Artificial genes and gene mutation methods

Abstract

To provide an artificial gene and a gene mutation method which are not subject to gene recombination regulations and enable the introduction of a local mutation into a target gene and enable the quantitation of the mutation at the time of mutation introduction.SOLUTION: An artificial gene has a 15N abundance ratio exceeding a natural abundance ratio in bases of at least a part of DNA. A gene mutation method includes: a first step of producing a state in which 15N is unevenly distributed in a prescribed DNA in a living cell; and a second step of irradiation with a proton beam at an energy at which 15N produces a resonant nuclear reaction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to artificial genes and methods for gene mutation. [Background technology]

[0002] The functions of the organisms involved in biodiversity, particularly natural products such as oils, polysaccharides, and pigments. The metabolic production functions of algae produced at high densities include energy, food and beverages, nutritional supplements, cosmetics, and It is attracting attention in a wide range of fields, including pharmaceuticals. Creating highly productive strains of algae involves those involved in production. Genome editing, which involves modifying a target gene, is used. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2018-000129 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] In the process of using wild animals for various purposes, humans have developed a system that better suits those purposes. By selecting strains that possess the current form, crossbreeding and breeding them, we can utilize organisms with traits advantageous for achieving the desired objective. In recent years, the genomic DNA sequences of these organisms have become relatively easy to analyze, and By understanding genetic information, we can uncover the reasons why those organisms possess beneficial phenotypes. Furthermore, advancements in genome editing technology have made it possible to arbitrarily modify specific genes and DNA regions. It is possible to change it. However, introducing and inserting DNA or genome editing methods into the cell nucleus is not possible. There are many organisms for which the technology to perform genetic modification and genome editing has not yet been established. The technique has not yet been established. Furthermore, the function of the gene region on the genomic DNA has not been elucidated. Since a region is only a part of a whole, it's not possible to modify even the most beneficial organisms exactly as desired. Genome editing technology has been regulated by the Cartagena Protocol (regulations on genetically modified organisms). Cartagena Protocol on Biosafety to the Convention on Biodiversity, January 2000, A ruling in Europe (adopted at the resumed meeting of the Special Conference of the Parties to the Convention on the Diversity of Materials) has resulted in the same treatment regulations being applied. The ruling was made by a judicial court, and discussions on how to handle it are ongoing in various countries.

[0005] Therefore, even today, there are methods that are not considered genetic modification, such as gamma ray or heavy ion beam irradiation and DNA repair. Breeding through mutation introduction using artificial reagents, etc., is essential for industrial applications (for example) (See Patent Document 1). However, conventional mutation breeding methods do not determine the extent to which they damage DNA. There is no quantitative method to directly evaluate this. Therefore, it is not possible to easily determine things like mortality rate or pigment formation. The irradiation dose and processing rate are determined based on the phenotypic changes, which are used as an indirect indicator of mutation efficiency. Furthermore, random mutations introduced by irradiation can lead to a vast number of strains possessing the desired phenotype. Screening is required from a treatment group of a certain size, which requires effort and time. Until the leaning results are obtained, it is unclear whether or not the target gene mutation has occurred. ru.

[0006] Therefore, the present invention is not subject to genetic modification regulations and does not involve localized mutations in the target gene. Artificial genes and gene mutations that introduce mutations and allow for the quantification of those mutations at the time of mutation introduction. Provide a method. [Means for solving the problem]

[0007] As will be described in detail later with reference to FIG. 5, , 15 , , , 15 , N( 1 H,α1γ) 12 In the C resonance nuclear reaction the 12.9686 MeV level of 16 O * emits an α particle and 12 C * de-excites to the first excited level of 11. <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​By determining this, the distribution from the surface to the interior within the target sample is determined. 15 For N, thoroughly 15 N( 1 H,α1γ) 12 We discovered that it may be possible to induce a 1C resonance nuclear reaction.

[0009] An artificial gene according to one aspect of the present invention, based on at least part of the above findings of the present inventors, At least in some DNA bases 15 The abundance of N exceeds its natural abundance. 15 N The relative abundance of each is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more. It is % or more, 95% or more, 96% or more, 97% or more, or 98% or more.

[0010] In the above embodiment, 15 N-unlabeled primer sequence, 15 N-labeled deoxyribonucleotide It may also contain citrate.

[0011] In the above embodiment, the artificial gene described in the above embodiment is ligated with another artificial gene to form It may be done. Other artificial genes may be used as vectors.

[0012] In the above embodiment, the system has multiple biomolecular binding sites, and of the multiple biomolecular binding sites, a small number Even if not entirely, the gene sequence may be mutated in some areas. Biomolecules are proteins. That's fine.

[0013] An artificial gene according to one aspect of the present invention is 15 An N-labeled artificial gene, comprising multiple biological components It has a binding site for multiple biomolecules, and at least some of the multiple binding sites have mutated gene sequences. It is located at one of several biomolecular binding sites, 15N-labeled biomolecules can bind to them. ru. 15 The biomolecule labeled with N may also be a protein. Of these, the parts of the gene sequence that have not mutated 15 Even if N-labeled biomolecules can bind to them, stomach.

[0014] A kit according to one aspect of the present invention is the above 15 N-unlabeled artificial gene and multiple biomolecular bonds Can be bonded to any of the bonding sites 15 It comprises an N-labeled biomolecule.

[0015] A method for mutating a gene according to one aspect of the present invention involves DNA 15 To label with N, 15 This includes irradiating DNA with a proton beam having the energy to cause a resonance nuclear reaction in which N occurs.

[0016] In the above embodiment, DNA 15 Labeling with N means that the N in DNA 15 Replace with N It may contain DNA. 15 Labeling with N is used to identify the vicinity of DNA. 15 N This may include labeling the DNA. 15 Labeling with N is possible for DNA 15 Signed with N This may also include binding the biomolecules. 15 N-labeled biomolecules It's fine if it's protein.

[0017] In the above embodiment, DNA in a living cell 15 It may also be labeled with N.

[0018] In the above embodiment, in cellular components other than DNA within living cells 15 Maintaining N at its natural abundance In DNA 15 The abundance of N may be greater than its natural abundance.

[0019] In the above embodiment, DNA in a living cell 15 To label with N, 15 N-labeled deoxy Even if it includes administering ribonucleotide and glutamine synthase inhibitors to living cells good.

[0020] In the above embodiment, DNA in a living cell 15 To label with N, 15 N-labeled deoxy This includes providing inhibitors of ribonucleotides and ribonucleotide reductase to living cells. It's okay to be there.

[0021] In the above embodiment, the DNA in a living cell 15 To label with N, 15 N Non-sign This may further include the delivery of lutamine to living cells.

[0022] In the above embodiment, the detection of a resonance nuclear reaction may be further included.

[0023] In the above embodiment, when detecting, a gamma ray dose of 4.43 MeV may be measured. stomach.

[0024] In the above embodiment, the number of mutations in the DNA is calculated based on the measured gamma ray dose. It may also include the following.

[0025] In the above embodiment, when calculating, 15 Resonance occurring in standard samples with a known N atom number You may also refer to the amount of gamma rays produced by nuclear reactions.

[0026] In the above embodiment, 15The energy at which N undergoes a resonant nuclear reaction may be changed. [Effects of the Invention]

[0027] According to the present invention, genetically modified organisms are not subject to regulations, and mutations localized to the target gene are not achieved. This invention introduces an artificial gene and gene mutation method that can quantify the mutation at the time of mutation introduction. We can provide the law. [Brief explanation of the drawing]

[0028] [Figure 1] This figure shows a DNA molecule in which the nitrogen atoms present in each base pair are replaced with 15N in a gene mutation method according to an embodiment of the present invention. [Figure 2] This figure shows how resonance nuclear reactions occur in DNA molecules. [Figure 3] This figure shows the case when using the conventional method of gene mutation by heavy ion beam irradiation. [Figure 4] This figure shows a standard sample of artificial DNA composed of 84 base pairs. [Figure 5] This is a diagram showing the 15N(1H,α1γ)12C resonance nuclear reaction. [Figure 6] This is a schematic diagram of a method for determining whether or not DNA is broken. [Figure 7] This diagram outlines a method for identifying how many breaks have occurred in one of the DNA strands. [Figure 8] This figure shows the synthesis of nitrogen-containing organic compounds within cells. [Figure 9] This figure illustrates a novel method for labeling only nucleic acids with 15N within cells when MSX and 15N-unlabeled glutamine are administered. [Figure 10] This diagram illustrates a method for labeling only DNA with 15N in the nucleic acid synthesis pathway. [Figure 11] This diagram illustrates a novel method for labeling only the target gene or its vicinity with 15N. [Figure 12]This figure shows the gene structure of the pUC4-KIXX circular plasmid. [Figure 13] This graph shows the resonance curve of the 15N(p,α1γ)12C resonance nucleus reaction of a 15N_pUC4-KIXX plasmid sample. [Figure 14] This graph shows the relationship between the amount of proton beam irradiation to a plasmid and gene damage. The proton-irradiated plasmid was introduced into E. coli, which was then cultured in a medium supplemented with ampicillin. [Figure 15] This graph shows the relationship between the amount of proton beam irradiation to a plasmid and gene damage. The proton-irradiated plasmid was introduced into E. coli, which was then cultured in a medium supplemented with kanamycin. [Figure 16] This graph shows the relationship between the amount of proton beam irradiation to a plasmid and gene damage. The proton-irradiated plasmid was introduced into E. coli, which was then cultured in a medium supplemented with ampicillin and kanamycin. [Modes for carrying out the invention]

[0029] Embodiments of the present invention will be described with reference to the attached drawings. Note that in each figure, the same Those marked with this symbol have the same or similar configuration.

[0030] The nitrogen element N in living organisms is scattered throughout cellular components, including proteins such as enzymes, and even DNA. In the gene mutation method according to this embodiment, the naturally occurring ratio of these is 0.364%. to be 15 We create a situation where the N isotope is concentrated and unevenly distributed in the target gene. 15 N and 1 H 15 N( 1 H ,α1γ) 12 By irradiating target genes with a proton beam using the energy that causes a 1C resonance nuclear reaction, It causes a major mutation in the target gene.

[0031] In the double strand of DNA that makes up genes, adenine (A) has 5 nitrogen atoms in purine It is bonded as a ring and an amino group, and thymine (T) has two nitrogen atoms bonded to the pyrimidine ring. In a double-stranded DNA adenine-thymine pair (AT pair), there are seven nitrogen atoms. Five nitrogen atoms are attached to guanine (G), and three are attached to cytosine (C), forming DNA. In a double-stranded guanine-cytosine pair (GC pair), there are eight nitrogen atoms.

[0032] Figure 1 shows the N present in each base pair. 15 This represents a DNA molecule substituted with N, and naturally 15 The isotope ratio in which N is present is concentrated 275.5 times. 15 This shows the state with N labeled. Oh, in the natural world 15 The abundance of N is 0.364%.

[0033] Figure 2 shows how resonance nuclear reactions occur in DNA molecules. 15 One of the N-labeled DNA of 15 N nucleus and proton 15 N( 1 H,α1γ) 12 When a 1C resonance nuclear reaction occurs, 15 N disappears, Before the reaction 15 Centered around the bond position of the N atom 4 He and 12 C nuclei are nearly isotropic in opposite directions. It is released, immediately captures electrons, and passes through the material as an ion. In nearby atoms, high-density electron excitation occurs.

[0034] The probability of receiving electron excitation decreases inversely proportional to the square of the distance from the reaction center. The farther away an atom is, the smaller it is. 15 When N undergoes a resonance nuclear reaction, 15 Bonds of N atoms The atoms near the conjunction position will be locally excited by a high probability. 15 Bonding position of N atom This makes it easier for localized mutations to occur in the surrounding DNA.

[0035] Figure 3 shows the case using the conventional heavy ion beam irradiation gene mutation method. In conventional gene mutation methods using heavy ion beam irradiation, heavy ions are located outside the DNA. Because heavy ions are incident at a constant density throughout the entire DNA, the atoms near the DNA along the heavy ion tracks... The probability of mutation occurring due to electron excitation is almost the same throughout the entire DNA molecule.

[0036] In contrast, the method of this embodiment involves the target gene or the vicinity of the target gene. 15 N same By increasing the tactile ratio, with a high probability, 15 This causes localized mutations in the DNA near the N atom's bonding site. It can be done.

[0037] In this embodiment, the target gene or its vicinity 15 Techniques to increase and enrich the nitrogen isotope ratio. It naturally exists in the environment at a concentration of 0.364%, and also exists in living cells at the same ratio. 15 N It is a technology that is often used. Also, in its DNA 15 It is produced by irradiating N with a proton beam. 15 N( 1 H,α1γ) 12 C-resonance nuclear reactions are the most abundant type of cosmic ray in the hadron component near the Earth's surface. The proton beams randomly rain down on surface organisms, and within their DNA... 15 Collision with N nucleus This is the same as the nuclear reaction that occurs, and it has a similar effect on living cells.

[0038] In other words, the target gene or its vicinity 15Technique for increasing and enriching the N isotope ratio, and that technique by 15 irradiating proton beams to N-labeled living cells 15 N( 1 H,α1γ) 12 C resonance nuclear reaction to cause and increase the mutation probability of the target gene is nothing but a technique to accelerate the process in which life has been naturally exposed and received mutations in its genetic information since the birth of life on Earth, and is the same as the conventional method of creating mutant strains by irradiation with radiation (UV, X-rays, γ-rays, heavy ions), and the basic principle of the mechanism does not change at all. Therefore, the gene mutation method according to this embodiment is fundamentally different in principle from gene recombination technology, and is not subject to the regulations on gene recombination technology, and has an excellent feature that it enables large mutations to be introduced into the target gene . The method is a technique with an excellent feature that enables large mutations to be introduced into the target gene, which is fundamentally different in principle from gene recombination technology and is not subject to the regulations on gene recombination technology .

[0039] For quantification of DNA mutations caused by irradiating proton beams to a DNA sample with an increased N isotope ratio in a target gene or near the target gene in a living cell, 15 construction of an N-labeled DNA standard sample with a clearly defined number of N atoms is necessary 15 . 15

[0040] 15 By constructing an N-labeled DNA standard sample, 15 it becomes possible to obtain calibration data between the 4.43 MeV γ-ray dose emitted by the N( 1 H,α1γ) 12 C resonance nuclear reaction and the N concentration in DNA . By obtaining the dose calibration value of the 4.43 MeV γ-ray, 15 it becomes possible to measure the 4.43 MeV γ-ray dose emitted by the resonance nuclear reaction simultaneously with the proton beam irradiation to N-labeled living cells 15 . ​​​It can measure and compare with the calibration value, enabling quantification of the number of mutations that occurred in DNA within living cells.

[0041] In the gene mutation method according to this embodiment, 15 DNA composed of N-labeled base pairs was used to construct a standard sample artificially synthesized by the polymerase chain reaction method. The constructed standard sample of artificial DNA is preferably composed of base sequences that are important in molecular biology, biochemistry, and biological functions. Figure 4 is a diagram showing a standard sample of artificial DNA composed of 84 base pairs. The artificial DNA standard sample is prepared, for example, by the following method. At the 5' end,

[0042] a primer OR_ with a TACGTTAAATC sequence structure containing N unlabeled, that is, 15 N-free, that is, 14 N-containing at a natural abundance ratio of 99.636%, and primer_F, and 15 N-labeled, that is, 15 N-containing deoxyribonucleoside triphosphate 15 N-labeled dNTPs (deoxyribonucle oside 5’-triphosphates) 73 pieces, from which oligonucleotide OR_WT_F is synthesized by the polymerase chain reaction method. Also, at the 5' end, 15 a primer OR_primer_R with an N-unlabeled TGCAACCATT sequence structure, and 15 N-labeled deoxyribonucleoside triphosphate 15 N-labeled dNTPs 74 pieces, from which oligonucleotide OR_WT_R is synthesized by the polymerase chain reaction method. Further, OR_WT_F and OR_WT_R form base pairs with each other by hydrogen bonding, and 15 N-labeled Recognition of oligonucleotide double-stranded DNA 15 N-labeled OR_DNA is formed. This will be used as the standard sample.

[0043] 15 N-labeled OR_DNA is, 15 N non-labeled primer OR_primer_F / _R, 15 N-labeled deoxyribonucleotide 15 N-labeled dNMPs (Deoxyri The bonucleotide 5'-monophosphates) together make up 84 It consists of 1 DNA base pair (84 bps). The breakdown of its base sequence is as follows: Excluding OR_WT_F at 73 bps, 15 N-labeled dAMP: 19 units , 15 N-labeled dTMP: 22 pieces, 15 N-labeled dGMP: 17 pieces , 15 There are 15 N-labeled dCMPs. Also, OR_WT excluding primers. In _R at 74 bps, 15 N-labeled dAMP: 23 units, 15 N-lab eled dTMP: 20 pieces, 15 N-labeled dGMP: 16 pieces, 15 N-lab There are 15 eled dCMP entries. These are summarized in Table 1 below.

[0044] [Table 1]

[0045] 15 N(N) included in the entire N-labeled OR_DNA 14 N+ 15 The total number of N) is 623. 15 N The standard purity of the label is 98% or higher, and according to that purity... 14 N and 15 Isotope abundance of N The rate changes. 15 If the N-labeling purity is 98%, the entire OR_DNA 15 N has 538.29 values. Ideally, 15 At 100% N-labeled purity, the entire OR_DNA 15 N has a maximum number of 54. There are 9.27 nitrogen atoms, which accounts for 88.17% of the total nitrogen atoms (see Table 1). 15 N labeled OR The molecular weight of DNA is also 15 Depending on the purity of the N label, it is 52,467 g / mol or higher. The maximum value is 52,477.95 g / mol.

[0046] Figure 5 shows 15 N( 1 H,α1γ) 12 It exhibits a 1C resonance nuclear reaction. 15 For a biological sample labeled with N When irradiating with a proton beam, 15 The binding energy of the two nuclei in a center-of-mass system of a nitrogen nucleus and a proton. -12.1277 MeV, 16 O * The second excited level of the composite nucleus is 12.9686 MeV. The energy difference is 0.8409 MeV (converted to the proton energy of a laboratory system, which is 0 When the two atomic nuclei collide at 0.987 MeV, they resonate and bond together. 16 O * Composite A nucleus is formed at a level of 12.9686 MeV. 16 O * It immediately emitted an alpha particle. 12 C * The first reaction channel de-excitates to the first excited level of 11.6007 MeV, and the alpha particle Release 12A second reaction channel de-excitates C to the ground state of 7.1616 MeV, and γ Emitting only lines 16 Three types of reaction channels, including a third reaction channel that de-excites O to its ground state of 0 MeV. The following types of reaction channels are generated: (p,α1γ), (p,α0), (p,γ0) and nuclear reaction. It will be written in the grammatical form.

[0047] A characteristic of the (p,α1γ) reaction channel used in this embodiment is its high ionization compared to proton beams. α( 4 He nucleus) and, 12 C and are released as reaction secondary particles, Each reaction 12 C * It de-excites from the first excited state to the ground state and emits a 4.43 MeV gamma ray. In the (p,α0) reaction channel, there is no emission of gamma rays, and also (p In the γ0) reaction channel, there is no emission of reaction secondary particles with high ionization properties. The emission of reactive secondary particles with a function is 15 If N is inside or near the DNA, 15 It exerts a high localized ionization effect on biomolecules near N, thereby ionizing the DNA for the desired genetic material. This has the effect of increasing the probability of producing sub-mutations. Furthermore, it is released with each nuclear reaction. The 4.43 MeV gamma rays produced are easily countable, and these gamma rays are produced by proton beam irradiation. This reflects the amount of DNA gene mutations that result. Therefore, 4.43 during beam irradiation. The effect is that the amount of gene mutations in DNA can be quantified by counting MeV gamma rays. It plays.

[0048] 16 O * The resonance energy with the second excitation level is the Coulomb barrier in the collision of the two atomic nuclei. It is far lower than the tensal. Therefore, the nuclear collision energies of the two are 16 O * composite atom The resonance energy width of 300 eV, defined as the second excitation level width of the nucleus, exceeds the resonance energy. When removed from the ghee, the reaction cross-section decreases rapidly. That is, 15 N-labeled OR_DNA When a proton beam is incident at resonance energy, fluctuations in the incident energy correspond to the resonance energy width. Only within the limits 15 N( 1 H,α1γ) 12 This causes a 1C resonance nuclear reaction.

[0049] In actual proton beam irradiation, the resonance energy width and the energy broadening of the proton beam are typically... The resonance energy width is determined by a convolution integral operation with approximately 1 keV, and the protons are... The energy loss during passage through the material is added to the resonance energy to determine the proton beam irradiation energy. To decide. 15 N-labeled DNA sample, or 15 Living cells containing N_DNA In proton beam irradiation of any biological sample, the energy of the proton beam is changed from the resonance energy to the resonance energy. Up to energies higher than energy, for example, by changing the energy in a constant energy step size. By doing so, the distribution within the target sample extends from the surface to the interior. 15 For N, thoroughly 15 N( 1 H,α1γ) 12 It may become possible to induce a 1C resonance nuclear reaction. Energy change The width and step size should be selected according to the target sample. 15 N( 1 H,α1γ) 12 C resonance nucleus The resonance curve of the reaction may be obtained and determined. See, for example, Figure 13.

[0050] 15 N( 1 H,α1γ) 12 Produced by 13C resonance nuclear reaction 12 C * α and α are emitted in opposite directions. Therefore, the kinetic energy reaches its maximum value E(max) when the proton beam is emitted in the same direction as the incident direction. And so, 12 C * E for each of α C (max) = 0.6252 MeV, E He (max= This becomes 1.2806 MeV. Also, 12 C * And α is emitted in the opposite direction to the direction of incidence of the proton beam. When this occurs, the kinetic energy reaches its minimum value E(min), and E C (min)=0.1437 MeV, E He (min) = 0.7991 MeV. 12 C * And α immediately after emission is electron Capture 12 C, 4 It becomes a He ion and travels through the substance.

[0051] 12 C and 4 He ions always have values ​​within these energy ranges. 15 N-labeled OR_D It passes through the NA sample. Nearby atoms along its range, especially those with a radius of 5 nm to 10 nm. The following neighboring atoms are electronically excited. The target sample is, 15 Not an N-labeled OR_DNA sample, but a eukaryotic sample. These are biological samples that possess cell walls and intracellular organelles, such as single-celled algae, and the cell walls and intracellular organelles are exposed to sunlight. Taking into account the energy loss when the child passes through, at an energy higher than the resonance energy Even when performing proton beam irradiation, the proton beam and 15 N-labeled DNA or DNA near the DNA 15 N-labeled test With the fee 15 N( 1 H,α1γ) 12 In 1C resonance nuclear reactions, regardless of the proton beam irradiation energy, 12 C and 4 The He reaction-produced ions are released with constant energy, and their trajectory affects nearby atoms. A key feature of this method is that it generates excitation at a constant intensity.

[0052] moreover, 12 C and 4 The electron excitation of the He reaction product ions is linear energy transfer. energy transfer, LET(MeVcm 2 / g)) is given, 12 C and 4 The He reaction product ions vary over the energy range, affecting the constituent elements of the target sample. LET to carbon C, one of the components, 12 2.51 × 10¹⁰ ions produced by the C reaction 3 MeVcm 2 / g or more, maximum 4.70 × 10 3 MeVcm 2 / g 4 In the He reaction product ions, , 1.73 × 10 3 MeVcm 2 / g or more, with a maximum of 1.99 × 10 3 MeVcm 2 / g Yes, this value is when the proton is at resonance energy. 15 LE when passing through N-labeled OR_DNA sample Compared to T, it is 17.2 times higher, and up to 27.1 times higher. Therefore, in this method, 15 N mark For biological samples, including DNA samples, 15 N( 1 H,α1γ) 12 Generation of C-resonance nuclear reaction It exhibits the characteristic that electron excitation by ions is overwhelmingly higher than electron excitation by proton beam irradiation. Target gene 15 N labeling makes it possible to selectively introduce large mutations. .

[0053] 12 C and 4 The LET value of the He reaction product ions is typical for heavy ion irradiation (e.g., 320). The LET to C in MeV C-ion irradiation is 0.58 × 10⁻¹⁰. 3 MeVcm 2 / g) Compared to that, it is more than 4.3 times higher, and up to 8.1 times higher. Furthermore, it is either inside or near the DNA. and others 12 C and 4 Since He reaction product ions are released, 15 N-labeled OR_DNA sample and D Near NA or DNA 15 In N-labeled living cells, one 15 N( 1 H,α1γ) 12 C By resonant nuclear reaction 15 Mutations occur with a high probability in the N-neighboring genes. Therefore, this method is effective. Reaction product nuclei 12 By measuring the 4.43 MeV gamma rays emitted from the first excitation level of C, the law of nature, 15 It is possible to directly quantify the number of gene mutations occurring in the vicinity of N.

[0054] By proton beam irradiation 15 Mutations occurring in N-labeled OR_DNA samples have DNA repair function Unlike biological samples, DNA repair does not occur. Therefore, 15 N sign OR_DN By examining the mutations in sample A, the purely physical mutations caused by proton beam irradiation will become clear. . 15 One method for examining mutations in N-labeled OR_DNA samples involves examining the cytoplasm of bacteria and other organisms. Using existing circular DNA and circular plasmid vectors, we can determine whether or not DNA is cleaved. This is a method for making distinctions.

[0055] Figure 6 shows an artificial gene obtained by ligating the artificial gene shown in Figure 4 to another artificial gene, and DNA cutting. This outlines a method for determining the presence or absence of [the substance]. It also describes β-lactam antibiotics used in the treatment of infectious diseases. By incorporating the gene of E. coli that is resistant to ampicillin, humans Industrially synthesized linear plasmid (T-vector pMD19, 2,692 base pairs) to 15 A plasmid vector, formed by ligating N-labeled OR_DNA into a circular shape, was added to the culture medium of E. coli. The circular plasmid vector is taken up by E. coli, and ampicillin-resistant cells are formed. E. coli bacteria multiply. 15 If N-labeled OR_DNA is pre-cleaved by proton beam irradiation The plasmid vector does not form a ring, and is degraded within E. coli cells to become a stable gene. Because it does not have resistance to ampicillin, E. coli is killed by it. 15 Ligation of N-labeled OR_DNA In the circular plasmid vector, the cleaved 15 It contains a large amount of N-labeled OR_DNA. The growth of E. coli will slow down accordingly. 15 N-labeled OR_DNA sample, 15 N-unlabeled OR_DNA assay Fees, 15 Each N-labeled OR_DNA sample, which was not irradiated with proton beams, was processed using T-vector pMD19. The cells were ligated, and each was placed in an ampicillin-containing medium to compare the number of E. coli colonies. 15 N-labeled OR_DNA sample 15 N( 1 H,α1γ) 12 DN by C-resonant nuclear reaction and proton beam A mutation occurs. 15 N-unlabeled OR_DNA samples undergo DNA mutations using only a proton beam. Therefore, samples not irradiated with proton beams do not show DNA mutations. This evaluation method allows for... 15 N labeled OR The relative frequency of DNA mutations introduced by proton beam irradiation of DNA will be revealed.

[0056] 15 A more direct method for investigating the state of N-labeled OR_DNA after it has been cleaved by proton irradiation. as, 32 A phosphate group containing the radioactive isotope P is added, and one base is decomposed by electrophoresis. There is a technique for separating fragments using this method. This technique allows us to determine how much of one strand of DNA can be cut. This makes it possible to identify whether a disconnection has occurred.

[0057] Figure 7 outlines a method for identifying how many breaks have occurred in one of the DNA strands. To demonstrate. First, 15 Before PCR sample synthesis of N-labeled OR_DNA, OR_primer_F or One of the OR_primer_R primers is phosphorylated with ATP, PC During R sample synthesis, a DNA fragment is synthesized in which a phosphate group is attached only to the 5' end of one of the DNA strands. After proton beam irradiation, [γ? 32 Using [P]-ATP, use the DNA without a phosphate group. Chain only 32 Label with P. Electrophoresis is performed using a thin gel with a low salt concentration and containing urea, under high voltage. By applying heat, the DNA is separated in a single-stranded state (denatured state), and a difference of one base is detected. DNA can be distinguished. Since a single base difference can be distinguished by gel electrophoresis, random cutting is possible. It is possible to determine whether cleavage is possible or whether only the vicinity of a specific base is susceptible to cleavage.

[0058] DNA within living cells 15 When labeling with N, nitrogen elements other than DNA, which make up living cells, are used. Intracellular molecules containing these molecules, such as proteins including enzymes that regulate cell formation and metabolic functions. Present nitrogen atoms, or ribosomal RNA that forms ribosomes, amino acids Transfer RNA that transports acid, and m that defines the amino acid sequence on ribosomes. The nitrogen element present in RNAs that have diverse functions within cells, such as e-sensor RNA, It distinguishes the nitrogen element that makes up DNA from the DNA itself, and extracts only the DNA. 15 Mark with N.

[0059] Nitrogen is essential for the formation of amino acids, nucleotides, proteins, nucleic acids (DNA, RNA), and It is used in a variety of nitrogen-containing organic compounds, such as phospholipids. These are all inorganic nitrogen ammonium ions (NH4). + But glutamine synthase produces glutamine The first organic nitrogen-containing compound reacts with an acid and is fixed to an organic compound as glutamine. This is produced (ammonia assimilation). Glutamate synthase converts glutamine to 2-oxygen. An amino group is transferred to soglutaric acid, producing two molecules of glutamic acid. The resulting glutamine and glutamic acid are transformed, or the amino group Numerous nitrogen-containing organic compounds are synthesized through transposition reactions.

[0060] Figure 8 shows the synthesis pathway of nitrogen-containing organic compounds within cells. Nitrogen-containing compounds are synthesized within cells. It is then broken down into ammonia and recycled. Amino acids are used in various structural applications. It is used in the synthesis of proteins, enzymes, nucleic acids, and lipids. On the other hand, DNA is deoxyribo DNA synthesis proceeds through the transmission of genetic information from nucleotides. 15When N-labeled deoxyribonucleotide is administered to the culture medium, it is taken up into the cells, The part is broken down and ammonium ions are synthesized, and protein synthesis begins. Only DNA is used. 15 To label with N, inhibit that process 15 N-labeled proteins are not synthesized. Sea urchin, MSX (methionine sulfoxazole), a glutamine synthase inhibitor. Administer imine.

[0061] Figure 9 shows MSX and 15 When N-unlabeled glutamine is administered, nucleic acids are produced in cells. Mi 15 We present a new method for N-labeling. Glutamate and A Ammonia acts as a mino group to synthesize glutamine, and glutamine is then synthesized from glutamine synthase. Luthamine is released. However, the structural formula of MSX has an HN=S=O structure, which is... Its structural formula is similar to that of luthamine, H2N-CH=O. Furthermore, MSX has a strong active site. When glutamic acid and ammonia bind together, they cannot detach from MSX, and glutamine synthesis stops. To stop. That is, glutamate is synthesized from glutamine by glutamate synthase. The circuit was cut off, 15 N-labeled proteins will no longer be synthesized. However, they are needed for a variety of purposes. To prevent the synthesis of the protein being produced from being interrupted, 15 N-unlabeled glutamine is administered to the culture medium. Therefore 14 Structural proteins, enzymes, and some DNA with a nitrogen (N) abundance of 99.637% NA is synthesized. It is taken into the cell. 15 Among N-labeled deoxyribonucleotides, The remaining ones not included in Monia synthesis 15 DNA is synthesized by N-labeled deoxyribonucleotides. Therefore, within the cell 15 the N-labeling will be limited to only DNA.

[0062] Furthermore, in the nucleic acid synthesis pathway within the cell, only DNA 15 the process of labeling with N is shown in more detail in Fig. 10. First To synthesize DNA labeled with N, (1) four types of bases are each 15 labeled with N and the deoxyribonucleotides are added to the medium to culture the cells. Next, within the cell, since deoxyribonucleotides for synthesizing DNA are synthesized only from ribonucleotides by ribonucleotide reductase ( each 15 RNR), (2) an inhibitor of RNR (i- RNR) is administered to create a condition where unlabeled deoxyribonucleotides are not synthesized within the cell. Further within the cell, (3) the bases are transposed between deoxyribonucleotides and ribonucleotides, and the N-labeled bases of the deoxyribonucleotides introduced from outside the cell are transposed to the bases of ribonucleotides within the cell and can be directly 15 used to synthesize N- RNA. Conversely, the <00…​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​When xylibonenucleotides are added to the culture medium, (3) 15 N_base is deoxyribonu The possibility of translocation from creotide to ribonucleotide arises. Therefore, 15 N...D NA synthesis efficiency is high, 15 To suppress N RNA synthesis, 15 N_Deoq The amount of siribonucleotide added can be adjusted as needed.

[0063] Figure 11 shows only the target gene or its vicinity. 15 We present a new method for N-labeling. (See Figure 4) 15 The base sequence of N-labeled OR_DNA is the lambda (λ) of a virus that parasitizes E. coli as a host. ) It is part of the phage's genes, and the lambda phage parasitizes the host E. coli without killing it. Therefore, it is a regulatory gene that inhibits self-replication. 15 N-labeled OR_DNA is 3 It has a Cro protein binding site, and the Cro protein will bind to one of these sites. This activates the regulatory gene. Of the three binding sites, the site where the gene sequence has mutated leads to... When this is introduced, the frequency of activation of regulatory genes is controlled. In the explanation in Figure 4, polymerase chain Chain reaction method 15 The method for preparing N-labeled OR_DNA was explained. In contrast, 84 base pairs OR_DNA 15 After synthesis without N labeling, the regulatory gene is added to the culture medium. The E. coli that had entered, 15 When N-labeled Cro protein is administered, 15 N-labeled Cro tamper The substance binds to the regulatory genes of E. coli. By irradiating with proton beams in this state, 15 N labeled C Near the specific gene to which the ro protein is bound 15 N( 1H,α1γ) 12 C resonance nuclear reaction occurs This method makes it possible to induce mutations in specific genes with a high probability.

[0064] [Example 1: 15 [Results of proton beam irradiation of N-labeled OR_DNA samples] (1) 15 Preparation of N-labeled OR_DNA samples [Phosphorylation of OR_primer_R] This section describes the phosphorylation process, assuming the evaluation of DNA rupture after proton beam irradiation. ATP(1 0 mM=mmol / L):16 μL, OR_primer_R(100 μM):1 0 μL, 10x dilution buffer: 16 μL, T4PNK: 8 μL, sterile water: 11 After reacting 0 μL at 37 °C for 35 minutes, the temperature is raised to 72 °C to stop the reaction, and PCI Extraction (Phenol Chloroform Isoamylalcohol (25: 24:1) After removing proteins and lipids by extraction, ethanol The material was fixed and extracted.

[0065] [By PCR method] 15 [Synthesis of N-labeled OR DNA] In addition, two types were synthesized: with and without phosphorylated primers. Heat-stable DNA polymerase ( PrimeSTAR: 0.5 μL, 5x diluted buffer solution: 10 μL, 15 N sign ( 15 N purity 98% or higher) dNTP: 6 μL, OR_primer_F (10 μM): 2.5 μL, OR_primer_RP (phosphorylated primer 10 μM): 7.5 μL, template: 1 μL, sterile water: 22.5 μL, thermostable DNA synthase (Pr ime STAR): 0.5 μL, 5x diluted buffer solution: 10 μL, 15 N sign (15 N purity 98% or higher) dNTP: 6 μL, OR_primer_F (10 μM): 2 0.5 μL, OR_primer_R (non-phosphorylated primer 10 μM): 2.5 μL of PCR, 1 μL of template, and 27.5 μL of sterile water were each subjected to PCR 35 cycles. The electrophoresis was completed after the following cycles: 98°C for 10 minutes, 31°C for 20 minutes, and 72°C for 30 minutes. We confirmed that 84 base pairs of DNA were synthesized using the dynamic method.

[0066] (2) Proton beam irradiation 15 N-labeled OR_DNA sample, 15 N-unlabeled OR_DNA sample was dropped onto an Au substrate and irradiated. A sample was prepared. The OR_DNA solution concentration was 31.2 ng / μL, and the drop volume was 13 μL. Area 0.23 cm 2 The dropped sample contained 4.7 × 10 12 Each D contains DNA NA has more than 538 15 N atoms are bonded. Converted to a per-sample area, 1.1 × 10 16 cm -2 The proton beam irradiation was carried out at the National Institute of Advanced Industrial Science and Technology (AIST). The 4MV Peletron Electrostatic Accelerator at the Tsukuba Center and the Research Infrastructure Center of the University of Tsukuba - This was performed using a 1MV tandem electrostatic accelerator. The beam current was 0.1 nA to 5 nA. A. The beam irradiation area is 0.071 cm². 2 That was the case. 15 N( 1 H,α1γ) 12 C-resonance nuclear reaction The 4.43 MeV gamma rays emitted are high-energy gamma rays containing Bi, which has atomic number 83. The most efficient detection method is BGO (Bi4Ge3O 12 Crystal scintillator, specific gravity 7.3 g / cm³ -3 A detector with a diameter of 76.2 mm × length of 79.2 mm was placed at a distance of 24 mm from the irradiated sample outside the vacuum for detection. The detectable solid angle of the detector is 10% of the omnidirectional angle . When the detection sensitivity for 4.43 MeV γ-rays is set to a maximum of 0.1, the detection efficiency for 4.43 MeV γ-rays is approximately 1% at maximum. 15 When proton beam irradiation is performed on the N-labeled OR_DNA sample at a certain energy, the count of 4.43 MeV γ-rays is 4,000 counts, and the total count for 8-point irradiation with an energy ranging from 8 keV is 25,000 counts . Considering the detection efficiency, at least 2.5×10 6 15 N-labeled OR_DN A 15 mutations due to N resonance nuclear reactions have occurred.

[0067] (3) 15 Ligation of N-labeled OR_DNA to a linear plasmid vector: 15 Creation of A overhangs at the ends of N-labeled OR_DNA Proton beam irradiated 15 N-labeled OR_DNA (9 ng / μL): 6.9 μL, buffer solution (Ex taq) 1 μL, Ex taq 0.1 μL, dATP (10 mM) 2 μL, unirradiated sample OR_DNA (8 ng / μL): 6.9 μL, 10-fold diluted buffer solution (Ex taq) 1 μL, Ex taq 0.1 μL, dATP (10 mM ) 2 μL, negative insert DNA sample: 6.9 μL, 10-fold diluted buffer solution (Ex_taq) 1 μL, Ex taq 0.1 μL, dATP (10 mM) 2 μL, control insert CI sample (10 ng / μL): 6.9 μL, 10-fold diluted sample ​1 μL of Buffer Solution (Ex_taq), 0.1 μL of Ex_taq, dATP (1 Four types of mixed solutions (0 mM) in 2 μL portions were prepared and reacted at 72 °C for 1 hour.

[0068] [Ligation with linear plasmid vectors] In a linear plasmid vector (T-Vector pMD 19, 2,692 base pairs), Mix the four types of mixed solutions on ice, leave for 30 minutes, then heat shock at 42°C for 30 seconds. The transformation efficiency was increased by adding [a specific ingredient].

[0069] [Ampicillin-resistant E. coli culture] Escherichia coli (JM109) cultured in M9 medium at 37°C with shaking at 180 rpm was subjected to 4 The molecules were inoculated into ampicillin-containing medium administered with various circular plasmid vectors, and JM109 Colony formation was compared. A circular plasmid vector, obtained by ligating a beam-unirradiated sample, was administered. In the culture medium, the colony count was 46 / 11 ngDNA, whereas in the beam-irradiated medium... 15 In the culture medium administered with a circular plasmid vector containing N-labeled OR_DNA, colonies The number of ngDNA molecules decreased to 13 out of 11, less than one-third of the original amount.

[0070] [Example 2: Results of proton beam irradiation of E. coli biological samples] Since proton beam irradiation is basically performed in a vacuum, first we freeze E. coli (JM109) The solution was dried and its resistance was examined. 10 μL of JM109 E. coli solution was placed in an Eppendorf tube. Place the samples in a container, and once they freeze at liquid nitrogen temperature, separate them into samples that freeze and samples that do not freeze at liquid nitrogen temperature. Place them in a vacuum dryer at -20°C and leave for 4 hours, then remove and leave at 4°C. The samples were then left for 24 hours to create freeze-dried samples. The JM109 colon was then prepared at this point. The bacterial survival rate was 26.8% for samples frozen with liquid nitrogen, and for samples that did not freeze with liquid nitrogen. The figure was 39.1%.

[0071] JM109 E. coli has 514 million base pairs of DNA, and this DNA is analyzed using two methods in 15 We placed an N marker. One of them was, 15 In a culture medium containing N-labeled ammonium ions, In JM109 E. coli cultured without administration of MSX, a protein synthase inhibitor, the protein Nucleic acid, nucleic acids, and all nitrogen within the details 15 It is labeled with N (sample F). The other one is, 15 N In a culture medium containing labeled deoxyribonucleotides, MSX and 15 Administer unlabeled glutamine. In the JM109 E. coli cultured with this method, only DNA and RNA were found. 15 N-labeled biological sample (Sample D).

[0072] 2 types 15 N-labeled JM109 E. coli and 15 N-unlabeled JM109 Escherichia coli (sample E) Three types of solutions were prepared to create proton beam irradiation samples. The concentration of E. coli in each irradiation sample was measured. , volume of solution dispensed, area of ​​application, number of cells dispensed, area density of cells, 15 The area density of N is as follows: It is.

[0073] [Sample D] E. coli concentration: 3.17×10 8 cells / mL, solution dropping volume: 10 μL, dropping surface Product: 1.26 cm 2 Number of cells dropped: 3.17 × 10 6 Cells, area density of cells Degree: 2.52 × 10 6 cells / cm 2 , 15N area density: 9.50×10 13 cm -2 .

[0074] [Sample E] E. coli concentration: 3.17×10 8 cells / mL, solution dropping volume: 10 μL, dropping surface Product: 1.20 cm 2 Number of cells dropped: 3.17 × 10 6 Cells, area density of cells Degree: 2.64 × 10 6 cells / cm 2 , 15 N area density: 3.66×10 11 cm -2 .

[0075] [Sample F] E. coli concentration: 8.62×10 7 cells / mL, solution dropping volume: 20 μL, dropping surface Product: 1.56 cm 2 Number of cells dropped: 1.72 × 10 6 Cells, area density of cells Degree: 1.11 × 10 6 cells / cm 2 , 15 N area density: 4.18×10 13 cm -2 .

[0076] From the 4.43 MeV gamma ray dose measured simultaneously with proton beam irradiation, the DNA and RN of sample D were determined. Only A 15 N-marked 15 The amount of N is the total amount of nitrogen throughout the cells of the F sample. 15 When labeled with N It was revealed that the probability is 1 in 44.6.

[0077] [Example 3: 15 [Results of proton beam irradiation of N-labeled drug resistance gene plasmid samples] (1) pUC4-KIXX plasmid and method for preparing it Figure 12 shows the gene structure of the pUC4-KIXX plasmid (plasmid DNA). This shows that the plasmid contains the ampicillin resistance gene (Ampicillin-R) and Ori The gene pUC4 (2605 bps (base pairs)) contains the kanamycin resistance gene (Ka A circular plasmid (3853 bp) into which namycin-R (1248 bps) is inserted. s) The gene size occupied by the promoters and transcription regions of the two types of drug resistance genes is The ampicillin resistance gene is 934 bps, and the kanamycin resistance gene is 953 bps. The region Ori, which is necessary for plasmid replication, is 621 bps. Types of base pairs in each region and 15 Table 2 shows the sample size (100% replacement rate). [Table 2]

[0078] By the following method, 15 Plasmids containing N in their natural abundance, 15 Contains over 98% N A plasmid containing the substance was generated. 15 NH4C containing N at its natural abundance (0.364%) A 250 mL minimum medium of M9 was prepared using 1 as the nitrogen source. 15 Contains over 98% N possess 15 A 250 mL minimum volume of M9 medium was prepared using NH4Cl as the nitrogen source. Specifically, Each of the M9 minimum mediums contains Na2HPO4 (15 g), KH2PO4 (7.5 g), and N aCl (1.25 g), NH4Cl (2.5 g), 1M (mol / L) MgSO4 (2 50 μL), 2.5 mL of 20% (w / v) glucose, and 1 M CaCl2 Dissolve (25 μL) in 250 mL of ultrapure water, autoclave, and then let cool to room temperature. Then, 250 μL of 1% Thiamine-HCl was added to prepare each sample. Escherichia coli (JM109) containing the pUC4-KIXX plasmid was cultured in a culture medium. Add 25 μL of E. coli to M9 minimal medium and incubate for 2 days at 37°C and 200 rpm. The cells were cultured with shaking. Afterward, E. coli were collected and extracted using the Plasmid Mi plasmid extraction kit. Plasmids were purified using the di kit (Qiagen). Plasmids were then purified in sterile water. Dissolve in the fluorescein and use NanoDrop (Thermo Fisher Science). e) The DNA concentration was quantified and adjusted to 115 ng / μL. , 15 This plasmid contains N in its naturally occurring proportions. 14 N_pUC4-KIXX and, 15 N to 9 Plasmids containing 8% or more 15 N_pUC4-KIXX was generated.

[0079] (2) Proton irradiation of pUC4-KIXX plasmid The two plasmid samples were dropped 1 μL onto a Si wafer substrate and dried, then placed in a vacuum chamber. The device was set up, and the sample was irradiated with a proton beam. The proton beam irradiation was performed by the National University Corporation University of Tsukuba. The experiment was conducted using the 1MV tandem electrostatic accelerator at the Research Infrastructure Center. The case is the same as in (2) of Example 2. Figure 13 shows, 15 N_pUC4-KIXX plasmid Sample 15 N( 1 H,α1γ) 12 The resonance curve for the 1C resonance nuclear reaction is shown. For reference, 15 N-mark Deo xylibonucleotide 15 Resonance curves obtained for N-labeled dGTP are also shown. Proton beam irradiation It changes the proton energy from 890 keV to 906 keV in 4 keV increments to create three types Irradiation was performed at different doses. The three different doses are shown in Table 3. [Table 3]

[0080] (3) Transformation of proton-irradiated plasmids 1 x 10 10 50 μL of E. coli cells / mL was treated with a proton-irradiated plasmid. In addition, plasmids are extracted from E. coli by electroporation. It was introduced into the cells. During the electroporation process, a DC electric field was applied with a voltage of 1500 V and an electrical resistance of 200. A current was applied for 3.5–3.7 ms using a Ω resistor and a capacitance of 25 μF. 1 mL of LB medium was added. Then, infiltration culture was performed for one hour (37°C, 200 rpm). During this culture process, proton beam therapy was performed. Plasmids damaged by irradiation, resulting in a linearized ring structure, enter the cells of E. coli. Because they are not replicated, only the proton-irradiated plasmids that maintain their circular shape are replicated. It is amplified and transformed.

[0081] (4) Assessment of the extent of damage to drug resistance genes Three types of culture plates were prepared, each containing two different drugs. (a) Ampicillin 100 (b) kanamycin 50 μg / mL, (c) ampicillin 100 μg / Each culture plate containing mL + kanamycin 50 μg / mL was irradiated with proton beams. We seeded 100 μL each of E. coli transformed with Sumido and examined the number of colonies formed. As control samples, three types of culture plates were used, one unirradiated and the other containing the same drug. 100 μL was sown in each of the three cultures. Figures 14, 15, and 16 show three types of cultures with added chemicals. This shows the number of colonies expressed in the culture plate. The vertical axis represents the number of colonies expressed in the control sample. This represents a quantity normalized by (=1.0) and shows the average of three measurements. The error is the result of the three measurements. The upper and lower limits are shown. In all figures, the number of colonies expressed at 10 μC was 0, The detection limit values ​​are plotted in the figure. The vertical axis represents the two types of plasmids after proton beam irradiation. This shows the percentage of individuals whose drug resistance genes function normally without damage. Total irradiation dose: 2.5 In μC, 15 The N_pUC4-KIXX plasmid was more irradiated than the margin of error. This represents the following: 15 N( 1 H,α1γ) 12 C-resonance nuclear reactions cause proton beam ionization This indicates that exceeding this level causes significant damage to genes. Note that in Figure 14, the total irradiation dose is approximately When the temperature exceeds 5.0 μC, it is thought that the gene has suffered nonspecific damage.

[0082] The embodiments described above are provided to facilitate understanding of the present invention and do not limit the present invention. It is not intended to be interpreted as such. Each element of the embodiment, as well as its arrangement, materials, and conditions. The shape and size are not limited to those exemplified and can be changed as appropriate. Furthermore, the configurations shown in different embodiments can be partially substituted or combined. It is Noh.

Claims

1. To prepare a gene having a Cro protein binding site, A Cro protein capable of binding to the Cro protein binding site, 15 Prepare a Cro protein labeled with N, The Cro protein is bound to the Cro protein binding site, The gene to which the Cro protein is bound at the Cro protein binding site, 15 Irradiating N with a proton beam having the energy to cause a resonant nuclear reaction, Methods for inducing gene mutations (excluding methods for inducing gene mutations within the human body), The aforementioned gene is a regulatory gene that allows lambda (λ) phages to suppress their own proliferation. method.

2. In the aforementioned Cro protein, 15 The method according to claim 1, wherein the abundance of N exceeds the natural abundance of 0.364%.

3. The method according to claim 1, further comprising detecting the resonance nuclear reaction.

4. The method according to claim 3, wherein the detection involves measuring a gamma ray dose of 4.43 MeV.

5. To prepare a gene that has multiple Cro protein binding sites, A Cro protein capable of binding to each of the plurality of Cro protein binding sites, 15 Prepare a Cro protein labeled with N, To bind the Cro protein to at least one of the plurality of Cro protein binding sites, In the gene to which the Cro protein is bound at at least one of the plurality of Cro protein binding sites, 15 Irradiating N with a proton beam having the energy to cause a resonant nuclear reaction, A method for controlling gene activity (excluding methods for controlling gene activity within the human body), including, The aforementioned gene is a regulatory gene that allows lambda (λ) phages to suppress their own proliferation. method.

6. In the aforementioned Cro protein, 15 The method according to claim 5, wherein the abundance of N exceeds the natural abundance of 0.364%.

7. The method according to claim 5, further comprising detecting the resonance nuclear reaction.

8. The method according to claim 7, wherein the detection involves measuring a gamma ray dose of 4.43 MeV.

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