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

Abstract

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 individual gene species. [Solution] A method for amplifying DNA, comprising the steps of: preparing a mixture of DNA which is circular DNA having a replication initiation sequence that can bind to an enzyme having DnaA activity; preparing a reaction gel solution containing the DNA mixture, a first group of enzymes that catalyze the replication of circular DNA, a second group of enzymes that synthesize two sister circular DNAs that form catenanes by catalyzing an Okazaki fragment ligation reaction, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, and a gelling agent; gelling the reaction gel solution to create an amplification gel plate containing circular DNA; and using the amplification gel plate to carry out a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product.

Description

Technical Field

[0001] The present invention relates to a method for selecting and isolating DNA species in a cell-free system. In particular, the present invention relates to a method for simultaneously amplifying, selecting and isolating a plurality of DNA species having a very small number of molecules in a cell-free system.

Background Art

[0002] Attempts have been made to prepare a pool of heterologous genes with different sequences, such as a DNA library, and screen for useful genes therefrom. Selection of individual genes from such a pool of heterologous genes is useful in various scenarios, including screening for target genes such as therapeutic genes, screening for disease risks associated with mutations, and elucidating interactions between DNA or proteins encoded thereby and other proteins, and may be applicable to diverse fields related to genome characterization, gene expression research, medical diagnosis, population genetics and the like.

[0003] Furthermore, unlike DNA isolated using cells, plasmids that have undergone the entire process from construction to amplification in a cell-free system contain plasmids having mutations generated during amplification, non-specific amplification products, and the like. Since this poses a problem when using such plasmids in subsequent experiments, isolation and selection of individual amplification products may be required.

[0004] As such a method for isolating and selecting heterologous genes, research on preparing individual gene colonies (so-called DNA colonies) from a pool of heterologous genes has been advanced. For example, Non-Patent Document 1 and Patent Document 1 (Example 15) disclose that colony-shaped "Polony (PCR + Colony)" formed by monoclonal DNA was prepared 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 single-molecule amplification of template DNA by LAMP (Loop-mediated isothermal amplification) in a microchamber containing polyacrylamide gel, primers, and polymerase. Non-Patent Document 4 is a review of single-molecule DNA amplification and analysis methods using microfluidics. Non-Patent Document 5 discloses the amplification of cell-derived DNA in a gel droplet using Phi29 DNA polymerase.

[0006] On the other hand, the RCR method is known as a method for amplifying DNA in a cell-free system (Patent Documents 2-4). As a method for isolating and selecting genes using the RCR method, a method has been attempted in which a pool of heterologous genes is ultradiluted and then amplified by the RCR method. However, ultradilution does not guarantee the isolation of a single molecule and is probabilistic, and a large amount of sample is required for dilution. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2000 / 053812 [Patent Document 2] International Publication No. 2016 / 080424 [Patent Document 3] International Publication No. 2017 / 199991 [Patent Document 4] International Publication No. 2018 / 159669 [Non-patent literature]

[0008] [Non-Patent Document 1] Mitra and Church, Nucleic Acids Research, 1999, vol.27, No.24, e34. [Non-Patent Document 2] Chetverina and Chetverin, Biochemistry (Moscow), 2008, Vol.73, No.13, p.1361-1387. [Non-Patent Document 3] Lam et al., Biomed Microdevices, 2008, Vol.10, p.539-546. [Non-Patent Document 4] Zhang and Xing, Chemical Reviews, 2010, Vol.110, No.8, p.4910-4947. [Non-Patent Document 5] Michikawa et al., Anal Biochem., 2008, Vol.383, No.2, p.151-158. [Overview of the project] [Problems that the invention aims to solve]

[0009] Conventional single-molecule DNA amplification methods, as described above, have problems that make them difficult to implement simply, such as the need to use temperature cycling, prepare fine compartments, and prepare large quantities of sample. Furthermore, there is also the problem that a single DNA molecule is not always isolated. Furthermore, selecting a target gene by confirming its phenotype (functional expression), as is done with blue-white selection using E. coli, had not been performed in colonies created using cell-free systems.

[0010] Against this backdrop, the primary objective of the present invention is to provide a method for easily isolating individual gene species from a plurality of 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 an indicator derived from the individual gene species. [Means for solving the problem]

[0011] The inventors have discovered that by performing the RCR method on a gel scaffold, it is possible to amplify a very low concentration of heterogeneous DNA molecules individually and separate and select them as single-sequence DNA colonies. Furthermore, they have found that functions such as protein expression linked to the DNA information can also be confirmed in the colonies within the gel, thus completing the present invention.

[0012] In other words, the methods, etc., according to the present invention are as follows [1] to

[20] . [1] A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising the aforementioned DNA mixture, a first group of enzymes that catalyzes the replication of circular DNA, a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, and a gelling agent. The process involves gelling the reaction gel solution to create an amplification gel plate containing DNA, The process involves using the amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted]. [2] A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence that can bind to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising: a first group of enzymes that catalyzes the replication of circular DNA; a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs; and a gelling agent. The process involves gelling the aforementioned reaction gel solution to create an amplification gel plate, The steps include: seeding the DNA mixture onto the amplification gel plate; The process involves using the amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted]. [3] The method according to [1] or [2], wherein the gelling agent is one or more gelling agents 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, and polyvinylpyrrolidone. [4] The method according to [3], wherein the gelling agent is agarose. [5] a step of preparing a mixture of DNA; a step of preparing a reaction gel solution comprising the mixture of DNA, agarose, and an enzyme for DNA amplification; a step of gelling the reaction gel solution to prepare an amplification gel plate containing DNA; a step of performing a DNA amplification reaction at a temperature of 80°C or lower using the amplification gel plate to obtain an amplified DNA product; A method for amplifying DNA, comprising: [6] a step of preparing a mixture of DNA; a step of preparing a reaction gel solution comprising agarose and an enzyme for DNA amplification; a step of preparing an amplification gel plate by gelling the reaction gel solution; a step of seeding the mixture of DNA onto the amplification gel plate; a step of performing a DNA amplification reaction at a temperature of 80°C or lower using the amplification gel plate to obtain an amplified DNA product; A method for amplifying DNA, comprising: [7] The method according to any one of [1] to [4], wherein the mixture of DNA comprises two or more types of DNA, and the amplified DNA product comprises two or more types of DNA colonies. [8] The method according to [7], further comprising a step of detecting the DNA colonies. [9] The method according to [7] or [8], further comprising a step of isolating the DNA colonies, and a step of performing a DNA amplification reaction using the isolated DNA colonies to obtain a colony-derived amplified DNA product.

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

[11] The method of

[10] further comprising the step of detecting the DNA colony.

[12] The method of

[10] or

[11] further comprising the steps of isolating the DNA colony and carrying out a DNA amplification reaction using the isolated DNA colony to obtain a colony-derived DNA amplified product.

[13] The amplification gel plate further comprises RNA polymerase, Furthermore, the process involves obtaining RNA by performing a transcription reaction using the amplified DNA as a template in the amplification gel plate, Any of the methods described in [1] to [4], [7] to [9] above, including the above.

[14] The amplification gel plate further comprises ribosomes, tRNA, and amino acids or derivatives thereof. Furthermore, the process involves performing a translation reaction using the RNA as a template in the amplification gel plate to obtain a protein, The method of

[13] , including the above.

[15] The amplification gel plate further comprises RNA polymerase, Furthermore, the process involves obtaining RNA by performing a transcription reaction using the amplified DNA as a template in the amplification gel plate, The method described in any of the preceding [5], [6],

[10] to

[12] , including the above.

[16] The amplification gel plate further comprises ribosomes, tRNA, and amino acids or derivatives thereof, Furthermore, the process involves performing a translation reaction using the RNA as a template in the amplification gel plate to obtain a protein, The method of

[15] , including the following:

[17] A step of preparing circular DNA having a replication initiation sequence that can bind to an enzyme having DnaA activity, A step of preparing a reaction solution comprising the aforementioned circular DNA, a first group of enzymes that catalyzes the replication of the circular DNA, a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a catenane, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, RNA polymerase, ribosomes, tRNA, and amino acids or their derivatives. A step of obtaining a translation product derived from circular DNA using the reaction solution, The step of detecting the aforementioned translation product, A method for detecting circular DNA, including [specific details omitted].

[18] A circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A first group of enzymes catalyzes the replication of circular DNA, a second group of enzymes catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and a third group of enzymes catalyzes the separation reaction of the two sister circular DNAs. RNA polymerase, ribosomes, and tRNA, A translation composition for circular DNA, including [the specified element].

[19] The process of preparing a mixture of DNA that codes for proteins, A step of preparing a gel plate containing the aforementioned DNA mixture, a gelling agent, a DNA amplification enzyme, RNA polymerase, ribosomes, and tRNA. A step of obtaining colonies of the DNA-derived translation product using the gel plate, A method for detecting DNA, including the detection of DNA.

[20] The process of preparing a mixture of DNA that codes for proteins and A process for creating a gel plate containing a gelling agent, a DNA amplification enzyme, RNA polymerase, ribosomes, and tRNA, The steps include: seeding the DNA mixture onto the gel plate; A step of obtaining colonies of the DNA-derived translation product using the gel plate, A method for detecting DNA, including the detection of DNA. [Effects of the Invention]

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

[0014] [Figure 1] Figure 1 shows an overview of Example 1. Figure 1 is a schematic diagram of the method for preparing an RCR plate and the DNA amplification method using the RCR plate. [Figure 2] Figure 2 shows the results of Example 1, which are images of RCR plates after amplification of DNA with different molecular sizes, taken using a fluorescence microscope. [Figure 3] Figure 3 shows DNA colonies created in Example 2 by spreading DNA onto an RCR plate that was prepared without DNA mixing. [Figure 4] Figure 4 shows the results of Example 3. Figure 4(A) shows DNA colonies prepared by pre-mixing 2.3kb to 23kb circular DNA with a gel (2% agarose). Figure 4(B) shows DNA colonies prepared by coating 93kb circular DNA onto an RCR plate (2% agarose). Figure 4(C) shows DNA colonies prepared by pre-mixing 2.3kb circular DNA with a gel (3% agarose). [Figure 5] Figure 5 is a schematic diagram of the experiment in Example 4. [Figure 6] Figure 6 is a graph showing the frequency of UMI sequences in each DNA colony obtained by NGS analysis in Example 4. [Figure 7] Figure 7 shows the results of Example 5. Figures 7(A) and 7(B) are red fluorescence microscope images (A) and green fluorescence microscope images (B) of circular DNA after reaction using a PURE-RCR plate, respectively. Figure 7(C) is an overlay image of Figures 7(A) and 7(B), and Figure 7(D) is an illustration of the photograph in 7(C). [Figure 8] Figure 8 shows a schematic diagram of the DNA size amplified when the two plasmid DNAs used in Example 5 are subjected to PCR using the same primers. [Figure 9] Figure 9 shows the results of electrophoresis of the RCR amplified product after colony picking, from the results of Example 5. [Figure 10] Figure 10 shows the results of Example 6. Figures 10(A) and (B) are red fluorescence microscope images (A) and green fluorescence microscope images (B) of circular DNA after reaction using a PURE-RCR plate, respectively. Figure 10(C) is an overlay image of Figures 10(A) and 10(B). [Modes for carrying out the invention]

[0015] The following describes specific embodiments for carrying out the present invention (hereinafter also referred to as these embodiments), but the present invention is not limited to these embodiments.

[0016] In one embodiment, A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising the aforementioned DNA mixture, a first group of enzymes that catalyzes the replication of circular DNA, a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, and a gelling agent. The process involves gelling the reaction gel solution to create an amplification gel plate containing DNA, The process involves using the amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, This relates to DNA amplification methods, including [specific method / technique].

[0017] This embodiment also, in one embodiment, A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising: a first group of enzymes that catalyzes the replication of circular DNA; a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs; and a gelling agent. The process involves gelling the aforementioned reaction gel solution to create an amplification gel plate, The steps include: seeding the DNA mixture onto the amplification gel plate; The process involves using the amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, This relates to DNA amplification methods, including [specific method / technique].

[0018] In the DNA amplification method of this embodiment, the DNA used is, in one embodiment, circular DNA, and any DNA having a circular structure may be single-stranded or double-stranded, but double-stranded is preferred. Its origin is not particularly limited, and examples include naturally occurring circular DNA such as the circular chromosomes of microorganisms, circular DNA formed by circularizing acyclic DNA, circular DNA formed by ligating multiple acyclic DNAs, and entirely artificially synthesized circular DNA. Examples of such acyclic DNA include naturally occurring circular DNA that has been cut by enzymatic treatment, naturally occurring acyclic DNA, and artificially synthesized acyclic DNA.

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

[0020] In one embodiment of this design, a mixture of DNA is prepared to contain multiple identical or different circular DNAs. For example, multiple types of circular DNAs with different sequences in some parts can be prepared. More specifically, a circular DNA library can be obtained by inserting multiple types of randomized fragments into the circular DNA, and the resulting circular DNA library can be prepared as a mixture of DNA. In one embodiment, preferably, the mixture contains multiple different types of circular DNAs.

[0021] The size of circular DNA is not particularly limited and can be, for example, 1 kb (1,000 base pairs) 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. It can also be 1 Mb (1 million base pairs) 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 embodiment, circular DNA has sizes of 1 kb to 100 kb, 1 kb to 10 kb, 1 kb to 8 kb, or 1 kb to 5 kb. A DNA mixture may contain circular DNA of different sizes.

[0022] In one embodiment of this invention, the reaction gel solution comprises a first group of enzymes that catalyze the replication of circular DNA, a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a catenane, a third group of enzymes that catalyze the separation reaction of the two sister circular DNAs, and a gelling agent.

[0023] The reaction gel solution contains the first, second, and third enzyme groups, and the circular DNA contains a replication initiation sequence (e.g., oriC) that can bind to an enzyme having DnaA activity, allowing DNA to be amplified using the Replication Cycle Reaction method (hereinafter referred to as the RCR method; see International Publication No. 2016 / 080424, International Publication No. 2017 / 199991, International Publication No. 2018 / 159669, etc.).

[0024] As the first group of enzymes that catalyze the replication of circular DNA, for example, the group of enzymes described in Kaguni JM & Kornberg A. Cell, 1984, vol.38, p.183-90. can be used. Specifically, the first group of enzymes can be one or more enzymes or groups of enzymes selected from the group consisting of an enzyme having DnaA activity, one or more nucleoid proteins, an enzyme or group of enzymes having DNA gyrase activity, a single-strand binding protein (SSB), an enzyme having DnaB-type helicase activity, an enzyme having DNA helicase loader activity, an enzyme having DNA primase activity, an enzyme having DNA clamp activity, and an enzyme or group of enzymes having DNA polymerase III* activity. All combinations of such enzymes or groups of enzymes can also be exemplified. In one embodiment, the first enzyme group 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.

[0025] As for enzymes possessing DnaA activity, there are no particular restrictions on their biological origin, as long as they are enzymes with initiator activity similar to that of DnaA, the initiator protein of Escherichia coli. For example, DnaA derived from Escherichia coli can be suitably used. DnaA derived from Escherichia coli may be present in the reaction gel solution as monomers in the range of 1 nM to 10 μM, preferably in the ranges of 1 nM to 5 μM, 1 nM to 3 μM, 1 nM to 1.5 μM, 1 nM to 1.0 μM, 1 nM to 500 nM, 50 nM to 200 nM, or 50 nM to 150 nM, but is not limited to these ranges.

[0026] A nucleoid protein refers to a protein contained within a nucleoid. The one or more nucleoid proteins used in this invention are not particularly limited in their biological origin, as long as they are enzymes having activity similar to that of the nucleoid protein of Escherichia coli. For example, IHF derived from Escherichia coli, i.e., a complex of IhfA and / or IhfB (heterodimer or homodimer), or HU derived from Escherichia coli, i.e., a complex of hupA and hupB, can be suitably used. The IHF derived from Escherichia coli may be present in the reaction gel solution as a hetero / homodimer in the range of 5 nM to 400 nM, preferably in the ranges of 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. E. coli-derived HU may be present in the reaction gel solution in a range of 1 nM to 50 nM, preferably in a range of 5 nM to 50 nM or 5 nM to 25 nM, but is not limited to this range.

[0027] The enzyme or group of enzymes having DNA gyrase activity is not particularly limited in its biological origin, as long as it has 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 present in the reaction gel solution as a heterotetramer in a range of 20 nM to 500 nM, preferably in the ranges of 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.

[0028] As for the single-stranded DNA-binding protein (SSB), there are no particular restrictions on its biological origin, as long as it is an enzyme having similar activity to the single-stranded DNA-binding protein of Escherichia coli. For example, SSB derived from Escherichia coli can be suitably used. The SSB derived from Escherichia coli may be present in the reaction gel solution as a homotetramer in the range of 20 nM to 1000 nM, preferably in the ranges 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, and 100 nM to 400 nM, but is not limited to these ranges.

[0029] As long as the enzyme having DnaB-type helicase activity has activity similar to that of DnaB from E. coli, there are no particular restrictions on its biological origin. For example, DnaB derived from E. coli can be suitably used. DnaB derived from E. coli may be present in the reaction gel solution as a homohexamer in a concentration of 5 nM to 200 nM, preferably in the ranges of 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited to these ranges.

[0030] As long as the enzyme having DNA helical orderer activity has activity similar to that of DnaC from E. coli, there are no particular restrictions on its biological origin. For example, DnaC derived from E. coli can be suitably used. DnaC derived from E. coli may be present in the reaction gel solution as a homohexamer in a concentration of 5 nM to 200 nM, preferably in the ranges of 5 nM to 100 nM, 5 nM to 50 nM, or 5 nM to 30 nM, but is not limited to these ranges.

[0031] As long as the enzyme having DNA primase activity has activity similar to that of DnaG from E. coli, there are no particular restrictions on its biological origin. For example, DnaG derived from E. coli can be suitably used. DnaG derived from E. coli may be present in the reaction gel solution as monomers in the range of 20 nM to 1000 nM, preferably in the ranges of 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 to these ranges.

[0032] As long as the enzyme having DNA clamp activity has activity similar to that of DnaN from E. coli, there are no particular restrictions on its biological origin. For example, DnaN derived from E. coli can be suitably used. DnaN derived from E. coli may be present in the reaction gel solution as a homodimer in the range of 10 nM to 1000 nM, preferably in the ranges of 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 to these ranges.

[0033] As long as the enzyme or group of enzymes having DNA polymerase III* activity has activity similar to that of the DNA polymerase III* complex of Escherichia coli, there are no particular restrictions on its biological origin. For example, an enzyme group containing any of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli can be suitably used. Preferably, an enzyme group containing a complex of DnaX, HolA, HolB, and DnaE derived from Escherichia coli can be suitably used. More preferably, an enzyme group containing a complex of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE derived from Escherichia coli can be suitably used. The E. coli-derived DNA polymerase III* complex may be present in the reaction gel solution as a heteromultimer in concentrations ranging from 2 nM to 50 nM, preferably in concentrations ranging from 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 to these ranges.

[0034] In this invention, the two sister circular DNAs that form a catenane refer to two circular DNAs synthesized by a DNA replication reaction that are linked together.

[0035] As a second group of enzymes that catalyze the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a catenane, examples 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 thereof. In one embodiment, the second group of enzymes preferably includes enzymes having DNA polymerase I activity and enzymes having DNA ligase activity.

[0036] As long as the enzyme possessing DNA polymerase I activity has activity similar to that of Escherichia coli (E. coli), there are no particular restrictions on its biological origin. For example, DNA polymerase I derived from E. coli can be suitably used. E. coli-derived DNA polymerase I may be present in the reaction gel solution as monomers in the range of 10 nM to 200 nM, preferably in the ranges of 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 to these ranges.

[0037] The enzyme having DNA ligase activity is not particularly limited in its biological origin, as long as it has activity similar to that of E. coli DNA ligase. For example, DNA ligase derived from E. coli or DNA ligase from T4 phage can be suitably used. E. coli DNA ligase may be present in the reaction gel solution as monomers in the range of 10 nM to 200 nM, preferably in the ranges of 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.

[0038] The enzyme possessing RNaseH activity is not particularly limited in its biological origin, as long as it has the activity to degrade the RNA strand of an RNA:DNA hybrid. For example, RNaseH derived from Escherichia coli can be suitably used. RNaseH derived from Escherichia coli may be present in the reaction gel solution as monomers in a range of 0.2 nM to 200 nM, preferably in the ranges of 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 to these ranges.

[0039] As a third group of enzymes that catalyze the separation reaction of two sister circular DNAs, for example, the group of enzymes described in Peng H & Marians KJ., PNAS, 1993, vol.90, p.8571-8575. can be used. Specifically, as the third group of enzymes, 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 thereof, can be exemplified. In one embodiment, preferably, the enzyme includes an enzyme having topoisomerase IV activity and / or an enzyme having topoisomerase III activity.

[0040] The enzyme possessing topoisomerase III activity is not particularly limited in its biological origin, as long as it has activity similar to that of Escherichia coli topoisomerase III. For example, topoisomerase III derived from Escherichia coli can be suitably used. Topoisomerase III derived from Escherichia coli may be present in the reaction gel solution as monomers in the range of 20 nM to 500 nM, preferably in the ranges of 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.

[0041] The enzyme having RecQ-type helicase activity is not particularly limited in its biological origin, as long as it has activity similar to that of RecQ from Escherichia coli. For example, RecQ derived from Escherichia coli can be suitably used. RecQ derived from Escherichia coli may be present in the reaction gel solution as monomers in the range of 20 nM to 500 nM, preferably in the ranges of 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 to these ranges.

[0042] As long as the enzyme possessing topoisomerase IV activity has activity similar to that of Escherichia coli topoisomerase IV, there are no particular restrictions on its biological origin. 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 present in the reaction gel solution as a heterotetramer in a concentration of 0.1 nM to 50 nM, preferably in concentrations of 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, 1 nM to 10 nM, or 1 nM to 5 nM, but is not limited to these ranges.

[0043] The reaction gel solution may contain further enzymes in addition to the first, second, and third enzyme groups. For example, if the circular DNA amplified by the RCR method has a pair of ter sequences inserted outward into a replication initiation sequence (e.g., oriC) that can bind to an enzyme having DnaA activity, the reaction gel solution may further contain a protein (e.g., Tus protein derived from E. coli) that has the activity to bind to the ter sequences and inhibit replication.

[0044] Each of the enzymes constituting the first, second, and third enzyme groups described above may be commercially available, or extracted from microorganisms and purified as necessary. The extraction and purification of enzymes from microorganisms can be carried out as appropriate using methods available to those skilled in the art.

[0045] When using enzymes other than those derived from E. coli as the first, second, and third enzyme groups, they can be used in a concentration range corresponding to the enzyme activity units relative to the concentration range specified for the E. coli-derived enzymes.

[0046] 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 considering 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, one or more gelling agents 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, and polyvinylpyrrolidone can be used as the gelling agent. Preferably, the gelling agent is selected from the group consisting of agarose, polyacrylamide, and agar, and more preferably agarose.

[0047] 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® Agarose LE 1-20K GAT (manufactured by TaKaRa), Agarose L (manufactured by Nippon Gene), etc.

[0048] In one embodiment, preferably, the gelling agent has a gelling temperature of 80°C or lower. This prevents the reaction gel plate from melting when the DNA amplification reaction after gelling is carried out at a temperature of 80°C or lower.

[0049] In one embodiment, preferably, the gelling agent has a gelation temperature of 60°C or lower, preferably 45°C or lower, from the viewpoint of ease of handling of the reaction gel solution before gelation, and from the viewpoint of carrying out the reaction below the inactivation temperature of the enzyme used in the amplification reaction.

[0050] The concentration of the gelling agent in the reaction gel solution can be appropriately adjusted according to the size of the DNA to be amplified. When the DNA size is small, increasing the gelling agent concentration can prevent the diffusion of DNA during and after amplification. For example, when amplifying DNA of a size of less than 4kb, 3kb or less, or 2.5kb or less using agarose as the gelling agent, the amount of agarose in the reaction gel solution can be set to more than 2%, for example, 3% or more. The above is merely one embodiment, and the reaction conditions, such as reaction temperature and reaction time, can also be appropriately adjusted.

[0051] The composition of the reaction gel solution is not particularly limited as long as it allows the gelation and DNA amplification reactions to proceed. For example, it may further contain, if necessary, one or more selected from buffer, ATP (adenosine triphosphate), GTP (guanosine triphosphate), CTP (cytidine triphosphate), UTP (uridine triphosphate), dNTP, magnesium ion source, and alkali metal ion source.

[0052] As the buffer solution, a buffer suitable for use at pH 7 to 9, preferably pH 8, can be used. Examples include Tris-HCl, Tris-OAc, Hepes-KOH, phosphate buffer, MOPS-NaOH, and Tricinene-HCl. The 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, for example, concentrations of 10 mM to 100 mM, 10 mM to 50 mM, and 20 mM can be selected.

[0053] The concentration of ATP contained in the reaction gel solution may be, for example, in the range of 0.1 to 3 mM, preferably in the range of 0.1 to 2 mM, 0.1 to 1.5 mM, or 0.5 to 1.5 mM.

[0054] The concentrations of GTP, CTP, and UTP contained in the reaction gel solution may be independently in the range of, for example, 0.1 mM to 3.0 mM, preferably in the ranges of 0.5 mM to 3.0 mM and 0.5 mM to 2.0 mM.

[0055] dNTPs are a collective term for deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP). The concentration of dNTPs 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 mM to 1 mM or 0.1 mM to 1 mM.

[0056] The magnesium ion source is magnesium ions (Mg) in the reaction gel solution. 2+ This is a substance that provides magnesium ions. Examples include Mg(OAc)2, MgCl2, and MgSO4. A preferred magnesium ion source is Mg(OAc)2. The concentration of the magnesium ion source contained in the reaction gel solution may be, for example, a concentration that provides magnesium ions in the reaction gel solution in the range of 5 to 50 mM.

[0057] An alkali metal ion source is a substance that provides alkali metal ions to the reaction gel solution. 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. Preferred alkali metal ion sources are potassium glutamate or potassium acetate. The concentration of the alkali metal ion source contained in the reaction gel solution may be, but is not limited to, a concentration that provides alkali metal ions in the reaction gel solution in the range of 100 mM or more, preferably in the range of 100 mM to 300 mM.

[0058] The reaction gel solution further contains non-specific protein adsorption inhibitors (bovine serum albumin, lysozyme, gelatin, heparin, casein, etc.), non-specific nucleic acid adsorption inhibitors (tRNA (transfer RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), glycogen, heparin, oligoDNA, poly(IC) (polyinosine-polycytidine), poly(dI-dC) (polydeoxyinosine-polydeoxycytidine), poly(A) (polyadenine), and poly(dA) (polydeoxyadenine), etc.), linear DNA-specific exonucleases (RecBCD, λ exonuclease, exonuclease III, exonuclease VIII, T5 exonuclease, T7 exonuclease, Plasmid-Safe® ATP-Dependent The reaction gel solution may contain DNase (e.g., epicentre), RecG-type helicase (e.g., RecG derived from E. coli), ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium acetate), and reducing agents (e.g., DTT, β-mercaptoethanol, glutathione). If the reaction gel solution contains DNA ligase derived from E. coli, it may also contain its cofactor, NAD (nicotinamide adenine dinucleotide).

[0059] In the DNA amplification method of this embodiment, a circular DNA amplification product is obtained by performing a circular DNA amplification reaction using a DNA amplification gel plate obtained by gelling the reaction gel solution. The circular DNA may be mixed with the reaction gel solution before gelling and then gelled, or the circular DNA may be seeded onto the amplification gel plate obtained by gelling the reaction gel solution. The gelling of the reaction gel solution can be carried out using known methods. Typically, an amplification gel plate can be obtained by pouring the reaction gel solution into a frame or container and lowering the temperature to below the gelling temperature of the gelling agent to induce gelling.

[0060] When mixing circular DNA into the reaction gel solution before gelation, the concentration of circular DNA can be adjusted as appropriate, depending on the size of the amplification gel plate to be obtained, so that the circular DNA is dispersed in the gel plate (preferably dispersed as individual molecules).

[0061] When seeding circular DNA onto an amplification gel plate after the reaction gel solution has gelled, the circular DNA can be appropriately diluted with a solvent before seeding, depending on the size of the gel plate on which the DNA is seeded, so that the circular DNA is dispersed on the gel plate (preferably dispersed as individual molecules).

[0062] The amplification reaction of circular DNA using a DNA amplification gel plate can be carried out at a temperature of 80°C or lower, preferably 65°C or lower. 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 methods such as covering the gel plate.

[0063] In isothermal amplification, there are no particular restrictions on the isothermal conditions as long as the DNA amplification reaction or DNA replication reaction can proceed. For example, it can be a constant temperature that falls within the optimal temperature range for DNA polymerase. Examples of isothermal conditions include constant temperatures of 15°C or higher, 16°C or higher, 20°C or higher, 25°C or higher, or 30°C or higher, and constant temperatures 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. Furthermore, isothermal conditions may be, 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 around 30°C. In this specification, "isothermal" in isothermal amplification means maintaining the temperature within a range of ±7°C, ±5°C, ±3°C, or ±1°C from the set temperature during the reaction. The reaction time for isothermal amplification can be appropriately set according to the size of the target double-stranded DNA, for example, from 20 minutes to 30 hours, preferably from 30 minutes to 24 hours, more preferably from 1 hour to 12 hours, and even more preferably from 2 hours to 9 hours.

[0064] In non-isothermal amplification, amplification can be performed under temperature conditions in which incubation is repeated under a temperature cycle at two temperatures below 80°C, preferably below 65°C. The first temperature in the temperature cycle is the temperature at which double-stranded DNA replication can begin, and the second temperature is the temperature at which replication initiation is suppressed and the DNA extension reaction proceeds. The first temperature may 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 at the first temperature is not particularly limited, but may be 10 seconds to 10 minutes per cycle, with 1 minute being preferred. The second temperature may be 27°C or lower, for example, 10°C to 27°C, 16°C to 25°C, or 24°C. The incubation at the second temperature is not particularly limited, but is preferably set according to the length of the circular DNA to be amplified, for example, 1 second to 10 seconds per 1000 bases per cycle. The number of temperature cycles is not particularly limited, but it may be 10 to 50 cycles, 20 to 45 cycles, 25 to 45 cycles, or 40 cycles.

[0065] The reaction gel solution may be pre-incubated prior to mixing with the circular DNA. Pre-incubation may be carried out by maintaining the temperature at a range of 0-40°C, 10-40°C, 15-37°C, or 16-30°C for 5-60 minutes, 5-45 minutes, 5-30 minutes, 10-60 minutes, 10-45 minutes, 10-30 minutes, 15-45 minutes, or 15-30 minutes. Pre-incubation is sufficient as long as the temperature of the reaction gel solution is kept within the aforementioned temperature range, and slight fluctuations during pre-incubation are permitted.

[0066] Although not constrained by theory, by amplifying circular DNA using RCR with an amplification gel plate, the circular DNA undergoes repeated replication cycles and preferably amplifies exponentially. According to the method of this embodiment, circular DNA can be produced by using the above-mentioned circular DNA as a template and amplifying it by at least 10x, 50x, 100x, 200x, 500x, 1000x, 2000x, 3000x, 4000x, 5000x, or 10000x. Furthermore, the amplified circular DNA has a supercoil structure similar to bacterial genomic DNA.

[0067] In one embodiment, The process of preparing a mixture of DNA, A step of preparing a reaction gel solution containing the aforementioned DNA mixture, agarose, and a DNA amplification enzyme, The process involves gelling the reaction gel solution to create an amplification gel plate containing DNA, The process involves using the aforementioned amplification gel plate to perform a DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, This relates to DNA amplification methods, including [specific method / technique].

[0068] In one embodiment, The process of preparing a mixture of DNA, A step of preparing a reaction gel solution containing agarose and a DNA amplification enzyme, The process involves creating an amplification gel plate by gelling the aforementioned reaction gel solution, The steps include: seeding the DNA mixture onto the amplification gel plate; The process involves using the aforementioned amplification gel plate to perform a DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, This relates to DNA amplification methods, including [specific method / technique].

[0069] The DNA used in the DNA amplification method of this embodiment may be 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 treated with enzymes, natural DNA with other DNA fragments ligated to it, and entirely artificially synthesized DNA.

[0070] In one embodiment of this model, a DNA mixture is prepared to contain multiple identical or different DNAs. For example, multiple types of DNA with different sequences in some parts can be prepared, and more specifically, multiple types of randomized fragments can be prepared and ligated or inserted into DNA to obtain a DNA library, and the obtained DNA library can be prepared as a DNA mixture. In one embodiment, preferably, the mixture contains multiple different types of DNA. The size of the DNA is not particularly limited and can be, for example, the same size as in the case of the circular DNA. The mixture may contain DNA of different sizes.

[0071] The type of agarose, gelation temperature, and concentration of the reaction gel solution are as described above for the gelling agent.

[0072] The DNA amplification enzyme contained in the reaction gel solution can be appropriately selected depending on the DNA amplification method used in the cell-free system. The DNA amplification method in the cell-free system can be any known technique in the art that can be carried out at 80°C or below, and may be isothermal or non-isothermal amplification. When using isothermal amplification in cell-free systems, various techniques are known (J. Li and J. Macdonald, Biosensors and Bioelectronics, 2015, vol.64, p.196-211), for example, in addition to the RCR method mentioned above, there are Helicase-dependent amplification (HDA) (Vincent, et al, EMBO Rep., 2004, vol.5 (8), p.795-800), Recombinase polymerase amplification (RPA) (Piepenburg, et al, PLoS. Biol., 2006, vol.4 (7), e204), Rolling circle amplification (RCA) (Fire, et al, Proc. Natl. Acad. Sci., 1995, vol.92 (10), p.4641-4645), Ramification amplification (RAM) (Zhang, et al, Mol. Diagn., Known techniques can be used, including, but not limited to, those described above, such as 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 amplification (LAMP) (Notomi, et al, Nucleic Acids Res., 2000, vol.28 (12), E63). Any known technique can be used, and each method can be carried out according to standard procedures. The choice of method can be appropriately selected depending on the shape of the DNA to be amplified.

[0073] The composition of the reaction gel solution is not particularly limited, as long as it allows for the gelation and DNA amplification reactions to proceed.

[0074] In the DNA amplification method of this embodiment, a DNA amplification product is obtained by carrying out the DNA amplification reaction using a DNA amplification gel plate obtained by gelling the reaction gel solution. The DNA may be mixed with the reaction gel solution before gelling and then gelled, or the DNA may be seeded on the amplification gel plate obtained by gelling the reaction gel solution. The gelling of the reaction gel solution, the method of mixing DNA with the reaction gel solution before gelling, and the method of seeding DNA on the amplification gel plate after the reaction gel solution has gelled can be carried out in the same way as in the circular DNA amplification method described above, depending on the DNA amplification method.

[0075] DNA amplification reactions using DNA amplification gel plates are carried out at temperatures below 80°C, preferably below 65°C. 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 methods such as covering the gel plate or maintaining humidity (e.g., maintaining saturated water vapor pressure).

[0076] In the DNA amplification method of this embodiment, a DNA amplification reaction of circular DNA or DNA (hereinafter also simply referred to as "DNA") is carried out using a DNA amplification gel plate obtained by gelling a reaction gel solution to obtain a DNA amplified product. In the amplification method of this embodiment, the aggregate of DNA amplified products obtained after amplification on the gel plate is called a "DNA colony". At the start of the reaction, each DNA in the DNA mixture is dispersed in the amplification gel plate, preferably dispersed into single molecules, so that after amplification proceeds in the gel, a DNA amplified product derived from one molecule is obtained as an aggregate that can be distinguished from DNA amplified products derived from other molecules. In one aspect of this embodiment, when the DNA mixture contains two or more types of DNA, preferably the DNA amplified product contains two or more types of DNA colonies, and more preferably each DNA colony is an aggregate of amplified products of one type of DNA.

[0077] In one embodiment, the DNA amplification method of this embodiment further includes a step of isolating DNA colonies. Colony isolation can be carried out by methods known to those skilled in the art, such as methods using chips or methods using colony pickers.

[0078] In one embodiment, the DNA amplification method of this embodiment includes, after the step of isolating the DNA colonies, a further step of performing a DNA amplification reaction to obtain a DNA amplified product derived from the colonies. This makes it easier to analyze the functional characteristics of the isolated DNA. The method of the DNA amplification reaction 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 embodiment, preferably, the above-described RCR method can be used in the case of circular DNA, and the PCR method can be used in the case of linear DNA.

[0079] In one embodiment, the amplification gel plate in the DNA amplification method of this embodiment further contains components necessary for the transcription reaction. In another embodiment, the amplification 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 can be simply added to the reaction gel solution before gelation, and by gelation, an amplification gel plate containing components necessary for the transcription reaction, or an amplification gel plate containing components necessary for both the transcription reaction and the translation reaction, can be obtained.

[0080] Components necessary for the transcription reaction include enzymes involved in transcription, such as RNA polymerase. For example, if T7 RNA polymerase is included, transcription proceeds from template DNA containing the T7 promoter. Components necessary for the translation reaction include ribosomes, tRNA, amino acids, or their derivatives. The amino acids or their derivatives can be appropriately selected depending on the DNA to be translated, and may be natural or unnatural amino acids. Examples of components necessary for transcription and translation reactions include those described in well-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, p.9-16; Shimizu et al., Nature Biotechnology (2001) vol.19, p.751-755; Ohashi et al., Biochem Biophys Res Commun. (2007) Vol.352, No.1, p.270-276; and Shimizu and Ueda, Methods Mol Biol. (2010) Vol.607, p.11-21.

[0081] For example, commercially available kits and their components used for in vitro transcription (IVT) and cell-free protein synthesis can be used as these components. Examples of such kits include, but are not limited to, PUREfrex® (manufactured by Gene Frontier), PURExpress® (manufactured by NEB), TNT® cell-free expression system (manufactured by Promega), myTXTL® (manufactured by Arbor Biosciences), and cell-free protein synthesis reagent kit (manufactured by NU Protein). For example, the reconstituted cell-free protein synthesis system (PUREsystem), which forms the basis of PUREfrex®, PURExpress®, etc., includes 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), termination factors (RF1, RF2, RF3), ribosome recycling factors, etc.), various aminoacyl-tRNA synthetases (ARS), methionyl-tRNA formyltransferase, purified ribosomes, E. coli tRNA, amino acids, NTPs, energy regeneration systems, etc., and some or all of these can be used as components necessary for transcription reactions or translation reactions.

[0082] If the amplification gel plate contains components necessary for the transcription reaction, including RNA polymerase, then, following the DNA amplification reaction, a transcription reaction using the obtained DNA amplification product as a template occurs on the amplification gel plate, yielding RNA derived from the DNA amplification product in addition to, or in place of, the DNA amplification product. In one embodiment, if the amplification gel plate contains components necessary for the transcription reaction, RNA colonies can be obtained.

[0083] If the amplification gel plate contains components necessary for the transcription reaction, such as RNA polymerase, as well as components necessary for the translation reaction, such as ribosomes, tRNA, amino acids, or their derivatives, then in the amplification gel plate, following the DNA amplification reaction, a transcription reaction occurs using the obtained DNA amplification product as a template, and then a translation reaction occurs using the obtained RNA as a template, resulting in the production of proteins derived from the DNA amplification product, either in addition to or in place of the DNA amplification product. In one embodiment, if the amplification gel plate contains components necessary for both the transcription reaction and the translation reaction, protein colonies can be obtained.

[0084] In one embodiment, the DNA amplification method of this embodiment further includes a step of detecting the DNA colonies. The step of detecting the DNA colonies may be performed, for example, before the step of isolating the DNA colonies. The detection of DNA colonies can be performed, for example, by fluorescence imaging detection using a DNA intercalator fluorescent dye (fluorescent dye for nucleic acid staining) or a fluorescent DNA probe, or by direct DNA detection using a phase-contrast microscope, or by detection of RNA transcribed from DNA or proteins transcribed and translated.

[0085] According to the DNA amplification method of this embodiment, each DNA can be easily separated from a mixture of heterologous DNAs, and each DNA can be selected either directly using a probe linked to its sequence, or linked to the function (including activity) of RNA and / or proteins.

[0086] One aspect of this embodiment is A step of preparing circular DNA having a replication initiation sequence that can bind to an enzyme having DnaA activity, The process involves preparing a reaction solution containing the aforementioned circular DNA, a first group of enzymes that catalyzes the replication of the circular DNA, a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a catenane, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, RNA polymerase, ribosomes, and tRNA. A step of obtaining a translation product derived from circular DNA using the reaction solution, The step of detecting the aforementioned translation product, This relates to a method for detecting circular DNA, including [specific type of DNA].

[0087] Each step can be carried out by referring to the circular DNA amplification method described above, 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 methods such as detecting the fluorescence intensity by encoding a fluorescent protein in the circular DNA, or detection using tags or probes can be used. This allows for the easy separation of each DNA from a mixture of heterologous DNAs, and enables the selection of each DNA in relation to the function of the protein it is linked to.

[0088] This embodiment also includes, A circular DNA having a replication initiation sequence capable of binding to an enzyme with DnaA activity, A first group of enzymes catalyzes the replication of circular DNA, a second group of enzymes catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and a third group of enzymes catalyzes the separation reaction of the two sister circular DNAs. Components necessary for the transcription reaction, and components necessary for the translation reaction, The invention also relates to translation compositions for circular DNA, including the above.

[0089] The components can be identified by referring to the descriptions in the DNA amplification methods for circular DNA mentioned above. The components necessary for the transcription reaction preferably include at least RNA polymerase. The components necessary for the translation reaction preferably include at least ribosomes and tRNA. Such compositions are useful for selecting each DNA molecule in relation to the function of the protein it is associated with.

[0090] In one embodiment, The process of preparing a mixture of DNA that codes for proteins. A step of preparing a gel plate containing the aforementioned DNA mixture, a gelling agent, a DNA amplification enzyme, components necessary for the transcription reaction, and components necessary for the translation reaction. A step of obtaining colonies of the DNA-derived translation product using the gel plate, This relates to a method for detecting DNA, including [specific details omitted].

[0091] In one embodiment, The process of preparing a mixture of DNA that codes for proteins. A process for creating a gel plate containing a gelling agent, a DNA amplification enzyme, components necessary for the transcription reaction, and components necessary for the translation reaction, The steps include: seeding the DNA mixture onto the gel plate; A step of obtaining colonies of the DNA-derived translation product using the gel plate, This relates to a method for detecting DNA, including [specific details omitted].

[0092] The components necessary for the transcription reaction preferably include at least RNA polymerase. The components necessary for the translation reaction preferably include at least ribosomes and tRNA, and more preferably include at least ribosomes, tRNA, and amino acids or their derivatives.

[0093] The protein encoded by the DNA is not particularly limited, but when the molecular weight of the protein to be expressed is small (not limited, but for example, 30 kDa or less), it is desirable to suppress diffusion within the gel. For example, the protein can be immobilized on DNA colonies by conferring DNA binding ability to it. Examples of protein domains (DNA binding domains) used to confer DNA binding ability include nucleoid proteins (HU); DNA binding domains of proteins involved in DNA replication, repair, and modification (such as the DnaA DNA binding domain); and DNA binding domains of transcription factors (such as TAL effectors). By using DNA that encodes such a DNA-binding protein, the translation product derived from the DNA binds to the DNA colony without diffusion, and the target DNA can be selected using the translation product as an indicator. In one embodiment, the protein is a protein having DNA binding ability, and more specifically, a protein having a DNA binding domain. [Examples]

[0094] [Example 1] Creation of DNA colonies An RCR plate was prepared by solidifying a solution containing circular DNA with oriC and an enzyme for RCR with agarose, and the RCR amplification reaction was carried out using the plate.

[0095] (Preparing the microscope slide) A microarray slide frame (Gene Frame, 25 μL (1.0 × 1.0 cm), manufactured by Thermo Scientific) was attached to a glass slide (Figure 1A).

[0096] (Preparation of circular DNA) Using the 2.8kb 6nM UPL fragment (SEQ ID NO: 1) and 2.0kb 6nM UPR fragment (SEQ ID NO: 2) shown in Tables 1 and 2, an RA ligation reaction was performed to obtain a 4.7kb ligate containing oriC. The ligation reaction was carried out by adding the 40-base pair overlap sequences attached to both ends of each fragment to an RA ligation reaction solution consisting of the following composition, and reacting at 42°C for 30 minutes. The obtained ligate was cloned using E. coli to obtain a 4.7kb plasmid.

[0097] Composition of RA coupling reaction solution: 1 μM wild-type RecA (prepared by purification from an E. coli RecA expression strain using 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), 4 mM creatine phosphate. Note that the concentration of each component in the RA reaction solution is the concentration relative to the total volume of the RA coupling reaction solution.

[0098] [Table 1]

[0099] [Table 2]

[0100] (Creating RCR plates) Agarose (PrimeGel® Agarose LMT 1-20K, manufactured by TaKaRa) was dissolved in ultrapure water by heating to prepare a 4% low-melting-point agarose. 30 μL (final concentration 2%) of the low-melting-point agarose was dispensed per sample and incubated at 50°C.

[0101] Next, a mixture for the RCR reaction was prepared to twice the concentration of the composition shown in Table 3 (2xRCR mix-1, total 28.4 μL), and pre-incubated at 33°C for 30 minutes. To 2xRCR mix-1, 1 μL of circular DNA was added so that the molecular count was 10, 50, or 100 molecules / rate per final RCR plate (25 μL), and then 0.6 μL of nucleic acid staining fluorescent dye (10 μM YOYO™-1 Iodide, Invitrogen) (final concentration 0.1 μM) was added and mixed by pipetting.

[0102] This solution was added to 4% low-melting-point agarose, which had been kept warm at 50°C, and thoroughly mixed 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 mixture was cooled at 4°C for 5 minutes to solidify the agarose.

[0103] (RCR amplification reaction using RCR plates) To protect from light, the prepared RCR plate was wrapped in aluminum foil and placed in a 37°C air incubator for 3 hours. After the reaction, the RCR plate was observed using a fluorescence microscope (Axio Observer Z1, Zeiss) and the number of colonies was measured.

[0104] The experimental overview 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 RCR plate after DNA amplification, as captured using a fluorescence microscope. As shown in Figure 2, we found that DNA is amplified into colonies when the RCR amplification reaction is performed using an RCR plate. Furthermore, the number of colonies correlated with the number of DNA molecules added.

[0105] [Table 3]

[0106] In Table 3, SSB is E. coli-derived SSB, IHF is a complex of E. coli-derived IhfA and IhfB, DnaG is E. coli-derived DnaG, DnaN is E. coli-derived DnaN, Pol III* is the DNA polymerase III* complex, which consists of E. coli-derived DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE, DnaB is E. coli-derived DnaB, DnaC is E. coli-derived DnaC, DnaA is E. coli-derived DnaA, RNaseH is E. coli-derived RNaseH, Ligase is E. coli-derived DNA ligase, Pol I is E. coli-derived DNA polymerase I, GyrA is E. coli-derived GyrA, GyrB is E. coli-derived GyrB, Topo IV is a complex of E. coli-derived ParC and ParE, Topo III represents Escherichia coli-derived topoisomerase III, and RecQ represents Escherichia coli-derived RecQ.

[0107] SSB was prepared by purifying an E. coli strain expressing SSB through a process including ammonium sulfate precipitation and ion-exchange column chromatography. IHF was prepared by purifying co-expression strains of E. coli containing IhfA and IhfB through a process including ammonium sulfate precipitation and affinity column chromatography. DnaG was prepared by purifying a DnaG-expressing E. coli strain through a process including ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography. DnaN was prepared by purifying a DnaN-expressing E. coli strain through a process including ammonium sulfate precipitation and anion exchange column chromatography. Pol III* was prepared by purifying E. coli co-expression strains of DnaX, HolA, HolB, HolC, HolD, DnaE, ​​DnaQ, and HolE through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaB and DnaC were prepared by purifying E. coli co-expressing strains of DnaB and DnaC through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. DnaA was prepared by purifying an E. coli strain expressing DnaA through a process including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography. GyrA and GyrB were prepared by purifying a mixture of E. coli strains expressing GyrA and GyrB through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo IV was prepared by purifying a mixture of ParC and ParE E. coli expression strains using a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. Topo III was prepared by purifying an Escherichia coli strain expressing Topo III through a process including ammonium sulfate precipitation and affinity column chromatography. RecQ was prepared by purifying an E. coli strain expressing RecQ through a process including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography. RNaseH, Ligase, and Pol I were obtained using commercially available enzymes derived from E. coli (manufactured by Takara Bio). Tus was prepared by purifying an E. coli expression strain of Tus using a process that included affinity column chromatography and gel filtration column chromatography.

[0108] [Example 2] DNA colony creation by DNA coating after RCR plate preparation. DNA colonies were created by not adding circular DNA during RCR plate preparation, but by spreading the circular DNA onto the plate after RCR plate preparation.

[0109] A DNA-free RCR plate was prepared using the same method as in Example 1, except that DNA was not added and 50 nM BOBO™-3 Iodide (Invitrogen) (final concentration 0.5 μM) was used as the fluorescent dye for nucleic acid staining.

[0110] 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 RCR amplification reaction was carried out using an RCR plate under the same conditions as in Example 1, and the reaction was observed using a fluorescence microscope afterward.

[0111] The results are shown in Figure 3. Even when an RCR plate without DNA was prepared and then DNA was spread onto the gel, it was possible to perform the RCR amplification reaction and create DNA colonies.

[0112] [Example 3] Creation of DNA colonies of different DNA sizes First, circular DNA with oriC of four different sizes, ranging from 2.3kb to 23kb, were prepared so that the final number of DNA molecules per RCR plate (25 μL) was 50. RCR plates (agarose concentration 2%) were then prepared using the same method as in Example 1. Subsequently, the RCR amplification reaction was carried out at 37°C for 4.5 hours. Fluorescence microscope images of the DNA colonies are shown in Figure 4A.

[0113] The four types of circular DNA were prepared as follows: A 2.3kb circular DNA containing oriC, pCLter_2 as described in Hasebe et al., Life (2018) Vol.8, No.43, was used. A 4.7kb circular DNA containing oriC was used: the same DNA used in Example 1. We used pOri8, a 9.4kb circular DNA containing oriC, as described in Su'etsugu et al., Nucleic Acids Res. (2017) Vol.45, pp.11525-11534. A 23kb circular DNA containing oriC was prepared using a pop-out method utilizing recombinant E. coli, similar to the pOri8 described in Su'etsugu et al., Nucleic Acids Res. (2017) Vol.45, pp.11525-11534., by E. coli genome regions containing 10kb upstream and downstream of oriC.

[0114] Next, DNA colonies were prepared using 93kb circular DNA containing oriC. Due to its long chain length, to minimize potential damage to the DNA during the mixing process with the melting gel, the same method as in Example 2 was adopted, where the DNA was coated after RCR plate preparation. (4x10) 6 The procedure was the same as in Example 2, except that molecular DNA was seeded on a plate and the RCR amplification reaction was carried out at 37°C for 9 hours. Fluorescence microscope images of DNA colonies taken using the same method as in Example 1 are shown in Figure 4(B).

[0115] The 93kb circular DNA used was a plasmid containing the *E. coli* genome region, prepared according to the method for preparing pOri93Zins described in International Publication No. 2020 / 027110, Example 7, by replacing the ligation fragment (KOZins fragment) with the CLter fragment (SEQ ID NO: 3). The CLter fragment contains the oriC and chloramphenicol resistance genes and was prepared by PCR using pCLter2k as a template. The sequence of the CLter fragment is shown below.

[0116] [Table 4]

[0117] Next, using 2.3kb circular DNA again, an RCR plate with a 3% agarose concentration was prepared, and DNA colonies were created using the same method as in Example 1. Fluorescence microscope images of the DNA colonies are shown in Figure 4(C).

[0118] 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 2.3kb circular DNA were diffuse and indistinct. Clear DNA colony formation was confirmed for circular DNA ranging from 4.7kb to 23kb. The larger the DNA size, the clearer and smaller the colony shape. As shown in Figure 4(B), DNA colonies consisting of 93kb circular DNA were even smaller. As shown in Figure 4(C), even with small 2.3kb circular DNA, clear colony formation was confirmed when the agarose concentration was increased (2% → 3%).

[0119] [Example 4] Isolation of DNA molecular species from a circular DNA library DNA colonies were created using a circular DNA library containing various UMIs (Unique Molecular Identifiers). Subsequently, each DNA colony was subjected to RCR amplification after colony picking, and the amplified products were sequenced using NGS (Next Generation Sequencing) to confirm whether the DNA colonies consisted of a single DNA molecular species.

[0120] (Creation of a circular DNA library containing UMI) A circular DNA library with random 7-nucleotide sequences (N=7) was created by self-linking and circularizing PCR fragments using the pUP-T7-GFP plasmid as a template. The pUP-T7-GFP plasmid was prepared by linking and circularizing a pUP fragment (4.4kb) and a T7-GFP fragment (1.1kb) using the In-Fusion® HD cloning kit (TaKaRa). The pUP fragment was obtained by PCR of the 4.4kb region containing oriC using the circular DNA (4.7kb) prepared in Example 1 as a template, using the primers shown in Table 5. The T7-GFP fragment has the sequence shown in Table 6.

[0121] [Table 5]

[0122] [Table 6]

[0123] Next, using pUP-T7-GFP as a template, PCR was performed using the primers listed in Table 7 to obtain the pUP-T7GFP-N7UMI fragment, which has a random 7-nucleotide sequence and contains restriction enzyme NheI recognition sites at both ends.

[0124] [Table 7]

[0125] The pUP-T7GFP-N7UMI fragment was cleaved with the restriction enzyme NheI, and then self-linked cyclization was performed using Ligation High Ver.2 (TOYOBO). Using 1.2 pg of this linked cyclization product, DNA colony formation was carried out according to the method of Example 1.

[0126] (Colony Pick Method) The fluorescence image of the DNA colonies was confirmed, and each colony was collected using a pipette tip and suspended in TE buffer. Next, 1 μL of colony pick solution was added to 9 μL of RCR solution having the composition shown in Table 3, and the RCR amplification reaction was carried out by 30 cycles of temperature cycling at 37°C for 1 minute, followed by 24°C for 29 minutes. As a control, 1 μL of the ligation product before DNA colony formation was added to the RCR solution, and the RCR amplification reaction was carried out similarly.

[0127] (Analysis using NGS) NGS analysis was performed on the RCR amplified reaction product of the colony-pick solution to analyze the sequence and frequency of each sequence in the UMI portion. An iSeq 100 (Illumina) was used for the NGS analysis.

[0128] A schematic diagram of the experiment is shown in Figure 5. Figure 6 shows a graph of the frequency of UMI sequences in each DNA colony. In Figure 6, the leftmost lane shows the frequency of each UMI sequence in the control (RCR amplification reaction product of UMI plasmid before colony formation), and 8061 different UMIs were identified. On the other hand, in the amplification products after picking DNA colonies, almost single UMIs (over 97%) were identified in 7 out of 8 samples (lanes 1-7). When DNA colonies were created using an RCR plate, monoclonal amplification derived from a single circular DNA molecule was achieved in each colony, demonstrating that DNA species can be isolated by colony picking. The minor UMI sequences mixed in samples in lanes 4-7 of Figure 6 are thought to be possible errors that occurred during RCR amplification or sequencing for NGS analysis. The sample in which no single UMI was identified (lane 8) is thought to be due to a colony picking error resulting in the selection of multiple colonies.

[0129] [Example 5] Selection and isolation of DNA molecular species based on gene expression using a PURE-RCR plate By combining the cell-free transcription and translation reaction system PUREfrex® with an RCR plate, the information encoded in DNA (protein) was presented to DNA colonies. The proteins were then detected as colonies, and the target DNA was selected and isolated based on these findings.

[0130] The plasmid DNA used for DNA colony creation was pET-HUmCdB-oriC and pET-HUGM-oriC, which encode the fluorescent proteins mCherry and GFP, respectively. Furthermore, these plasmids were fused with the DNA-binding protein HU to these fluorescent proteins.

[0131] pET-HUGM was created by cloning the hupA gene from E. coli MG1655 as an sfGFP fusion into pET21a (Novagen). The sequence of the sfGFP fusion hupA is shown in Table 8.

[0132] [Table 8]

[0133] pET-HUGM-oriC was prepared by inserting a PoriC_terG16 cassette, shown in Table 10, which had overlap sequences added using the ColE1_Fw primer and ColE1_Rv primer shown in Table 9, into pET-HUGM via homologous recombination. The homologous recombination was carried out using the method of Example 1 in International Publication No. 2023 / 038145.

[0134] [Table 9]

[0135] [Table 10]

[0136] pET-HUmCdB-oriC was created by replacing the sfGFP region (uppercase portion in Table 8) of pET-HUGM-oriC with the mCdB region. The mCdB region contains the mCherry and dBroccoli sequences. The sequences are shown in Table 11.

[0137] [Table 11]

[0138] (Creation of DNA colonies using a combination of PUREfrex® and RCR) Ultra-low gelling temperature agarose (Sigma) was mixed with ultrapure water, heated, and dissolved to prepare a 4% agarose solution. 30 μL (final concentration 2%) of the agarose solution was dispensed per sample and incubated at 45°C.

[0139] Next, 2xRCRmix-2 was prepared. 2xRCRmix-2 is a twice-concentrated solution of the enzyme group composition shown in Table 3 (Total 24.5uL). The reaction buffer portion was replaced with 0.8 times the concentration of Solution I (included with GeneFrontier's PUREfrex® 2.0), and 0.2 mM dNTPs were added to each. This 2xRCRmix-2 was pre-incubated at 33°C for 15 minutes.

[0140] 1.5 μL of Solution II and 3 μL of Solution III from PUREfrex® (GeneFrontier) were added to 2xRCRmix-2. Each 25 μL plate contained 100 molecules of pET-HUmCdB-oriC and 100 molecules of pET-HUGM-oriC, respectively.

[0141] The resulting mixture was added to a 4% agarose solution kept warm at 45°C and thoroughly mixed by pipetting. Of the total 60 μL, 45 μL was poured into a Gene Frame attached to a glass slide, as in Example 1. Then, a cover was placed on the Gene Frame and cooled at 4°C for 5 minutes to solidify the agarose, creating a PURE-RCR plate.

[0142] After a reaction at 37°C for 3 hours, the fluorescence images of colonies exhibiting red fluorescence (mCherry colonies) and colonies exhibiting green fluorescence (GFP colonies) were detected using a fluorescence microscope. Subsequently, three mCherry colonies and one GFP colony were selected using the same method as in Example 4. For each sample, an RCR amplification reaction was carried out at 33°C for 6 hours.

[0143] (Identification of molecular species by PCR) After colony picking, the RCR products were amplified by PCR using the primers shown in Table 12, and then the DNA molecular species were identified by size comparison using gel electrophoresis.

[0144] [Table 12]

[0145] A schematic diagram of the sequence (partial) and the results are shown in Figures 7-9. Figures 7(A) and 7(B) are red fluorescence microscopy images (A) and green fluorescence microscopy images (B) of colonies after reaction of circular DNA using a PURE-RCR plate, respectively. Figure 7(C) is a superimposed image of the microscopy images of Figures 7(A) and 7(B). mCherry colonies displaying the mCherry fluorescent protein (Figure 7(A)) and GFP colonies displaying the GFP fluorescent protein (Figure 7(B)) were observed at 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 plasmid DNAs used were PCR using the same primers (Table 12). When pET-HUGM-oriC was PCR, a 1.2kb fragment was amplified. When pET-HUmCdB-oriC was PCR, a 2.0kb fragment was amplified. Figure 9 shows the results of size analysis after PCR amplification of each colony-picked sample. Lanes 1-3 are from the three samples in Figure 7(A), and lane 4 is from the single sample in Figure 7(B). A 2.0kb fragment was amplified from the mCherry colony, confirming that the plasmid containing the mCherry gene was selected and isolated. Similarly, a 1.2kb fragment was amplified from the GFP colony, confirming that the plasmid containing the GFP gene was selected and isolated. It was confirmed that by using the PURE-RCR plate, it is possible to select and isolate the target DNA molecule from multiple types of DNA molecules by linking it to the function of the protein expressed from the gene encoded by the plasmid DNA.

[0146] [Example 6] Colony formation of mRNA transcribed from DNA colonies We investigated whether mRNA transcribed from DNA colonies could be detected as presented colonies on DNA, similar to the case of proteins in Example 5. mRNA aptamers were used for fluorescence detection of mRNA.

[0147] Of the plasmid DNAs used to create DNA colonies in Example 5, pET-HUmCdB-oriC encodes the fluorescent protein mCherry and also encodes the fluorescent RNA aptamer dBroccoli (Grigory et al., J. Am. Chem. Soc., 2014, vol.136, No.46, p.16299-16308). Using pET-HUmCdB-oriC, a PURE-RCR plate was prepared in which RCR amplification and transcription-translation reactions proceeded using the same method as in Example 5. At this time, 10 μM of DHFBI (Lucerna), a fluorescence detection reagent for dBroccoli, was added to the plate composition. DNA amplification, transcription, and translation reactions were carried out using the PURE-RCR plate as in Example 5. Subsequently, fluorescence microscopy images of red mCherry protein and green mRNA (dBroccoli-DHFBI) were detected.

[0148] Figure 10 shows the results confirmed by fluorescence microscopy. Figure 10(A) shows the red fluorescence image derived from the fluorescent protein mCherry, and from the results of Example 5, it is known that mCherry colonies are presented on DNA colonies. The green fluorescence image shown in Figure 10(B) is derived from DHFBI, which specifically binds to the Broccoli structure of mRNA and emits green fluorescence. The confirmation of green fluorescence colonies indicates that mRNA also forms colonies. In the merged image of the red and green fluorescence images shown in Figure 10(C), the colonies overlapped, confirming that mRNA transcribed from DNA colonies can be detected as colonies presented on template DNA, along with the expressed protein.

Claims

1. A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising: a mixture of DNA; a first group of enzymes that catalyzes the replication of circular DNA; a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs; and a gelling agent. The process involves gelling the reaction gel solution to create an amplification gel plate containing DNA, The process involves using the aforementioned amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted].

2. A step of preparing a mixture of DNA, wherein the DNA in the mixture is circular DNA having a replication initiation sequence capable of binding to an enzyme having DnaA activity, A step of preparing a reaction gel solution comprising: a first group of enzymes that catalyzes the replication of circular DNA; a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes; a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs; and a gelling agent. The process involves gelling the aforementioned reaction gel solution to create an amplification gel plate, The steps include: seeding the DNA mixture onto the amplification gel plate; The process involves using the aforementioned amplification gel plate to perform a circular DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted].

3. The method according to claim 1 or 2, wherein the gelling agent is one or more gelling agents 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, and polyvinylpyrrolidone.

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

5. The process of preparing a mixture of DNA, A step of preparing a reaction gel solution containing the aforementioned DNA mixture, agarose, and a DNA amplification enzyme, The process involves gelling the reaction gel solution to create an amplification gel plate containing DNA, The process involves using the aforementioned amplification gel plate to carry out a DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted].

6. The process of preparing a mixture of DNA, A step of preparing a reaction gel solution containing agarose and a DNA amplification enzyme, The process involves creating an amplification gel plate by gelling the aforementioned reaction gel solution, The steps include: seeding the DNA mixture onto the amplification gel plate; The process involves using the aforementioned amplification gel plate to carry out a DNA amplification reaction at a temperature of 80°C or lower to obtain a DNA amplified product, A method for amplifying DNA, including [specific details omitted].

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

8. Furthermore, the method according to claim 7, further comprising the step of detecting the DNA colony.

9. Furthermore, the process of isolating the DNA colony, The method according to claim 7, comprising the step of performing a DNA amplification reaction using isolated DNA colonies to obtain a colony-derived DNA amplified product.

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

11. Furthermore, the method according to claim 10, further comprising the step of detecting the DNA colony.

12. Furthermore, the process of isolating the DNA colony, The method according to claim 10, comprising the step of performing a DNA amplification reaction using isolated DNA colonies to obtain a colony-derived DNA amplified product.

13. The amplification gel plate further contains RNA polymerase, Furthermore, the process involves obtaining RNA by performing a transcription reaction using the DNA amplification product as a template in the amplification gel plate, The method according to claim 1 or 2, including the method described in claim 1 or 2.

14. The amplification gel plate further comprises ribosomes, tRNA, and amino acids or their derivatives. Furthermore, the process involves performing a translation reaction using the RNA as a template in the amplification gel plate to obtain a protein, The method according to claim 13, including the method described in claim 13.

15. The amplification gel plate further contains RNA polymerase, Furthermore, the process involves obtaining RNA by performing a transcription reaction using the DNA amplification product as a template in the amplification gel plate, The method according to claim 5 or 6, including the method described in claim 5 or 6.

16. The amplification gel plate further comprises ribosomes, tRNA, and amino acids or their derivatives. Furthermore, the process involves performing a translation reaction using the RNA as a template in the amplification gel plate to obtain a protein, The method according to claim 15, including the method described in claim 15.

17. A step of preparing circular DNA having a replication initiation sequence that can bind to an enzyme having DNAA activity, A step of preparing a reaction solution comprising the aforementioned circular DNA, a first group of enzymes that catalyzes the replication of the circular DNA, a second group of enzymes that catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form a catenane, a third group of enzymes that catalyzes the separation reaction of the two sister circular DNAs, RNA polymerase, ribosomes, tRNA, and amino acids or their derivatives. A step of obtaining a translation product derived from circular DNA using the reaction solution, The step of detecting the aforementioned translation product, A method for detecting circular DNA, including [specific DNA type].

18. A circular DNA having a replication initiation sequence capable of binding to an enzyme with DNAA activity, A first group of enzymes catalyzes the replication of circular DNA, a second group of enzymes catalyzes the Okazaki fragment ligation reaction to synthesize two sister circular DNAs that form catenanes, and a third group of enzymes catalyzes the separation reaction of the two sister circular DNAs. RNA polymerase, ribosomes, and tRNA, A translation composition for circular DNA, including [the specified element].

19. The process of preparing a mixture of DNA that codes for proteins, A step of preparing a gel plate containing the aforementioned DNA mixture, a gelling agent, a DNA amplification enzyme, RNA polymerase, ribosomes, and tRNA. A step of obtaining colonies of the DNA-derived translation product using the gel plate, A DNA detection method, including the following.

20. The process of preparing a mixture of DNA that codes for proteins and A process for creating a gel plate containing a gelling agent, a DNA amplification enzyme, RNA polymerase, ribosomes, and tRNA, The steps include: seeding the DNA mixture onto the gel plate; A step of obtaining colonies of the DNA-derived translation product using the gel plate, A DNA detection method, including the following.

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