase. For example, E. coli-derived DNA ligase or T4 phage DNA ligase can be suitably used. The E. coli-derived DNA ligase may be contained in the reaction gel solution as a monomer in a range of 10 nM to 200 nM, preferably 15 nM to 200 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, or 20 nM to 80 nM, but is not limited thereto.

[0042] The enzyme having RNase H activity is not particularly limited in terms of its biological origin, as long as it has the activity of degrading the RNA strand of an RNA:DNA hybrid. For example, RNase H derived from Escherichia coli can be suitably used. The RNase H derived from Escherichia coli may be contained in the reaction gel solution as a monomer in a range of 0.2 nM to 200 nM, preferably 0.2 nM to 200 nM, 0.2 nM to 100 nM, 0.2 nM to 50 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 50 nM, or 10 nM to 50 nM, but is not limited thereto.

[0043] Examples of the third enzyme group that catalyzes the separation reaction of the two sister circular DNAs include the enzyme group described in Peng H & Marians KJ., PNAS, 1993, vol. 90, pp. 8571-8575. Specifically, the third enzyme group may include one or more enzymes selected from the group consisting of enzymes having topoisomerase IV activity, enzymes having topoisomerase III activity, and enzymes having RecQ-type helicase activity, or a combination of such enzymes. In one embodiment, the third enzyme group preferably includes an enzyme having topoisomerase IV activity and / or an enzyme having topoisomerase III activity.

[0044] The biological origin of the enzyme having topoisomerase III activity is not particularly limited, as long as it has activity similar to that of Escherichia coli topoisomerase III. For example, Escherichia coli-derived topoisomerase III can be suitably used. Escherichia coli-derived topoisomerase III may be contained in the reaction gel solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0045] The biological origin of the enzyme having RecQ helicase activity is not particularly limited, as long as it has activity similar to that of Escherichia coli RecQ. For example, RecQ derived from Escherichia coli can be suitably used. RecQ derived from Escherichia coli may be contained in the reaction gel solution as a monomer in a range of 20 nM to 500 nM, preferably 20 nM to 400 nM, 20 nM to 300 nM, 20 nM to 200 nM, 20 nM to 100 nM, or 30 to 80 nM, but is not limited thereto.

[0046] The enzyme having topoisomerase IV activity is not particularly limited in terms of its biological origin, as long as it has activity similar to that of Escherichia coli topoisomerase IV. For example, Escherichia coli-derived topoisomerase IV, which is a complex of ParC and ParE, can be suitably used. Escherichia coli-derived topoisomerase IV may be contained in the reaction gel solution as a heterotetramer in a range of 0.1 nM to 50 nM, preferably 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, 1 nM to 10 nM, or 1 nM to 5 nM, but is not limited thereto.

[0047] The reaction gel solution may contain an additional enzyme in addition to the first, second, and third enzyme groups. For example, when the circular DNA to be amplified by PCR has a pair of ter sequences inserted facing outward relative to a replication origin sequence (e.g., oriC) capable of binding to an enzyme having DnaA activity, the reaction gel solution may further contain a protein (e.g., Tus protein derived from Escherichia coli) that has the activity of binding to the ter sequence and inhibiting replication.

[0048] The enzymes constituting the first, second, and third enzyme groups may be commercially available or may be extracted from microorganisms and purified as necessary. Extraction and purification of enzymes from microorganisms can be carried out appropriately using techniques available to those skilled in the art.

[0049] When enzymes other than those derived from Escherichia coli are used as the first, second, and third enzyme groups, they can be used in a concentration range corresponding in terms of enzyme activity units to the concentration range specified for the enzymes derived from Escherichia coli.

[0050] The gelling agent contained in the reaction gel solution is not particularly limited as long as it does not interfere with the DNA amplification reaction, and can be selected from known gelling agents taking into consideration the properties of the DNA to be amplified, the properties of the enzyme contained in the reaction gel solution, the temperature during the subsequent DNA amplification reaction, etc. For example, the gelling agent can be selected from the group consisting of agarose, polyacrylamide, agar, carrageenan, alginic acid, alginate, gellan gum, pectin, collagen, gelatin, gluten, polyvinyl alcohol, polyethylene glycol, polyacrylamide, agaropectin, polyacrylic acid, polyvinylpyrrolidone, and combinations of one or more thereof. Preferably, the gelling agent is selected from the group consisting of agarose, polyacrylamide, agar, and combinations of one or more thereof, more preferably agarose, and particularly preferably agarose.

[0051] In one embodiment, when agarose is used as a gelling agent, the agarose is not particularly limited, and examples of commercially available agarose include Ultra-low Gelling Temperature (manufactured by Sigma-Aldrich), Agarose H14 (manufactured by TaKaRa), PrimeGel (trademark) Agarose LE 1-20K GAT (manufactured by TaKaRa), and Agarose L (manufactured by Nippon Gene Co., Ltd.).

[0052] In one aspect, the gelling agent preferably has a gelling temperature of 80° C. or lower. This can prevent the reaction gel plate from melting when the DNA amplification reaction after gelling is carried out at 80° C. or lower.

[0053] In one embodiment, the gelling agent preferably has a gelling temperature of 60° C. or lower, preferably 45° C. or lower, from the viewpoint of ease of handling the reaction gel solution before gelation and from the viewpoint of carrying out the reaction at a temperature equal to or lower than the inactivation temperature of the enzyme used in the amplification reaction. The lower limit of the gelling temperature is not particularly limited, but is, for example, 5° C. or higher. Depending on the type, it may be 8° C. or higher.

[0054] The concentration of the gelling agent in the reaction gel solution can be adjusted appropriately depending on the size of the DNA to be amplified. When the DNA size is small, increasing the gelling agent concentration can prevent DNA diffusion during and after amplification. For example, when using agarose as a gelling agent to amplify DNA of a size less than 4 kb, 3 kb or less, or 2.5 kb or less, the agarose concentration in the reaction gel solution can be more than 2%, for example, 3% or more. The above is merely one embodiment, and the concentration can also be adjusted appropriately depending on reaction conditions such as reaction temperature and reaction time.

[0055] The composition of the reaction gel solution is not particularly limited as long as it allows gelation and DNA amplification reactions to proceed, and may, for example, further contain one or more selected from a buffer solution, ATP (adenosine triphosphate), GTP (guanosine triphosphate), CTP (cytidine triphosphate), UTP (uridine triphosphate), dNTP, a magnesium ion source, and an alkali metal ion source, as necessary.

[0056] The buffer solution may be a buffer solution suitable for use at pH 7 to 9, preferably pH 8, and examples thereof 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. In the case of Tris-HCl or Tris-OAc, the concentration can be selected from, for example, 10 mM to 100 mM, 10 mM to 50 mM, or 20 mM.

[0057] The concentration of ATP contained in the reaction gel solution may be, for example, in the range of 0.1 to 3 mM, and preferably in the range of 0.1 to 2 mM, 0.1 to 1.5 mM, or 0.5 to 1.5 mM.

[0058] The concentrations of GTP, CTP, and UTP contained in the reaction gel solution may be, independently, for example, in the range of 0.1 mM to 3.0 mM, preferably in the range of 0.5 mM to 3.0 mM, or 0.5 mM to 2.0 mM.

[0059] dNTP is a collective term for deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The concentration of dNTP contained in the reaction gel solution may be, for example, in the range of 0.01 to 1 mM, preferably in the range of 0.05 to 1 mM, or 0.1 to 1 mM.

[0060] The magnesium ion source is a magnesium ion (Mg ２＋ ) is a substance that gives Mg(OAc) ２ , MgCl ２ , and MgSO ４ A preferred magnesium ion source is Mg(OAc) ２ The concentration of the magnesium ion source contained in the reaction gel liquid may be, for example, a concentration that provides magnesium ions in the reaction gel liquid in the range of 5 to 50 mM.

[0061] The alkali metal ion source is a substance that provides alkali metal ions to the reaction gel liquid. Examples of alkali metal ions include sodium ions (Na ＋ ), potassium ions (K ＋ ) Examples of alkali metal ion sources include potassium glutamate (KGlu), potassium aspartate, potassium chloride, potassium acetate (KOAc), 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 gel solution may be a concentration that provides alkali metal ions in the reaction gel solution at 100 mM or more, preferably in the range of 100 mM to 300 mM, but is not limited thereto.

[0062] The reaction gel solution may further contain a non-specific adsorption inhibitor for proteins (bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.), a non-specific adsorption inhibitor for nucleic acids (tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), glycogen, heparin, oligo-DNA, poly(IC) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyribonucleotide), etc.), and a non-specific adsorption inhibitor for nucleic acids (tRNA, rRNA, ribosomal RNA, mRNA, glycogen, heparin, oligo-DNA, poly(IC) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyribonucleotide), etc.). poly(A) (poly(A) (poly(A)-poly(A)), poly ... The reaction gel solution may contain a DNA ligase derived from Escherichia coli, a single-stranded DNA-specific exonuclease (exonuclease I, RecJ, exonuclease T, etc.), an ammonium salt (ammonium sulfate, ammonium chloride, ammonium acetate, etc.), a reducing agent (DTT, β-mercaptoethanol, glutathione, etc.), and when the reaction gel solution contains a DNA ligase derived from Escherichia coli, it may also contain its cofactor, NAD (nicotinamide adenine dinucleotide).

[0063] In the DNA amplification method of this embodiment, an amplification reaction is performed on circular DNA using a DNA amplification gel plate obtained by gelling the reaction gel solution to obtain amplified DNA (amplified circular DNA). The circular DNA may be mixed with the reaction gel solution before gelling and then gelled, or the circular DNA may be seeded on the amplification gel plate obtained by gelling the reaction gel solution. The gelling of the reaction gel solution can be performed using known techniques. Typically, the amplification gel plate can be obtained by gelling the reaction gel solution poured into a frame or container by lowering the temperature to a temperature below the gelling temperature of the gelling agent.

[0064] When mixing circular DNA with the reaction gel solution before gelation, the concentration of circular DNA can be adjusted appropriately depending on the size of the amplification gel plate to be obtained so that the circular DNA is dispersed in the gel plate (preferably so that it is dispersed one molecule at a time).

[0065] When plating circular DNA on an amplification gel plate after gelation of the reaction gel solution, the circular DNA can be diluted with a solvent as appropriate before plating so that the circular DNA is dispersed on the gel plate (preferably so that it is dispersed one molecule at a time) depending on the size of the gel plate on which the DNA is plated.

[0066] The amplification reaction of circular DNA using a DNA amplification gel plate can be carried out at a temperature of 80° C. or less, preferably 65° C. or less. The amplification reaction may be either non-isothermal or isothermal. During the DNA amplification reaction, it is desirable to prevent evaporation from the gel surface by using a technique such as covering the gel plate.

[0067] In the case of isothermal amplification, the isothermal conditions are not particularly limited as long as they allow the DNA amplification reaction or DNA replication reaction to proceed. For example, a constant temperature within the optimal temperature range of the DNA polymerase can be used. Examples of isothermal conditions include a constant temperature of 15°C or higher, 16°C or higher, 20°C or higher, 25°C or higher, or 30°C or higher, and a constant temperature of 80°C or lower, 75°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, 50°C or lower, 45°C or lower, 40°C or lower, 35°C or lower, or 33°C or lower. Further, isothermal conditions include, for example, a constant temperature within the range of 15 ° C. to 80 ° C., 16 ° C. to 80 ° C., or 20 ° C. to 80 ° C., a constant temperature within the range of 15 ° C. to 75 ° C., 16 ° C. to 75 ° C., or 20 ° C. to 75 ° C., a constant temperature within the range of 15 ° C. to 70 ° C., 16 ° C. to 70 ° C., or 20 ° C. to 70 ° C., a constant temperature within the range of 15 ° C. to 65 ° C., 16 ° C. to 65 ° C., or 20 ° C. to 65 ° C., a constant temperature within the range of 25 ° C. to 50 ° C., a constant temperature within the range of 25 ° C. to 40 ° C., a constant temperature within the range of 30 ° C. to 33 ° C., or about 30 ° C. In this specification, "isothermal" in isothermal amplification means maintaining a temperature within a temperature range of ± 7 ° C., ± 5 ° C., ± 3 ° C., or ± 1 ° C. relative to the temperature set during the reaction. The reaction time for isothermal amplification can be appropriately set depending on the size of the target double-stranded DNA, and can be, for example, 20 minutes to 30 hours, preferably 30 minutes to 24 hours, more preferably 1 hour to 12 hours, and even more preferably 2 hours to 9 hours.

[0068] Non-isothermal amplification can be performed under temperature cycle conditions, in which incubation is repeated at two temperatures below 80°C, preferably below 65°C. The first temperature in the temperature cycle is a temperature at which replication initiation of double-stranded DNA can be initiated, and the second temperature is a temperature at which replication initiation is suppressed and DNA extension reaction proceeds. The first temperature can be 30°C or higher, for example, 30°C to 80°C, 30°C to 50°C, 30°C to 40°C, or 37°C. The incubation time at the first temperature is not particularly limited, but may be 10 seconds to 10 minutes, or 30 seconds to 5 minutes per cycle, with approximately 1 minute being preferred. The second temperature can be 27°C or lower, for example, 10°C to 27°C, 16°C to 25°C, or 24°C. The incubation time at the second temperature is not particularly limited, but is preferably set according to the length of the circular DNA to be amplified, and may be, for example, 1 second to 10 seconds per 1,000 bases per cycle. The number of temperature cycles is not particularly limited, but may be 10 to 50 cycles, 20 to 45 cycles, 25 to 45 cycles, or 40 cycles.

[0069] The reaction gel solution may be pre-incubated prior to mixing with the circular DNA. Pre-incubation may be carried out by incubating at a temperature ranging from 0 to 40°C, 10 to 40°C, 15 to 37°C, or 16 to 30°C for 5 to 60 minutes, 5 to 45 minutes, 5 to 30 minutes, 10 to 60 minutes, 10 to 45 minutes, 10 to 30 minutes, 15 to 45 minutes, or 15 to 30 minutes. The temperature of the reaction gel solution during pre-incubation may be maintained within the above-mentioned temperature range, and may fluctuate slightly during pre-incubation.

[0070] Without being bound by theory, amplification of circular DNA by PCR using an amplification gel plate results in repeated replication cycles of the circular DNA, preferably exponentially amplified. According to the method of this embodiment, the above-described circular DNA can be used as a template to amplify it at least 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or 10,000-fold to produce circular DNA. Furthermore, the amplified circular DNA has a supercoiled structure similar to bacterial genomic DNA.

[0071] In one aspect, the present embodiment relates to a method for amplifying DNA, comprising: preparing a mixture of DNA; preparing a reaction gel solution containing the mixture, agarose, and one or more DNA amplification enzymes; gelling the reaction gel solution to prepare an amplification gel plate containing DNA; and performing a DNA amplification reaction on the DNA using the gel plate at a temperature of 80°C or less to obtain amplified DNA.

[0072] In one aspect, the present embodiment relates to a method for amplifying DNA, comprising: preparing a mixture of DNA; preparing a reaction gel solution containing agarose and one or more DNA amplification enzymes; gelling the reaction gel solution to create an amplification gel plate; plating the mixture on the gel plate; and performing a DNA amplification reaction on the DNA using the gel plate at a temperature of 80°C or less to obtain amplified DNA.

[0073] The DNA used in the DNA amplification method of this embodiment may be either single-stranded or double-stranded. It may also be linear or circular. Its origin is not particularly limited, and examples include natural DNA, natural DNA that has been subjected to enzyme treatment, natural DNA to which another DNA fragment has been linked, and artificially synthesized DNA.

[0074] In one aspect of this embodiment, a mixture of DNA is prepared to contain multiple identical or different DNA fragments. For example, multiple types of DNA with different partial DNA sequences can be prepared. More specifically, multiple types of randomized fragments can be prepared and ligated or inserted into DNA to obtain a DNA library, and the resulting DNA library can be prepared as a DNA mixture. In one aspect, the mixture preferably contains multiple different types of DNA. The size of the DNA is not particularly limited and can be, for example, the same size as that of the circular DNA. The mixture may contain DNA of different sizes.

[0075] The type, gelling temperature, and concentration of agarose contained in the reaction gel solution are as described above for the gelling agent.

[0076] The DNA amplification enzyme contained in the reaction gel solution can be appropriately selected depending on the cell-free DNA amplification method to be adopted. The cell-free DNA amplification method can utilize a technique known in the art that can be performed at 80°C or less, and can be either isothermal or non-isothermal amplification. When using isothermal amplification in a cell-free system, various techniques are known (J. Li and J. Macdonald, Biosensors and Bioelectronics, 2015, vol. 64, pp. 196-211). For example, in addition to the PCR method described above, there are Helicase-Dependent Amplification (HDA) (Vincent, et al., EMBO Rep., 2004, vol. 5 (8), pp. 795-800), Recombinase Polymerase Amplification (RPA) (Piepenburg, et al., PLoS. Biol., 2006, vol. 4 (7), e204), and Rolling Circle Amplification (RCA) (Fire, et al., Proc. Natl. Acad. Sci., 1995, vol.92 (10), p.4641-4645), Ramification amplification (RAM) (Zhang, et al, Mol. Diagn., 2001, vol.6 (2), p.141-150), Multiple displacement amplification (MDA) (Dean, et al, Genome Res., 2001, vol.11 (6), p.1095-1099, and Spits, et al, Nat. Protoc., 2006, vol.1 (4), p.1965-1970), Loop-mediated isothermal Known techniques can be used, including, but not limited to, loop-mediated amplification (LAMP) (Notomi, et al., Nucleic Acids Res., 2000, vol. 28 (12), E63), etc. Any of these methods can be performed according to a standard method, and the method to be used can be appropriately selected depending on the shape of the DNA to be amplified, etc.

[0077] The composition of the reaction gel solution is not particularly limited as long as it allows gelation and DNA amplification reaction to proceed.

[0078] In the DNA amplification method of this embodiment, an amplification reaction is performed on DNA using a DNA amplification gel plate obtained by gelling the reaction gel liquid to obtain amplified DNA. The DNA may be mixed with the reaction gel liquid before gelling to gel it, or the DNA may be seeded on the amplification gel plate obtained by gelling the reaction gel liquid. The gelling of the reaction gel liquid, the method of mixing the DNA with the reaction gel liquid before gelling, and the method of seeding the DNA on the amplification gel plate after gelling the reaction gel liquid can be performed in the same manner as in the above-mentioned method of amplifying circular DNA, depending on the DNA amplification method.

[0079] The DNA amplification reaction using a DNA amplification gel plate can be carried out at a temperature of 80°C or less, preferably 65°C or less. The amplification reaction may be non-isothermal or isothermal. During the DNA amplification reaction, it is desirable to prevent evaporation from the gel surface by using techniques such as covering the gel plate or maintaining humidity (e.g., maintaining saturated water vapor pressure).

[0080] In the DNA amplification method of this embodiment, a DNA amplification gel plate obtained by gelling the reaction gel solution is used to carry out an amplification reaction of circular DNA or DNA (hereinafter also simply referred to as "DNA"), thereby obtaining amplified DNA, which is the amplified DNA. In the amplification method of this embodiment, the collection of DNA amplified products obtained after amplification in the gel plate is also referred to as "DNA colonies." At the start of the reaction, each DNA in the DNA mixture is dispersed in the amplification gel plate, preferably so that it separates into single molecules. After amplification progresses in the gel, amplified DNA derived from a single molecule is obtained as a collection that is distinguishable from amplified DNA derived from other molecules. In one aspect of this embodiment, when the DNA mixture contains two or more types of DNA, preferably the amplified DNA contains two or more types of DNA colonies, and more preferably each DNA colony is a collection of amplified products of one type of DNA.

[0081] In one aspect, the DNA amplification method of the present embodiment further includes isolating DNA colonies. Colony isolation can be performed by a method known to those skilled in the art, such as a method using a tip or a method using a colony picker.

[0082] In one aspect, the DNA amplification method of this embodiment includes isolating the DNA colonies and then performing a DNA amplification reaction to obtain amplified DNA from the colonies. This makes it easier to analyze the functional properties of the isolated DNA. The method of DNA amplification using the isolated DNA colonies is not particularly limited as long as the DNA is amplified, and the reaction temperature is also not limited. In one aspect, preferably, the above-mentioned PCR method can be used in the case of circular DNA, and the PCR method can be used in the case of linear DNA.

[0083] In one aspect, the amplification gel plate in the DNA amplification method of this embodiment further contains components necessary for the transcription reaction. In another aspect, the gel plate further contains components necessary for the transcription reaction and components necessary for the translation reaction. The components necessary for the transcription reaction and the components necessary for the translation reaction are conveniently added to the reaction gel solution before gelation, and then gelled to obtain an amplification gel plate containing the components necessary for the transcription reaction or an amplification gel plate containing the components necessary for the transcription reaction and the translation reaction.

[0084] Components necessary for the transcription reaction include enzymes involved in transcription, such as RNA polymerase. For example, when T7 RNA polymerase is included, transcription proceeds from a template DNA containing a T7 promoter. Components necessary for the translation reaction include ribosomes, tRNA, and amino acids and / or amino acid derivatives. Amino acids and amino acid derivatives can be selected appropriately depending on the DNA to be translated, and may be natural or unnatural amino acids. Preferred examples of amino acids include natural amino acids. Preferred examples of amino acid derivatives include homoalanine (the methyl group of alanine is replaced with an ethyl group), methylated lysine (the amino group of lysine is methylated), homoserine (the hydroxyl group of serine is replaced with an ethyl group), nitroanilide derivatives (the carboxyl group is replaced with a natril group), fluoroamino acids (the hydrogen atom is replaced with a fluorine atom), etc., but are not limited to these.

[0085] Components necessary for transcription reactions and components necessary for translation reactions include those described in known literature on in vitro transcription (IVT) and cell-free protein translation, such as Pavlov and Ehrenberg, Arch Biochem Biophys. (1996) Vol. 328, No. 1, pp. 9-16; Shimizu et al., Nature Biotechnology (2001) Vol. 19, pp. 751-755; Ohashi et al., Biochem Biophys Res Commun. (2007) Vol. 352, No. 1, pp. 270-276; Shimizu and Ueda, Methods Mol Biol. (2010) Vol. 607, pp. 11-21.

[0086] For example, commercially available kits and their components used for in vitro transcription (IVT), cell-free protein synthesis, etc. can be used as these components. Examples of such kits include, but are not limited to, PUREfrex (registered trademark) (Gene Frontier), PURExpress (registered trademark) (NEB), TNT (registered trademark) Cell-Free Expression System (Promega), myTXTL (registered trademark) (Arbor Biosciences), Cell-Free Protein Synthesis Reagent Kit (NU Protein), and Mu-Cell-Kun (Taiyo Nippon Sanso). For example, the reconstituted cell-free protein synthesis system (PURE system), which is the basis for PUREfrex (registered trademark), PURExpress (registered trademark), and the like, contains T7 RNA polymerase necessary for transcription reactions, protein factors necessary for E. coli translation (initiation factors (IF1, IF2, IF3), elongation factors (EF-Tu, EF-Ts, EF-G), release factors (RF1, RF2, RF3), ribosome recycling factors, etc.), various aminoacyl-tRNA synthetases (ARS), methionyl-tRNA formyltransferases, purified ribosomes, E. coli tRNA, amino acids, NTPs, an energy regeneration system, and the like, and some or all of these can be used as components necessary for transcription reactions or translation reactions.

[0087] When the amplification gel plate contains components necessary for the transcription reaction, including RNA polymerase, a transcription reaction using the amplified DNA as a template occurs in the amplification gel plate following the DNA amplification reaction, and RNA derived from the amplified DNA is obtained in addition to or instead of the amplified DNA. In one aspect, when the amplification gel plate contains the components necessary for the transcription reaction, RNA colonies can be obtained.

[0088] When an amplification gel plate contains components necessary for a translation reaction, such as ribosomes, tRNA, amino acids, and / or amino acid derivatives, in addition to components necessary for a transcription reaction, such as RNA polymerase, a transcription reaction occurs in the amplification gel plate following a DNA amplification reaction, using the amplified DNA as a template, and a translation reaction occurs using the RNA obtained by transcription as a template, resulting in the production of proteins derived from the amplified DNA in addition to, or instead of, the amplified DNA. In one aspect, when an amplification gel plate contains components necessary for a transcription reaction and components necessary for a translation reaction, protein colonies can be obtained.

[0089] When a transcription reaction and a translation reaction are carried out using the amplified DNA as a template following a DNA amplification reaction, the reaction temperature and reaction time are not particularly limited as long as they are conditions that allow each reaction to occur. The conditions for the DNA amplification reaction are as described above. Furthermore, when using commercially available kits and their components for the transcription and translation reactions, the instructions for each kit can be referred to. In one embodiment, the amplification reaction and / or transcription-translation reaction can be carried out at a temperature of, for example, 15°C or higher, 16°C or higher, 20°C or higher, 25°C or higher, or 30°C or higher, and 80°C or lower, 75°C or lower, 70°C or lower, 65°C or lower, 60°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower. The reaction time can be, for example, 20 minutes to 30 hours, preferably 30 minutes to 24 hours, and more preferably 1 hour to 12 hours. For example, the DNA amplification reaction, transcription reaction, and translation reaction can be carried out by incubating at around 37°C for about 1 to 4 hours.

[0090] In one aspect, the DNA amplification method of this embodiment further includes detecting the DNA colonies. Detection of the DNA colonies may be performed, for example, before isolating the DNA colonies. Detection of the DNA colonies can be performed, for example, by fluorescent image detection using a DNA intercalating fluorescent dye (a fluorescent dye for staining nucleic acids) or a fluorescent DNA probe, or by direct DNA detection using a phase-contrast microscope, or by detecting RNA transcribed from DNA or a protein transcribed and translated from the DNA.

[0091] According to the DNA amplification method of this embodiment, each DNA can be easily separated from a mixture of heterogeneous DNAs, and each DNA can be selected directly using a probe linked to its sequence, or linked to the function (including activity) of RNA and / or protein.

[0092] One aspect of this embodiment relates to a method for detecting circular DNA, comprising: preparing a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction solution containing the circular DNA, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, an RNA polymerase, a ribosome, tRNA, and an amino acid and / or an amino acid derivative; translating the circular DNA using the reaction solution to obtain a translation product; and detecting the translation product.

[0093] The above detection method can be carried out with reference to the above-mentioned circular DNA amplification method, except that the reaction solution does not necessarily contain a gelling agent. The method for detecting the translation product is not particularly limited, and known techniques can be used, such as encoding a fluorescent protein in the circular DNA and detecting using fluorescence intensity, or detecting using tags or probes. This allows each DNA to be easily separated from a mixture of heterogeneous DNAs, and each DNA can be selected by linking it to the function of the protein.

[0094] The present embodiment also relates to a composition for translating a circular DNA, more specifically, a composition for amplifying and translating a circular DNA, and a composition for amplifying, transcribing, and translating a circular DNA, comprising: a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; a first group of enzymes that catalyze the replication of the circular DNA, 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 components necessary for a transcription reaction and components necessary for a translation reaction.

[0095] For each component, the description of the DNA amplification method for circular DNA can be referred to. Components necessary for the transcription reaction preferably include at least RNA polymerase. Components necessary for the translation reaction preferably include at least ribosomes and tRNA. Such a composition is useful for linking each DNA to a protein function and selecting them.

[0096] In one aspect, the present embodiment relates to a method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, wherein the DNA encodes a protein; creating a gel plate containing the mixture, a gelling agent, one or more types of DNA amplification enzymes, components necessary for a transcription reaction, and components necessary for a translation reaction; and translating the two or more types of DNA using the gel plate to obtain colonies of translation products.

[0097] In one aspect, the present embodiment relates to a method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, wherein the DNA encodes a protein; preparing a gel plate containing a gelling agent, one or more types of DNA amplification enzymes, components necessary for a transcription reaction, and components necessary for a translation reaction; plating the mixture on the gel plate; and translating the two or more types of DNA using the gel plate to obtain colonies of translation products.

[0098] The above-mentioned method for detecting DNA may further include detecting the DNA by detecting the translation product. The method for detecting the translation product is not particularly limited, and the above-mentioned methods can be used.

[0099] Components necessary for the transcription reaction preferably include at least RNA polymerase, and components necessary for the translation reaction preferably include at least ribosomes and tRNA, and more preferably include at least ribosomes, tRNA, and amino acids and / or amino acid derivatives.

[0100] The protein encoded by the DNA is not particularly limited, but when the molecular weight of the protein to be expressed is small (for example, but not limited to, 30 kDa or less), it is desirable to suppress diffusion within the gel. For example, the protein can be conferred with DNA-binding ability and immobilized to DNA colonies. Protein domains (DNA-binding domains) used to confer DNA-binding ability include nucleoid protein HU; DNA-binding domains of proteins involved in DNA replication, repair, and modification (e.g., DnaA DNA-binding domain); and DNA-binding domains of transcription factors (e.g., TAL effectors). By using DNA encoding such DNA-binding proteins, the translation product obtained by translating the DNA binds to the DNA colonies without diffusing, allowing the target DNA to be selected using the translation product as an indicator. In one embodiment, the protein is a protein with DNA-binding ability, more specifically, a protein with a DNA-binding domain.

[0101] This embodiment also relates to a gel plate for amplifying circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA; a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs; and agarose, wherein the circular DNA to be amplified has a replication initiation sequence that can bind to an enzyme having DnaA activity. This embodiment also relates to an amplification gel plate that further includes, in addition to the components of the amplification gel plate, a circular DNA to be amplified, the circular DNA having a replication initiation sequence that can bind to an enzyme having DnaA activity. Each component is as described above. Using such a plate, a circular DNA amplification reaction can be performed at a temperature of 80°C or less, preferably 65°C or less.

[0102] This embodiment also relates to a gel plate for translating circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA; 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; agarose; an RNA polymerase, a ribosome, and tRNA; wherein the circular DNA to be translated has a replication initiation sequence that can bind to an enzyme with DnaA activity. This embodiment also relates to a translation gel plate that further comprises the above-mentioned translation gel plate, wherein the circular DNA to be translated has a replication initiation sequence that can bind to an enzyme with DnaA activity. Each component is as described above. Using such a plate, amplification reactions and transcription-translation reactions can be performed at appropriate temperatures to obtain transcription products.

[0103] Example 1 Preparation of DNA Colonies A solution containing circular DNA containing oriC and an enzyme for PCR was solidified with agarose to prepare a PCR plate, and a PCR amplification reaction was carried out using the plate.

[0104] (Preparation of Slide Glass) A microarray slide frame (Gene Frame, 25 μL (1.0×1.0 cm), manufactured by Thermo Scientific) was attached to a slide glass ( FIG. 1A ).

[0105] (Preparation of circular DNA) RA ligation was performed using the 6 nM 2.8 kb UPL fragment (SEQ ID NO: 4) and the 6 nM 2.0 kb UPR fragment (SEQ ID NO: 5) shown in Tables 1 and 2 to obtain a 4.7 kb ligate containing oriC. The ligation reaction was carried out by adding each fragment to an RA ligation reaction solution consisting of the following composition, utilizing the 40 base pair overlap sequences added to both ends of each fragment, and reacting at 42°C for 30 minutes. The resulting ligate was cloned using E. coli to obtain a 4.7 kb plasmid.

[0106] Composition of the RA ligation reaction solution: 1 μM wild-type RecA (purified from an E. coli expression strain of RecA through a process including polyethyleneimine precipitation, ammonium sulfate precipitation, and affinity column chromatography), 80 mU / μL exonuclease III (2170A, TaKaRa Bio), 1 U / μL exonuclease I (M0293, New England Biolabs), 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM tetramethylammonium chloride (TMAC), 5% by mass PEG8000, 10% by volume DMSO, 20 ng / μL creatine kinase (10127566001, Sigma-Aldrich), and 4 mM creatine phosphate. The concentration of each component in the RA reaction solution is the concentration relative to the total volume of the RA ligation reaction solution.

[0107]

[0108]

[0109] (Preparation of RCR Plate) Ultrapure water was added to agarose (PrimeGel™ Agarose LMT 1-20K, manufactured by TaKaRa) and dissolved by heating to prepare 4% low-melting-point agarose. 30 μL (final concentration 2%) of low-melting-point agarose was dispensed per sample and kept at 50°C.

[0110] Next, an RCR reaction mixture was prepared to have a concentration twice that shown in Table 3 (2xRCR mix-1, total 28.4 μL), and pre-incubated at 33°C for 30 minutes. 1 μL of circular DNA was added to the 2xRCR mix-1 so that the final number of molecules per RCR plate (25 μL) was 10, 50, or 100 molecules / rate, and 0.6 μL (final concentration 0.1 μM) of fluorescent nucleic acid staining dye (10 μM YOYO™-1 Iodide, Invitrogen) was added and mixed by pipetting.

[0111] This solution was added to 4% low-melting-point agarose that had been warmed at 50°C and mixed thoroughly by pipetting. 45 μL of the total 60 μL was poured into a Gene Frame attached to a glass slide. A plastic cover was then attached to the Gene Frame to prevent evaporation, and the Gene Frame was cooled at 4°C for 5 minutes to solidify the agarose (final agarose concentration: 2%).

[0112] (RCR amplification reaction using RCR plate) The prepared RCR plate was wrapped in aluminum foil to block light, and placed in an air incubator at 37°C for reaction for 3 hours. After the reaction, the RCR plate was observed under a fluorescence microscope (Axio Observer Z1, manufactured by Zeiss) and the number of colonies was counted.

[0113] The experimental outline and results are shown in Figures 1 and 2. Figure 1 shows a schematic diagram of DNA colony formation using an RCR plate. Figure 2 shows the results of photographing the RCR plate after DNA amplification using a fluorescence microscope. As shown in Figure 2, it was found that DNA was amplified in the form of colonies by performing an RCR amplification reaction using an RCR plate. Furthermore, the number of colonies correlated with the number of DNA molecules added.

[0114]

[0115] In Table 3, SSB is SSB derived from E. coli, IHF is a complex of IhfA and IhfB derived from E. coli, DnaG is DnaG derived from E. coli, DnaN is DnaN derived from E. coli, Pol III* is a DNA polymerase III* complex which is a complex consisting of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from E. coli, DnaB is DnaB derived from E. coli, DnaC is DnaC derived from E. coli, DnaA is DnaA derived from E. coli, RNaseH is RNaseH derived from E. coli, Ligase is DNA ligase derived from E. coli, Pol I is DNA polymerase I derived from E. coli, GyrA is GyrA derived from E. coli, GyrB is GyrB derived from E. coli, Topo IV represents a complex of ParC and ParE derived from E. coli, Topo III represents topoisomerase III derived from E. coli, and RecQ represents RecQ derived from E. coli.

[0116] SSB was purified and prepared from an E. coli strain expressing SSB using a process including ammonium sulfate precipitation and ion exchange column chromatography. IHF was purified and prepared from an E. coli strain co-expressing IhfA and IhfB using a process including ammonium sulfate precipitation and affinity column chromatography. DnaG was purified and prepared from an E. coli strain expressing DnaG using a process including ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography. DnaN was purified and prepared from an E. coli strain expressing DnaN using a process including ammonium sulfate precipitation and anion exchange column chromatography. Pol III* was purified and prepared from an E. coli strain co-expressing DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaB and DnaC were purified and prepared from an E. coli strain co-expressing DnaB and DnaC using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaA was purified and prepared from an E. coli strain expressing DnaA using a process including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography. GyrA and GyrB were purified and prepared from a mixture of an E. coli strain expressing GyrA and an E. coli strain expressing GyrB using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo IV was purified and prepared from a mixture of an E. coli strain expressing ParC and an E. coli strain expressing ParE using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo III was purified and prepared from a Topo III-expressing E. coli strain through a process including ammonium sulfate precipitation and affinity column chromatography. RecQ was purified and prepared from a RecQ-expressing E. coli strain through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. RNase H, Ligase, and Pol I were commercially available enzymes derived from E. coli (Takara Bio).Tus was purified and prepared from an E. coli expression strain of Tus through a process including affinity column chromatography and gel filtration column chromatography.

[0117] Example 2 Preparation of DNA colonies by spreading DNA after preparation of RCR plates DNA colonies were prepared by spreading circular DNA on the plates after preparation of the RCR plates without adding circular DNA during the preparation of the RCR plates.

[0118] A PCR plate containing no DNA was prepared using the same method as in Example 1, except that no DNA was added and 50 nM BOBO™-3 Iodide (Invitrogen) (final concentration: 0.5 μM) was used as the fluorescent dye for nucleic acid staining.

[0119] 1 μL of circular DNA (4.7 kb, same as in Example 1) diluted to 100 molecules / μL was dropped onto the obtained plate and spread using the side of the tip. An PCR amplification reaction was carried out using the PCR plate under the same conditions as in Example 1, and after the reaction, the reaction was observed using a fluorescence microscope.

[0120] The results are shown in Figure 3. Even when a PCR plate containing no DNA was prepared and then DNA was spread on the gel, PCR amplification reaction was carried out and DNA colonies could be produced.

[0121] Example 3: Creation of DNA colonies of different DNA sizes. First, circular DNAs containing oriC of four different sizes, ranging from 2.3 kb to 23 kb, were prepared so that the final number of DNA molecules per PCR plate (25 μL) was 50, and PCR plates (agarose concentration 2%) were prepared in the same manner as in Example 1. PCR amplification reactions were then carried out at 37°C for 4.5 hours. Fluorescence microscopic images of the DNA colonies are shown in Figure 4A.

[0122] Four types of circular DNA were prepared as follows: 2.3 kb circular DNA containing oriC: pCLter_2 described in Hasebe et al., Life (2018) Vol. 8, No. 43 was used. 4.7 kb circular DNA containing oriC: The same DNA used in Example 1 was used. 9.4 kb circular DNA containing oriC: pOri8 described in Su'etsugu et al., Nucleic Acids Res. (2017) Vol. 45, pp. 11525-11534 was used. 23-kb circular DNA containing oriC: In a similar manner to pOri8 described in Su'etsugu et al., Nucleic Acids Res. (2017) Vol. 45, pp. 11525-11534, an E. coli genomic region containing 10 kb upstream and downstream of oriC was prepared by the pop-out method using E. coli recombination.

[0123] Next, DNA colonies were prepared using 93 kb circular DNA containing oriC. Because the circular DNA was long, the DNA was applied to the PCR plate after preparation, as in Example 2, in order to prevent damage to the DNA during mixing with the melted gel. ６ The same procedure as in Example 2 was used, except that the DNA of the molecule was plated and PCR amplification reaction was carried out for 9 hours at 37° C. A fluorescent microscope image of the DNA colony taken using the same procedure as in Example 1 is shown in FIG.

[0124] The 93 kb circular DNA used is a plasmid containing the E. coli genome region and was prepared by changing the ligation fragment (KOZins fragment) to a CLter fragment (SEQ ID NO: 6) according to the preparation method of pOri93Zins described in WO 2020 / 027110, Example 7. The CLter fragment is a fragment containing oriC and chloramphenicol resistance genes and was prepared by PCR using pCLter2k as a template. The sequence of the CLter fragment is shown below.

[0125]

[0126] Next, a PCR plate with a 3% agarose concentration was prepared using the 2.3 kb circular DNA again, and DNA colonies were prepared in the same manner as in Example 1. A fluorescent microscope image of the DNA colonies is shown in Figure 4(C).

[0127] As shown in Figure 4, it was confirmed that DNA colonies can be created using a wide range of DNA sizes. As shown in Figure 4(A), at an agarose concentration of 2%, DNA colonies consisting of 2.3 kb circular DNA were diffuse and unclear. Clear DNA colony formation was confirmed for circular DNA ranging from 4.7 kb to 23 kb. The larger the DNA size, the clearer and smaller the colony shape. As shown in Figure 4(B), DNA colonies consisting of 93 kb circular DNA were even smaller. As shown in Figure 4(C), clear colonies were confirmed to form even with the small 2.3 kb circular DNA when the agarose concentration was increased (from 2% to 3%).

[0128] Example 4 Isolation of DNA molecular species from a circular DNA library DNA colonies were generated using a circular DNA library containing various UMIs (Unique Molecular Identifiers). Each DNA colony was then picked and PCR-amplified, and the amplified product was sequenced by next-generation sequencing (NGS) to confirm whether the DNA colony consisted of a single DNA molecular species.

[0129] (Creation of a circular DNA library containing UMI) A circular DNA library containing seven random bases (N=7) was created by self-ligation and circularization of PCR fragments using the pUP-T7-GFP plasmid as a template. The pUP-T7-GFP plasmid was created by ligating and circularizing the pUP fragment (4.4 kb) and the T7-GFP fragment (1.1 kb) using the In-Fusion® HD Cloning Kit (TaKaRa). The pUP fragment was obtained by PCR of a 4.4 kb region containing oriC using the circular DNA (4.7 kb) created in Example 1 as a template and the primers shown in Table 5. The sequence of the T7-GFP fragment is shown in Table 6.

[0130]

[0131]

[0132] Next, PCR was performed using pUP-T7-GFP as a template and the primers listed in Table 7 to obtain a pUP-T7GFP-N7UMI fragment having a random 7-base sequence and containing restriction enzyme NheI recognition sites at both ends.

[0133]

[0134] The pUP-T7GFP-N7UMI fragment was cleaved with the restriction enzyme NheI and then self-ligated and circularized using Ligation High Ver. 2 (manufactured by TOYOBO Co., Ltd.). DNA colonies were formed using 1.2 pg of this ligated circularized product according to the method of Example 1.

[0135] (Colony Pick Method) Fluorescent images of DNA colonies were confirmed, and each colony was picked with a pipette tip and suspended in TE buffer. Next, 1 μL of colony pick solution was added to 9 μL of PCR solution having the composition shown in Table 3, and PCR amplification reaction was carried out by performing 30 temperature cycles of 37°C for 1 minute, followed by 24°C for 29 minutes. As a control, 1 μL of the ligation product before DNA colony creation was added to the PCR solution, and PCR amplification reaction was carried out in the same manner.

[0136] (NGS analysis) NGS analysis was performed using the PCR amplification reaction product of the colony pick solution, and the sequence and frequency of each sequence were analyzed for the UMI portion. For the NGS analysis, iSeq 100 (manufactured by Illumina) was used.

[0137] A schematic diagram of the experiment is shown in Figure 5. Figure 6 also shows a graph of the frequency of UMI sequences in each DNA colony. The leftmost lane in Figure 6 shows the frequency of each UMI sequence in the control (the PCR amplification product of the UMI plasmid before colony formation), and 8,061 types of UMI were identified. Meanwhile, for the amplification products after DNA colony picking, nearly a single UMI (97% or more) was identified in seven of the eight samples (lanes 1-7). When DNA colonies were created using the PCR plate, monoclonal amplification from a single circular DNA molecule was achieved in each colony, demonstrating the feasibility of isolating DNA species using colony picking. The minor UMI sequences present in the samples in lanes 4-7 in Figure 6 may be due to errors that occurred during PCR amplification or sequencing for NGS analysis. The sample in which a single UMI was not identified (lane 8) was likely due to multiple colonies being picked due to a colony pick error.

[0138] Example 5 Selection and isolation of DNA molecular species based on gene expression using PURE-RCR plate By combining PUREfrex (registered trademark), a cell-free transcription-translation reaction system, with an RCR plate, DNA-encoded information (protein) was displayed by DNA colonies, and the proteins were detected as colonies, and the target DNA was selected and isolated based on this.

[0139] The plasmid DNAs used to prepare DNA colonies were pET-HUmCdB-oriC and pET-HUGM-oriC, which encode the fluorescent proteins mCherry and GFP, respectively, and further comprise plasmids in which the DNA-binding protein HU is fused to these fluorescent proteins.

[0140] pET-HUGM was prepared by cloning the hupA gene derived from Escherichia coli MG1655 as an sfGFP-fused gene into pET21a (Novagen). The sequence of the sfGFP-fused hupA gene is shown in Table 8.

[0141]

[0142] pET-HUGM-oriC was prepared by inserting the PoriC_terG16 cassette (a cassette having a pair of mutant ter sequences inserted facing outward relative to oriC) shown in Table 10, to which overlapping sequences were added using the ColE1_Fw and ColE1_Rv primers shown in Table 9, into pET-HUGM by homologous recombination. The homologous recombination reaction was carried out using the method described in Example 1 of WO 2023 / 038145.

[0143]

[0144]

[0145] pET-HUmCdB-oriC was created by replacing the sfGFP region (in uppercase letters in Table 8) of pET-HUGM-oriC with the mCdB region. The mCdB region contains the mCherry and dBroccoli sequences. The sequence is shown in Table 11.

[0146]

[0147] (Creation of DNA colonies by combining PUREfrex® and PCR) Ultrapure water was added to agarose (Ultra Low Gelling Temperature Agarose, Sigma), and the mixture was heated and dissolved to prepare a 4% agarose solution. 30 μL of the agarose solution (final concentration 2%) was dispensed per sample and kept at 45°C.

[0148] Next, 2xRCRmix-2 was prepared. 2xRCRmix-2 is a 2x concentrated solution of the enzyme group portion of the composition in Table 3 (Total 24.5 uL), and its reaction buffer portion was replaced with 0.8x concentrated Solution I (supplied with PUREfrex (registered trademark) 2.0 manufactured by GeneFrontier), and 0.2 mM of each dNTP was added. This 2xRCRmix-2 was pre-incubated at 33°C for 15 minutes.

[0149] To 2xRCRmix-2, 1.5 μL of PUREfrex (registered trademark) (GeneFrontier) Solution II and 3 μL of Solution III were added. Each DNA was added so that 100 molecules of pET-HUmCdB-oriC and 100 molecules of pET-HUGM-oriC were present per 25 μL plate.

[0150] The resulting mixture was added to a 4% agarose solution kept at 45°C and mixed well by pipetting. 45 μL of the total 60 μL was poured into a Gene Frame attached to a glass slide in the same manner as in Example 1. Then, a cover was placed on the Gene Frame, and the mixture was cooled at 4°C for 5 minutes to solidify the agarose, thereby preparing a PURE-RCR plate.

[0151] After 3 hours of incubation at 37°C, the fluorescent images of colonies displaying red fluorescence (mCherry) (mCherry colonies) and colonies displaying green fluorescence (GFP) (GFP colonies) were detected using a fluorescence microscope. Subsequently, three samples of mCherry colonies and one sample of GFP colonies were picked using the same method as in Example 4. PCR amplification reactions were carried out for each sample at 33°C for 6 hours.

[0152] (Discrimination of molecular species by PCR) After colony picking, PCR products were amplified by PCR using the primers in Table 12, and then DNA molecular species were identified by size comparison using gel electrophoresis.

[0153]

[0154] Schematic diagrams of the sequences (partial) and the results are shown in Figures 7 to 9. Figures 7(A) and (B) are red fluorescent microscopic images (A) and green fluorescent microscopic images (B), respectively, of colonies after reaction of circular DNA using a PURE-PCR plate. Figure 7(C) is a superposition of the microscopic images of Figures 7(A) and 7(B). An image of an mCherry colony displaying the mCherry fluorescent protein (Figure 7(A)) and an image of a GFP colony displaying the GFP fluorescent protein (Figure 7(B)) were observed in different positions. Figure 7(D) is an illustration of the photograph in Figure 7(C). Figure 8 shows a schematic diagram of the DNA size amplified when the two types of plasmid DNA used were subjected to PCR using the same primers (Table 12). When pET-HUGM-oriC was subjected to PCR, a 1.2 kb fragment was amplified. When pET-HUmCdB-oriC was subjected to PCR, a 2.0 kb fragment was amplified. Figure 9 shows the results of size analysis of each colony-picked sample after PCR amplification. Lanes 1 to 3 are from the three samples in Figure 7(A), and lane 4 is from one sample in Figure 7(B). A 2.0 kb fragment was amplified from the mCherry colony, confirming that a plasmid containing the mCherry gene had been selected and isolated. Similarly, a 1.2 kb fragment was amplified from the GFP colony, confirming that a plasmid containing the gfp gene had been selected and isolated. It was confirmed that the use of the PURE-PCR plate makes it possible to select and isolate a target DNA molecule species from multiple types of DNA molecules, in association with the function of the protein expressed from the gene encoded by the plasmid DNA.

[0155] [Example 6] Colony formation of mRNA transcribed from DNA colonies We investigated whether mRNA transcribed from DNA colonies could be detected as colonies displayed on DNA, similar to the case of proteins in Example 5. An mRNA aptamer was used for fluorescent detection of mRNA.

[0156] Among the plasmid DNAs used to create DNA colonies in Example 5, pET-HUmCdB-oriC is a plasmid that encodes the fluorescent protein mCherry and the fluorescent RNA aptamer dBroccoli (Grigory et al., J. Am. Chem. Soc., 2014, vol. 136, No. 46, pp. 16299-16308). Using pET-HUmCdB-oriC, a PURE-RCR plate was created using the same method as in Example 5, allowing PCR amplification and transcription-translation reactions to proceed. Note that 10 μM DHFBI (Lucerna), a fluorescent detection reagent for dBroccoli, was added to the plate composition. DNA amplification, transcription, and translation reactions were carried out using the PURE-RCR plate in the same manner as in Example 5. Thereafter, a fluorescent microscope image of green mRNA (dBroccoli-DHFBI) was detected together with a fluorescent microscope image of red mCherry protein.

[0157] The results of observation using a fluorescence microscope are shown in Figure 10. Figure 10(A) shows a red fluorescent image derived from the fluorescent protein mCherry, and the results of Example 5 indicate that mCherry colonies are formed when displayed on DNA colonies. The green fluorescent image shown in Figure 10(B) is derived from DHFBI, which specifically binds to the structure of Broccoli mRNA and emits green fluorescence. The observation of green fluorescent colonies indicated that mRNA also forms colonies. In the merged image shown in Figure 10(C), in which the red and green fluorescent images are superimposed, the colonies overlap, confirming that mRNA transcribed from DNA colonies, along with the expressed protein, can be detected as colonies displayed on template DNA.

Claims

1. A method for amplifying DNA, comprising: preparing a mixture of DNA, the DNA being circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing the mixture, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to create an amplification gel plate containing DNA; and using the gel plate to perform a circular DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA.

2. A method for amplifying DNA, comprising: preparing a mixture of DNA, the DNA being circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction gel solution containing a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to prepare an amplification gel plate; plating the mixture on the gel plate; and using the gel plate to perform a circular DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA.

3. The method of claim 1 or 2, wherein the gelling agent is selected from the group consisting of agarose, polyacrylamide, agar, carrageenan, alginic acid, alginates, gellan gum, pectin, collagen, gelatin, gluten, polyvinyl alcohol, polyethylene glycol, polyacrylamide, agaropectin, polyacrylic acid, polyvinylpyrrolidone, and combinations of one or more thereof.

4. The method of claim 1 or 2, wherein the gelling agent is agarose.

5. A method for amplifying DNA, comprising: preparing a mixture of DNA; preparing a reaction gel solution containing the mixture, agarose, and one or more DNA amplification enzymes; gelling the reaction gel solution to create an amplification gel plate containing DNA; and using the gel plate to perform a DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA.

6. A method for amplifying DNA, comprising: preparing a mixture of DNA; preparing a reaction gel solution containing agarose and one or more DNA amplification enzymes; creating an amplification gel plate by gelling the reaction gel solution; plating the mixture on the gel plate; and using the gel plate to perform a DNA amplification reaction on the DNA at a temperature of 80°C or less to obtain amplified DNA.

7. The method of claim 1 or 2, wherein the mixture comprises two or more types of DNA and the amplified DNA comprises two or more types of DNA colonies.

8. The method of claim 7, further comprising detecting at least one of said two or more DNA colonies.

9. The method of claim 7, further comprising: isolating at least one of said two or more DNA colonies; and performing a DNA amplification reaction on the isolated DNA colony to obtain amplified DNA from the colony.

10. The method of claim 5 or 6, wherein the mixture comprises two or more types of DNA and the amplified DNA comprises two or more types of DNA colonies.

11. The method of claim 10, further comprising detecting at least one of said two or more DNA colonies.

12. The method of claim 10, further comprising: isolating at least one of said two or more DNA colonies; and performing a DNA amplification reaction on the isolated DNA colony to obtain amplified DNA from the colony.

13. The method according to claim 1 or 2, wherein the gel plate further contains an RNA polymerase, and further comprising: carrying out a transcription reaction in the gel plate using the amplified DNA as a template to obtain RNA.

14. The method according to claim 13, wherein the gel plate further comprises ribosomes, tRNA, and amino acids and / or amino acid derivatives, and further comprising carrying out a translation reaction in the gel plate using the RNA as a template to obtain a protein.

15. The method according to claim 5 or 6, wherein the gel plate further contains an RNA polymerase, and further comprising: carrying out a transcription reaction in the gel plate using the amplified DNA as a template to obtain RNA.

16. The method according to claim 15, wherein the gel plate further contains ribosomes, tRNA, and amino acids and / or amino acid derivatives, and further comprises carrying out a translation reaction in the gel plate using the RNA as a template to obtain a protein.

17. A method for detecting circular DNA, comprising: preparing a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; preparing a reaction solution containing the circular DNA, a first group of enzymes that catalyze the replication of the circular DNA, a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs, RNA polymerase, ribosomes, tRNA, and amino acids and / or amino acid derivatives; translating the circular DNA using the reaction solution to obtain a translation product; and detecting the translation product.

18. A composition for translating circular DNA, comprising: a circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity; a first group of enzymes that catalyze the replication of the circular DNA; 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; an RNA polymerase, a ribosome, and a tRNA.

19. A method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, the DNA encoding a protein; creating a gel plate containing the mixture, a gelling agent, one or more types of DNA amplification enzymes, an RNA polymerase, ribosomes, and tRNA; and using the gel plate to translate the two or more types of DNA to obtain colonies of translation products.

20. A method for detecting DNA, comprising: preparing a mixture of two or more types of DNA, the DNA encoding a protein; creating a gel plate containing a gelling agent, one or more types of DNA amplification enzymes, an RNA polymerase, ribosomes, and tRNA; plating the mixture on the gel plate; and using the gel plate to translate the two or more types of DNA to obtain colonies of translation products.

21. A gel plate for amplifying circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA; a second group of enzymes that catalyze an Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyze a separation reaction of the two sister circular DNAs; and agarose, wherein the circular DNA has a replication initiation sequence that can bind to an enzyme having DnaA activity.

22. A gel plate for translating circular DNA, comprising: a first group of enzymes that catalyze the replication of circular DNA, 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; agarose; RNA polymerase, ribosomes, and tRNA; wherein the circular DNA has a replication initiation sequence that can bind to an enzyme having DnaA activity.

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