Artificial gene and gene mutation method

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 an artificial gene and a method for gene mutation. [Background technology]

[0002] The functions of the organisms involved in biodiversity, especially their natural products such as oils, polysaccharides, and pigments The metabolic function of high density algae production is essential for the production of energy, food and beverages, nutritional foods, cosmetics, and It is attracting attention in a wide range of fields, including pharmaceuticals. Genome editing is used to modify the desired gene (target gene). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-000129 Summary of the Invention [Problem to be solved by the invention]

[0004] In the process of using wild animals for various purposes, humans have developed a system that is more suited to those purposes. Select strains with the current type, crossbreed and breed them, and use organisms with advantageous characteristics to achieve the goal. In recent years, it has become relatively easy to analyze the genome DNA sequences of these organisms, and Understanding genetic information can help us understand why these organisms have beneficial phenotypes. Furthermore, advances in genome editing technology have made it possible to arbitrarily modify specific genes and DNA regions. However, it is possible to introduce or insert DNA or genome editing tools into the cell nucleus. There are many organisms for which the technology has not yet been established, and the techniques of gene recombination and genome editing have not yet been established for all organisms. Furthermore, the function of gene regions on genomic DNA has not been elucidated. Since the area is part of a whole, not every beneficial organism can be modified as desired. The genome editing technology developed by the company has been approved by the Cartagena Protocol (Protocol on the Regulation of Genetically Modified Organisms) Cartagena Protocol on Biosafety to the Convention on Biological Diversity, January 2000, The European Court of Justice has ruled that the same restrictions apply to the Convention on Biological Diversity (adopted at the resumed meeting of the Special Conference of the Parties). The decision was made by a judicial court, and discussions on how to handle it are ongoing in each country.

[0005] Therefore, even today, there are some methods that are not considered genetically modified, such as gamma ray or heavy ion beam irradiation and DNA modification. Mutation breeding using additives is essential for industrial applications (e.g. (See Patent Document 1.) However, conventional mutation breeding methods do not know to what extent DNA is damaged. Therefore, there is no quantitative method to directly evaluate the mortality rate or pigmentation. The irradiation dose and treatment amount are determined based on the phenotypic change, which is an indirect indicator of mutation efficiency. Furthermore, randomly induced mutations by irradiation allow for the creation of a large number of strains with the desired phenotype. It is necessary to spend time and effort to screen a large number of individuals from the treatment group. Until the screening results are obtained, it is unclear whether the target gene mutation has occurred. do.

[0006] Therefore, the present invention is a method for producing a gene that is not subject to genetic recombination regulations and that does not cause localized mutations in a target gene. Artificial genes and gene mutations that can be introduced and quantified upon mutation introduction A method is provided. [Means for solving the problem]

[0007] As will be explained in more detail below with reference to FIG. 15 N( 1 H,α1γ) 12 C resonance nuclear reaction 12.9686 MeV level 16 O * emits alpha particles 12 C * First excited level of 11. In the (p,α1γ) reaction channel, which de-excites to 6007 MeV, the α( 4 He nuclei) and 12 C and are emitted as secondary reaction particles. In addition, for each reaction 12 C * It de-excites from the first excited level to the ground state and emits γ After extensive research, the inventors have discovered that the radiation emitted from the reaction secondary particles has a high ionizing effect. teeth, 15 If N is located inside or near the DNA, 15 For biomolecules near N , which exerts a high local ionization effect and has a high probability of causing the desired genetic mutation in DNA. The inventors also found that the 4.43 MeV released per nuclear reaction The gamma rays of V can be easily counted, and the gamma rays are generated by proton irradiation of DNA. Since it reflects the amount of genetic mutation, it is possible to count 4.43 MeV gamma rays during beam irradiation. We found that this method makes it possible to quantify the amount of genetic mutations in DNA.

[0008] In addition, the present inventors 15 N-labeled DNA samples, or 15 Living cells containing N_DNA In proton beam irradiation of biological samples such as For example, the energy can be varied in fixed energy increments up to a high energy level exceeding the By determining the concentration of ions, the target sample is distributed from the surface to the interior. 15 For N, thoroughly 15 N( 1 H,α1γ) 12 It has been found that it may be possible to produce C resonant nuclear reactions.

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

[0010] In the above aspect, 15 N unlabeled primer sequence; 15 N-labeled deoxyribonucleoside It may also include a tide.

[0011] In the above embodiment, the artificial gene according to the above embodiment is formed by ligating another artificial gene. Other artificial genes may be vectors.

[0012] In the above-mentioned embodiment, the biomolecule-binding agent has a plurality of biomolecule-binding sites, and The gene sequence may be mutated at least in part. That's fine.

[0013] An artificial gene according to one aspect of the present invention comprises: 15 N Untagged artificial genes, which can be expressed in multiple organisms The biomolecular binding site is a molecule binding site, and at least a part of the binding site has a mutation in the gene sequence. It has multiple biomolecule binding sites. 15N-labeled biomolecules can bind do. 15 The N-labeled biomolecule may be a protein. Among these, the gene sequence is not mutated 15 N-labeled biomolecules may be capable of binding stomach.

[0014] The kit according to one aspect of the present invention comprises the above-mentioned 15 N-unlabeled artificial genes and multiple biomolecular bonds capable of binding to either of the binding sites 15 and an N-labeled biomolecule.

[0015] A method for mutating a gene according to one embodiment of the present invention comprises: 15 Labeling with N and 15 and irradiating the DNA with a proton beam having an energy such that N undergoes a resonant nuclear reaction.

[0016] In the above embodiment, DNA 15 Labeling with N is the process of replacing the N in DNA with 15 Substituting with N Alternatively, the DNA may be 15 Labeling with N allows the DNA to 15 N The DNA may be labeled. 15 Labeling with N gives DNA 15 Labeled with N The method may include binding the biomolecule to the target protein. 15 N-labeled biomolecules It may also be protein.

[0017] In the above embodiment, DNA in living cells is 15 It may be labeled with N.

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

[0019] In the above-mentioned embodiment, DNA is extracted from living cells. 15 Labeling with N 15 N-labeled deoxy Even though it involves administering ribonucleotides and inhibitors of glutamine synthetase to living cells, good.

[0020] In the above-mentioned embodiment, DNA is extracted from living cells. 15 Labeling with N 15 N-labeled deoxy This involves administering ribonucleotides and inhibitors of ribonucleotide reductase to living cells. It's fine.

[0021] In the above-mentioned embodiment, the DNA is 15 Labeling with N 15 N Unlabeled group The method may further comprise providing glutamin to living cells.

[0022] The above-described embodiment may further include detecting a resonant nuclear reaction.

[0023] In the above embodiment, the detection may measure the amount of 4.43 MeV gamma rays. stomach.

[0024] In the above-mentioned embodiment, the number of mutations that have occurred in DNA is calculated based on the measured amount of gamma rays. It may further include.

[0025] In the above aspect, in the calculation, 15 Resonance occurring in a standard sample with a known number of N atoms It may also refer to the amount of gamma rays produced by nuclear reactions.

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

[0027] According to the present invention, a method for producing a gene that is not subject to genetic recombination regulations and has a localized mutation in a target gene is provided. and a method for quantification of mutations during mutation introduction. It can provide a law. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 shows a DNA molecule in which N present in each base pair has been substituted with 15N in a gene mutation method according to an embodiment of the present invention. [Figure 2] FIG. 1 illustrates how resonant nuclear reactions occur in DNA molecules. [Figure 3] FIG. 1 is a diagram showing a case where a gene mutation method using heavy ion beam irradiation, which is a conventional technique, is used. [Figure 4] FIG. 1 shows an artificial DNA standard sample consisting of 84 base pairs. [Figure 5] FIG. 1 shows the 15N(1H,α1γ)12C resonance nuclear reaction. [Figure 6] FIG. 1 is a schematic diagram of a method for determining the presence or absence of DNA breaks. [Figure 7] FIG. 1 is a diagram showing an overview of a method for identifying how much cleavage has occurred in one of the DNA strands. [Figure 8] FIG. 1 shows the synthesis of nitrogen-containing organic compounds in cells. [Figure 9] FIG. 1 is a diagram illustrating a new method for 15N-labeling only nucleic acids in cells when MSX and 15N-unlabeled glutamine are administered. [Figure 10] FIG. 1 is a diagram illustrating a method for labeling only DNA with 15N in a nucleic acid synthesis pathway. [Figure 11] This figure explains a new method for labeling only the target gene or its vicinity with 15N. [Figure 12]FIG. 1 shows the genetic structure of pUC4-KIXX circular plasmid. [Figure 13] 1 is a graph showing the resonance curve of the 15N(p,α1γ)12C resonance nuclear reaction of the 15N_pUC4-KIXX plasmid sample. [Figure 14] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the damage to the gene. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium containing ampicillin. [Figure 15] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the damage to the gene. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium containing kanamycin. [Figure 16] This is a graph showing the relationship between the amount of proton irradiation to a plasmid and the genetic damage. The proton-irradiated plasmid was introduced into E. coli, and the E. coli was cultured in a medium supplemented with ampicillin and kanamycin. DETAILED DESCRIPTION OF THE INVENTION

[0029] An embodiment of the present invention will be described with reference to the accompanying drawings. Those marked with the same or similar symbols have the same or similar configurations.

[0030] Nitrogen (N) in living organisms is scattered throughout cellular components, including proteins such as enzymes and DNA. In the gene mutation method according to this embodiment, the natural abundance ratio of the gene is 0.364%. exists 15 Create a situation where N isotopes are concentrated and distributed unevenly in the target gene, 15 N and 1 H 15 N( 1 H ,α1γ) 12 By irradiating the target gene with proton beams with the energy to cause a C resonance nuclear reaction, It causes large mutations in the target gene.

[0031] In the double-stranded DNA that makes up genes, adenine (A) has five nitrogen atoms that are purine. thionine (T) has two nitrogen atoms attached to the pyrimidine ring. Therefore, there are seven nitrogen atoms in the adenine-thymine pair (AT pair) of the double-stranded DNA. Five nitrogen atoms are attached to guanine (G) and three 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 represents a DNA molecule substituted with N, naturally 15 The isotope ratio of N is enriched by 275.5 times. 15 The state indicated by N is shown. Oh, in nature 15 The abundance ratio of N is 0.364%.

[0033] Figure 2 shows how resonant nuclear reactions occur in a DNA molecule. 15 1 in N-labeled DNA of 15 N nucleus and proton 15 N( 1 H,α1γ) 12 When a C resonance nuclear reaction occurs, 15 N has disappeared, Before reaction 15 Centered on the bonding position of the N atom 4 He and 12 C nuclei are nearly isotropic in opposite directions The electrons are released into the atmosphere, and they immediately capture electrons and pass through the material as ions. Nearby atoms undergo high density electronic excitations.

[0034] The probability of electron excitation decreases inversely with the square of the distance from the reaction center. The farther away an atom is from the center, the smaller it is. 15 When N undergoes a resonant nuclear reaction, 15 N atom bond The atoms near the junction will be locally excited with a high probability, 15 Bonding position of N atom This makes it easier for local mutations to occur in the DNA near the site.

[0035] FIG. 3 shows the case where gene mutation using heavy ion beam irradiation, which is a conventional technique, is used. In the conventional gene mutation method using heavy ion beam irradiation, heavy ions enter the DNA from the outside. Since the heavy ions are incident on the entire DNA at a constant density, the atoms near the DNA along the heavy ion track The probability of undergoing electronic excitation and resulting in a mutation is roughly the same across the entire DNA region.

[0036] In contrast, in the method of this embodiment, 15 N same Increase the position ratio, with high probability, 15 It causes local mutations in the DNA near the N atom binding site. It can be done.

[0037] In this embodiment, the target gene or its vicinity 15 Techniques for increasing and enriching N isotope ratios exists in nature at 0.364% and is also present in living cells at the same ratio. 15 N is the It is a technology that utilizes the DNA as it is. 15 Generated by irradiating N with protons 15 N( 1 H,α1γ) 12 C-resonance nuclear reactions are the most abundant hadronic component of cosmic rays near the Earth's surface. The proton beams randomly fall on surface organisms, and the DNA 15 Collision with N nucleus This is the same as the nuclear reaction that occurs when a substance is released from a substance, and has similar effects on living cells.

[0038] That is, at or near the target gene 15Techniques for increasing and enriching N isotope ratios, and the techniques By 15 N-labeled living cells were irradiated with protons. 15 N( 1 H,α1γ) 12 C resonance nuclear reaction Since the birth of life on Earth, the creation of new genes has led to the creation of new genes that increase the probability of mutation. It is merely a method to accelerate the process of genetic mutations that have occurred in the past. A method for producing mutant strains by irradiation (UV, X-rays, gamma rays, heavy ions) and a basic The principle of the mechanism remains unchanged. The law is based on a fundamentally different principle from genetic engineering technology, and It has the excellent feature of enabling large mutations to be introduced into target genes without being subject to regulations. This is a useful technique.

[0039] In or near a target gene in a living cell 15 DNA test with increased N isotope ratio To quantify DNA mutations caused by proton irradiation of materials, 15 The number of N atoms is clearly prescribed 15 It is necessary to construct N-labeled DNA standards.

[0040] 15 By constructing N-labeled DNA standard samples, 15 N( 1 H,α1γ) 12 Emitted by C resonance nuclear reaction The 4.43 MeV gamma ray dose and the 15 Obtaining calibration data with N concentration By obtaining the dose calibration value of 4.43 MeV gamma rays, 15 N-labeled raw The dose of 4.43 MeV gamma rays emitted by a resonant nuclear reaction simultaneously with proton irradiation of somatic cells is By measuring and comparing with a calibration value, the number of mutations that have occurred in the DNA within living cells can be quantified.

[0041] In the gene mutation method according to this embodiment, 15 DNA consisting of N-labeled base pairs is By polymerase chain reaction The artificial DNA standard sample was constructed using the DNA of a molecular biologist. It is desirable that the sequences be composed of biologically, biochemically, and biologically important base sequences.

[0042] Figure 4 shows an artificial DNA standard sample consisting of 84 base pairs. The standard sample is prepared, for example, by the following method. 15 N unlabeled, i.e. 14 N Primer OR_ containing the TACGTTAAATC sequence structure with a natural abundance of 99.636% primer_F and 15 N-label, i.e. 15 Deoxyribonucleic acid containing N at an abundance of 98% or more leucoside triphosphate 15 N-labeled dNTPs (deoxyribonucle side 5'-triphosphates) and 73 other 5'-triphosphates, polymerase chain reaction The oligonucleotide OR_WT_F is synthesized by the method described above. 15 N non-standard Primer OR_primer_R has the known TGCAACCATT sequence structure, 15 N mark Deoxynucleoside triphosphates 15 Polynucleotides were synthesized from 74 N-labeled dNTPs. The oligonucleotide OR_WT_R is synthesized by the polymerase chain reaction. OR_WT_F and OR_WT_R form base pairs with each other through hydrogen bonds, 15 N mark Identified oligonucleotide double-stranded DNA 15 N-labeled OR_DNA is formed. This is used as the standard sample.

[0043] 15 N-labeled OR_DNA 15 N unlabeled primer OR_primer_F / _R, 15 N-labeled deoxyribonucleotides 15 N-labeled dNMPs (Deoxyri 84 total The DNA sequence consists of 84 base pairs (84 bps). Excluding OR_WT_F, at 73 bps, 15 N-labeled dAMP: 19 units , 15 N-labeled dTMP: 22 units, 15 N-labeled dGMP: 17 units , 15 N-labeled dCMP: 15 units. OR_WT excluding primers At 74 bps of _R, 15 N-labeled dAMP: 23 units, 15 N-lab eled dTMP: 20 pieces, 15 N-labeled dGMP: 16 units, 15 N-lab eled dCMP: 15. These are summarized in Table 1 below.

[0044] [Table 1]

[0045] 15 N-labeled OR_N contained in the whole DNA ( 14 N+ 15 The total number of N) is 623. 15 N The standard purity of the label is 98% or more, and the label is 14 N and 15 N isotope abundance ratio The rate changes. 15 When the N-labeling purity is 98%, the total OR_DNA 15 N is 538.29 Ideally, 15 At 100% N-labeling purity, the total OR_DNA 15 N is the maximum number of 54 The number of nitrogen atoms is 9.27, which is 88.17% of the total number of nitrogen atoms (see Table 1). 15 N labeled OR The molecular weight of DNA is also 15 Depending on the N-labeling purity, it can reach 52,467 g / mol or more. The maximum is 52,477.95 g / mol.

[0046] Figure 5 shows 15 N( 1 H,α1γ) 12 C resonance nuclear reaction. 15 For N-labeled biological samples When irradiating with proton beams, 15 Binding energy of N nuclei and protons in the center-of-mass system -12.1277 MeV, 16 O * Second excited level of compound nuclei 12.9686 MeV The energy difference between the two is 0.8409 MeV (0.0 ... When the two nuclei collide at an energy of 0.987 MeV, they resonantly bond together 16 O * composite source A child nucleus is formed. 16 O * immediately emits an alpha particle 12 C * The first reaction channel de-excites the α particle to the first excited level of 11.6007 MeV. Release the 12The second reaction channel de-excites the C ground state to 7.1616 MeV, and the γ Emitting only lines 16 The third reaction channel de-excites the O ground state to 0 MeV, and the three species The reaction channels are (p,α1γ), (p,α0), and (p,γ0), respectively. It is written in the form of a formula.

[0047] The (p,α1γ) reaction channel used in this embodiment is characterized by a higher ionization rate than that of proton beams. α( 4 He nuclei) and 12 C and are emitted as reaction secondary particles; For each reaction 12 C * It de-excites from the first excited level to the ground state and emits a 4.43 MeV gamma ray. In the (p,α0) reaction channel, no gamma rays are emitted, and (p In the γ0 reaction channel, there is no emission of highly ionizing secondary particles. The release of reactive secondary particles with 15 If N is located inside or near the DNA, 15 It exerts a high local ionization effect on biomolecules in the vicinity of N, and induces the desired genetic This has the effect of increasing the probability of generating mutations. The 4.43 MeV gamma rays can be easily counted, and the gamma rays are generated by proton irradiation. This reflects the amount of DNA mutations that occur during beam irradiation. The effect of being able to quantify the amount of genetic mutations in DNA by counting MeV gamma rays It plays a key role.

[0048] 16 O * The resonance energy with the second excited level is the Coulomb barrier potential in the collision of the two nuclei. Therefore, the collision energy between the two nuclei is 16 O * composite atom The resonance energy width, which is defined as the width of the second excited level of the nucleus, exceeds 300 eV, and the resonance energy When the reaction deviates from the energy, the cross section of the reaction decreases rapidly. 15 For N-labeled OR_DNA The proton beam incident at the resonance energy has fluctuations in the incident energy that are proportional to the resonance energy width. Only as long as it fits 15 N( 1 H,α1γ) 12 C resonance nuclear reaction occurs.

[0049] In actual proton irradiation, the resonance energy width and the energy spread of the proton beam, typically The resonance energy width is determined by the convolution integral calculation with about 1 keV, and the proton The proton beam irradiation energy is calculated by adding the energy loss during passage through the medium to the resonance energy. Determine the 15 N-labeled DNA samples, or 15 Living cells containing N_DNA In proton beam irradiation of any biological sample, the energy of the proton beam is converted from the resonance energy to the resonance energy. For example, the energy can be changed in fixed energy increments up to a high energy level exceeding 1000kJ / s. By this, the target sample is distributed from the surface to the interior. 15 For N, thoroughly 15 N( 1 H,α1γ) 12 It may be possible to produce a C resonant nuclear reaction. The width and step size can be selected depending on the target sample. 15 N( 1 H,α1γ) 12 C resonance nucleus This may be determined by obtaining a resonance curve for the reaction, see, for example, FIG.

[0050] 15 N( 1 H,α1γ) 12 Produced in a C resonant nuclear reaction 12 C * and α are emitted in opposite directions. When the proton beam is emitted in the same direction as the incident direction, the kinetic energy reaches its maximum value E(max) And 12 C * and α respectively. C (max)=0.6252 MeV, E He (max)= It becomes 1.2806 MeV. Also, 12 C * and α are emitted in the opposite direction to the incident direction of the proton beam. When the force is applied, the kinetic energy reaches a minimum value E(min), and E C (min)=0.1437 MeV, E He (min)=0.7991 MeV. 12 C * and α is the electron immediately after emission Capture 12 C. 4 It becomes a He ion and travels through the material.

[0051] 12 C and 4 He ions always have values ​​in these energy ranges, 15 N-labeled OR_D NA passes through the sample. Nearby atoms along the path, especially those with a radius of 5 nm to 10 nm The following nearby atoms are excited electronically: 15 N-labeled OR_DNA samples, but not eukaryotic Biological samples that have cell walls and intracellular organelles, such as unicellular algae, are used to detect positively the cell walls and intracellular organelles. Considering the energy loss when electrons pass through, the electrons are generated at energies higher than the resonance energy. Even when using proton beam irradiation, 15 N-labeled DNA or DNA nearby 15 N-labeled test with fee 15 N( 1 H,α1γ) 12 In C resonance nuclear reactions, regardless of the proton beam irradiation energy, 12 C and 4 He reaction product ions are emitted with a certain energy and align nearby atoms along their trajectories. A feature of this method is that excitation is performed 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)), 12 C and 4 The energy range of the He reaction product ions emitted varies, and the constituent elements of the target sample The LET to carbon C, which is one of the 12 C reaction product ions: 2.51 × 10 3 MeVcm 2 / g or more, up to 4.70 × 10 3 MeVcm 2 / g, 4 He reaction product ions , 1.73×10 3 MeVcm 2 / g or more, up to 1.99 × 10 3 MeVcm 2 / g This value is the resonance energy of the proton. 15 LE when passing through N-labeled OR_DNA sample It is 17.2 times higher and up to 27.1 times higher than T. 15 N mark For biological samples, including DNA samples, 15 N( 1 H,α1γ) 12 C-resonance nuclear reaction production The electron excitation caused by ions is overwhelmingly higher than that caused by proton 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 the same as that of typical heavy ion irradiation (e.g., 320 The LET of C in MeV C ion irradiation is 0.58×10 3 MeVcm 2 / g) It is 4.3 times higher and up to 8.1 times higher than the original. and others 12 C and 4 He reaction product ions are released, 15 N-labeled OR_DNA samples and D Near NA or DNA 15 In N-labeled living cells, 15 N( 1 H,α1γ) 12 C by resonant nuclear reaction 15 There is a high probability that mutations will occur in genes near N. Therefore, this method Reaction product nuclei 12 By measuring the 4.43 MeV gamma ray emitted from the first excited level of C, the law of nature, 15 It is possible to directly quantify the number of gene mutations occurring near N.

[0054] By proton irradiation 15 The mutations that occurred in the N-labeled OR_DNA samples were detected by DNA repair. Unlike biological samples, DNA repair does not occur. 15 N indicator OR_DN By examining the mutations in sample A, the purely physical mutation status caused by proton irradiation will become clear. . 15 One method for examining mutations that have occurred in N-labeled OR_DNA samples is to use the DNA with a circular shape and circular plasmid vectors are used to detect DNA cleavage. This is a method for determining

[0055] Figure 6 shows the artificial gene in which the artificial gene shown in Figure 4 is ligated to another artificial gene, and the DNA fragment. The following shows an outline of the method for determining the presence or absence of β-lactam antibiotics, which are used to treat infectious diseases. It is a drug that has been developed by incorporating an E. coli gene that confers resistance to ampicillin, a drug used in humans. An engineered linearized plasmid (T-vector pMD19, 2,692 base pairs) to 15 Plasmid vector made circular by ligating N-labeled OR_DNA is introduced into E. coli culture medium. The circularized plasmid vector is taken up by E. coli and becomes ampicillin-resistant. E. coli grows. 15 N-labeled OR_DNA previously cleaved by proton irradiation However, the plasmid vector does not become circular and is degraded in E. coli cells to become a stable gene. Therefore, E. coli does not tolerate ampicillin and dies. 15 Ligation of N-labeled OR_DNA In the circular plasmid vector, 15 Contains a large amount of N-labeled OR DNA The longer the incubation period, the slower the growth of E. coli. 15 N-labeled OR_DNA sample, 15 N-unlabeled OR_DNA assay Fee, 15 Each N-labeled OR_DNA sample that was not irradiated with protons was cloned into T-vector pMD19 Each was placed on an ampicillin-containing medium and the number of E. coli colonies was compared. 15 N-labeled OR_DNA samples were 15 N( 1 H,α1γ) 12 C resonance nuclear reaction and DN by proton beam A mutation occurs. 15 The N-unlabeled OR_DNA sample shows that DNA mutations are induced by the proton beam alone. In samples that have not been irradiated with protons, no DNA mutations occur. 15 N labeled OR The relative frequency of DNA mutations introduced by proton irradiation of DNA will be revealed.

[0056] 15 A more direct method for examining the state of N-labeled OR_DNA cleaved by proton irradiation As, 32 Add a phosphate group containing a P radioisotope and separate one base by electrophoresis. There is a technique for separating the fragments using the enzyme. This technique allows us to determine how much of one of the DNA strands is cut. It becomes possible to identify whether a disconnection has occurred.

[0057] Figure 7 outlines how we can identify how much breakage has occurred in one of the DNA strands. First, 15 Before PCR sample synthesis of N-labeled OR_DNA, OR_primer_F or Either one of the primers OR_primer_R was phosphorylated with ATP, and PC R During sample synthesis, a DNA fragment with a phosphate group attached only to the 5' end of one DNA strand is synthesized. After proton beam irradiation, [γ? 32 P]-ATP is used to bind the DNA without phosphate groups. Chain only 32 It is labeled with P. It is electrophoresed at high voltage in a thin gel with low salt concentration and containing urea. By applying heat and separating the DNA in a single-stranded state (denatured state), the difference between the two is one base. It is possible to distinguish between DNA fragments. A single base difference can be distinguished by gel electrophoresis. It is possible to distinguish whether cleavage is possible or whether only the vicinity of a particular base is susceptible to cleavage.

[0058] DNA in living cells 15 When labeling with N, nitrogen atoms other than DNA that make up living cells Intracellular molecules containing ATP, such as proteins and enzymes that control cell formation and metabolic functions Nitrogen atoms present, or ribosomal RNA that forms ribosomes, amino transfer RNA, which transports the acid, and m, which specifies the amino acid sequence on the ribosome The nitrogen element is present in RNAs that have various functions within cells, such as messenger RNA. Distinguishing between the nitrogen element that makes up DNA and DNA alone 15 Label with N.

[0059] Within cells, nitrogen is used to form amino acids, nucleotides, proteins, nucleic acids (DNA, RNA), and These nitrogen-containing organic compounds are used in a variety of applications, including phospholipids. Both are inorganic nitrogen ammonium ions NH4 + glutamine is synthesized by glutamine synthetase By reacting with acid and fixing it to an organic compound as glutamine, it becomes the first organic nitrogen-containing compound. (ammonia assimilation) 2-oxo-glutamine is produced by glutamate synthase. An amino group is transferred to glutaric acid, producing two molecules of glutamic acid. The glutamine and glutamic acid produced are transformed as they are, or by the amino group A number of nitrogen-containing organic compounds are synthesized by rearrangement reactions.

[0060] Figure 8 shows the synthesis pathway of nitrogen-containing organic compounds in cells. Amino acids are used in a variety of applications, including as structural proteins. It is used in the synthesis of proteins, enzymes, nucleic acids, and lipids. DNA synthesis proceeds through the hereditary transmission of genetic information from nucleotides. 15When N-labeled deoxyribonucleotides are administered to the culture medium, they are taken up into the cells and The DNA is then decomposed to form ammonium ions, and protein synthesis begins. 15 To label N, you need to inhibit that process. 15 N-labeled proteins are not synthesized In addition, MSX (Methionine sulfoxidase), an inhibitor of glutamine synthetase, Administer HCl (HCl).

[0061] Figure 9 shows the MSX and 15 When N-unlabeled glutamine was administered, the nucleic acid Mio 15 We present a new method for N-labeling of glutamine synthetase. Ammonia as an amino group binds to synthesize glutamine, and glutamine is released from glutamine synthetase. However, the structural formula of MSX contains the HN=S=O structure, which is The structural formula of MSX is similar to glutamin's H2N-CH=O. MSX also has a strong active site. When glutamic acid and ammonia bind, they cannot separate from MSX, and glutamine synthesis stops. In other words, glutamic acid is synthesized from glutamine by glutamate synthase. The circuit is broken, 15 N-labeled proteins are no longer synthesized. However, they are needed for various purposes. To prevent the synthesis of proteins that are 15 Administer N-unlabeled glutamine to the culture medium. As a result 14 Structural proteins, enzymes, and some DNA R with an N abundance of 99.637% NA is synthesized and taken up into the cell 15 Among N-labeled deoxyribonucleotides, The remaining parts that are not included in the Monia synthesis 15 DNA is synthesized using N-labeled deoxyribonucleotides Therefore, within the cell 15 N-labeling is limited to DNA only.

[0062] Furthermore, in the intracellular nucleic acid synthesis pathway, only DNA is synthesized. 15 The process of labeling with N is shown in Figure 10. Here is a more detailed explanation: 15 To synthesize N-labeled DNA, (1) the four bases are Each 15 N-labeled deoxyribonucleotides are added to the medium and the cells are cultured. In the cell, deoxyribonucleotides that synthesize DNA are converted by ribonucleotide reductase ( (2) RNR inhibitors (i -RNR) was administered, 15 Deoxyribonucleotides that are not N-labeled are synthesized in cells. In addition, (3) deoxyribonucleic acid is produced in the cell. The bases are transferred between deoxyribonucleic acid and ribonucleotides, and the extracellular input deoxyribonucleic acid is converted to ribonucleotides. Leotide 15 The N-labeled base is transferred to the base of ribonucleotide in the cell and then 15 N_ RNA can be synthesized, and conversely, ribonucleotides 15 The bases not labeled with N are denoted However, (4) intracellular 14 N_Ami It is metabolized from fatty acids 14 The amount of N-ribonucleotides is such that RNR in (2) is inhibited. and so on, 15 N_The amount of base transition from deoxyribonucleotide to ribonucleotide It can be suppressed. 15 Excessive amounts exceed those required for DNA synthesis 15 N_Deo When xylisothiazolinone is added to the medium, in (3) 15 N_ base is deoxyribonucleic acid There is a possibility that a nucleotide may be translocated to a ribonucleotide. 15 N_D High NA synthesis efficiency, 15 To suppress the synthesis of N_RNA, 15 N_Deoki The amount of ribonucleotides added can be adjusted as appropriate.

[0063] Figure 11 shows the results of analyzing only the target gene or its vicinity. 15 A new method for N labeling is shown in Figure 4. 15 The base sequence of N-labeled OR_DNA is a nucleotide sequence of the virus lambda (λ) that parasitizes E. coli. ) is part of the phage's genes, and allows λ phage to parasitize the host E. coli without killing it. It is a regulatory gene that inhibits the proliferation of the cell itself. 15 N-labeled OR_DNA was It has three Cro protein binding sites, and the Cro protein binds to one of these sites. When the gene sequence is mutated, the regulatory gene is activated. In Figure 4, the polymerase chain reaction is shown. Chain reaction method 15 A method for preparing N-labeled OR_DNA was described. OR_DNA 15 After synthesizing N-unlabeled proteins, they are added to the culture medium and are then taken up by the control genes. The E. coli bacteria that were introduced 15 When N-labeled Cro protein is administered, 15 N-labeled Cro protein The protein binds to the regulatory gene of E. coli. When exposed to proton beams in this state, 15 N labeled C near specific genes to which ro protein binds 15 N( 1H,α1γ) 12 C resonance nuclear reaction occurs This method makes it possible to generate mutations in specific genes with a high probability.

[0064] Example 1 15 Proton irradiation results for N-labeled OR_DNA samples] (1) 15 Preparation of N-labeled OR_DNA samples [OR_primer_R phosphorylation] The phosphorylation process of 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 0 μL was reacted at 37°C for 35 minutes, then the reaction was stopped by heating to 72°C. Extraction (Phenol Chloroform Isoamylalcohol (25: 24:1) extraction) to remove proteins and lipids, and then ethanol The solution was fixed and extracted.

[0065] [By PCR method 15 Synthesis of N-labeled OR DNA] In addition, two types of primers were synthesized, one with and one without phosphorylated primers. 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, template: 1 μL, sterile water: 27.5 μL, each for 35 PCR cycles The electrophoresis was completed by heating at 98°C for 10 minutes, 31°C for 20 minutes, and 72°C for 30 minutes. The synthesis of 84 base pair DNA was confirmed by the ELISA method.

[0066] (2) Proton beam irradiation 15 N-labeled OR_DNA sample, 15 The N-unlabeled OR_DNA sample was dropped onto the Au substrate and irradiated. The concentration of the OR_DNA solution was 31.2 ng / μL, the amount of dripping was 13 μL, and the amount of dripping was 13 μL. Area 0.23 cm 2 The sample dropped contains 4.7 × 10 12 Contains DNA, each D NA has more than 538 15 N atoms are bonded. Converted to per sample area, it is 1.1 x 10 16 cm -2 The proton beam irradiation was carried out by the National Institute of Advanced Industrial Science and Technology. 4MV Pelletron electrostatic accelerator at the Center and the Research Infrastructure Center at the University of Tsukuba The beam current was 0.1 nA to 5 nA. A, beam irradiation area is 0.071 cm 2 It was. 15 N( 1 H,α1γ) 12 C resonance nuclear reaction The 4.43 MeV gamma ray emitted by the atom is a high-energy gamma ray containing Bi, which has atomic number 83. The most efficient detection is BGO (Bi4Ge3O 12 Crystalline scintillator, specific gravity 7.3 gcm -3 The detector (76.2 mm diameter x 79.2 mm length) was placed 24 mm away from the sample. The detector was installed outside the vacuum and the detection was carried out. The detectable solid angle of the detector was 10% of the total azimuth angle. If the maximum detection sensitivity for 4.43 MeV gamma rays is 0.1, then The detection efficiency for gamma rays is up to about 1%. 15 N-labeled OR DNA sample When proton irradiation was performed at a constant energy, the count of 4.43 MeV gamma rays was 4,000 cou The total count was 25,000 counts, with 8 points irradiated at 8 keV. Considering the detection efficiency, it was at least 2.5 × 10 6 of 15 N indicator OR_DN A 15 This means that a mutation occurred due to an N resonance nuclear reaction.

[0067] (3) 15 Ligation of N-labeled OR_DNA into a linearized plasmid vector: [ 15 Creation of A-overhangs in N-labeled OR DNA Proton beam irradiation 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, 10x dilution buffer Ex taq 1 μL, Ex taq 0.1 μL, dATP (10 mM ) 2 μL, Negative insert DNA sample: 6.9 μL, 10x 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, 10x dilution Dilution buffer solution (Ex_taq) 1 μL, Ex_taq 0.1 μL, dATP (1 Four types of mixed solutions, each containing 2 μL of 0 mM HCl, were prepared and reacted at 72° C. for 1 hour.

[0068] [Ligation with linear plasmid vector] A linearized plasmid vector (T-Vector pMD 19, 2,692 base pairs) The above four mixed solutions were mixed on ice and left for 30 minutes, then subjected to thermal shock at 42°C for 30 seconds. The transformation efficiency was increased by adding

[0069] [Ampicillin-resistant E. coli culture] E. coli (JM109) cultured in M9 medium at 37°C with shaking at 180 rpm was The cells were inoculated with one of the circular plasmid vectors and inoculated onto ampicillin-containing medium, and the resulting cells were then placed in a medium containing JM109. The colony formation was compared between the unirradiated and non-irradiated samples and the ligated circular plasmid vector. In the medium treated with beam irradiation, the number of colonies was 46 / 11 ng DNA. 15 In the medium administered with the circular plasmid vector ligated with N-labeled OR_DNA, colonies The number of cells decreased to less than one-third, at 13 cells / 11 ng DNA.

[0070] [Example 2: Results of proton irradiation on Escherichia coli biological samples] Since proton beam irradiation is basically performed in a vacuum, the E. coli (JM109) is first frozen. The resistance of the JM109 E. coli solution was then examined by drying it. The samples were placed in a container and divided into two groups: one that was frozen at liquid nitrogen temperature and one that was not frozen in liquid nitrogen. Each was placed in a vacuum dryer at -20°C and left for 4 hours, then taken out and stored at 4°C. The JM109 colon was then left for another 24 hours to prepare a freeze-dried sample. The survival rate of bacteria was 26.8% for samples frozen with liquid nitrogen and 10.4% for samples not frozen with liquid nitrogen. The rate was 39.1%.

[0071] JM109 E. coli has 514 million base pairs of DNA, which can be separated in two ways. in 15 N labeling was performed. 15 In a medium containing N-labeled ammonium ions, In JM109 E. coli cultured without administration of MSX, an inhibitor of ATP synthase, All nitrogen in the cells, including proteins and nucleic acids, 15 N-labeled (sample F). 15 N In a medium containing labeled deoxynucleotides, MSX and 15 N-unlabeled glutamine was added. In JM109 E. coli cells cultured with the 15 N-labeled biological samples (Sample D).

[0072] Two types 15 N-labeled JM109 E. coli and 15 N unlabeled JM109 E. coli (sample E) Three types of solutions were prepared to create proton beam irradiated samples. The E. coli concentration in each irradiated sample was , solution dripping amount, dripping area, number of dripped cells, area density of cell number, 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 cell number Degrees: 2.52 x 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 cell number Degrees: 2.64 x 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 cell number Degrees: 1.11 x 10 6 cells / cm 2 , 15 N area density: 4.18×10 13 cm -2 .

[0076] The 4.43 MeV gamma ray dose measured simultaneously with proton irradiation revealed that the DNA and RNA of sample D Only A 15 N-labeled 15 The amount of N was calculated by dividing the total amount of nitrogen in the F sample by the total amount of nitrogen in the F sample. 15 When labeled with N It was revealed that the probability was 1 in 44.6.

[0077] Example 3 15 Proton irradiation results for N-labeled drug resistance gene plasmid samples] (1) pUC4-KIXX Plasmid and Its Construction Method Figure 12 shows the genetic structure of pUC4-KIXX plasmid (plasmid DNA). The plasmid contains the ampicillin resistance gene (Ampicillin-R) and Ori The gene pUC4 (2605 bps (base pairs)) carrying the kanamycin resistance gene (Ka namycin-R, 1248 bp) inserted into a circular plasmid (3853 bp The gene size occupied by the promoter and transcription region of the two drug resistance genes is , ampicillin resistance gene 934 bps, kanamycin resistance gene 953 bps The region Ori required for plasmid replication is 621 bps. The types of base pairs in each region and 15 Table 2 shows the N number (100% substitution rate). [Table 2]

[0078] By the following method, 15 a plasmid containing N at natural abundance; 15 Contains 98% or more N A plasmid carrying the gene was generated. 15 NHC containing N at its natural abundance (0.364%) 250 mL of M9 minimal medium containing 1 sucrose as a nitrogen source was prepared. 15 Contains more than 98% N have 15 250 mL of M9 minimal medium containing NH4Cl as a nitrogen source was prepared. Each M9 minimal medium contained NaHPO (15 g), KHPO (7.5 g), 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 1M CaCl2 (25 μL) was dissolved in 250 mL of ultrapure water, autoclaved, and then cooled to room temperature. Then, 250 μL of 1% Thiamine-HCl was added. E. coli (JM109) carrying the pUC4-KIXX plasmid was cultivated in the medium. For the incubation, 25 μL of E. coli was added to M9 minimal medium and incubated at 37°C and 200 rpm for 2 days. The E. coli was then harvested and extracted using the Plasmid Mi kit. The plasmid was purified using a DI kit (Qiagen). The solution was dissolved in 100 ml of fluorimeter NanoDrop (Thermo Fisher Scientific) and analyzed by fluorimeter. The DNA concentration was quantified in e) and adjusted to 115 ng / μL. , 15 It is a plasmid containing N at natural abundance. 14 N_pUC4-KIXX and 15 N to 9 It is a plasmid containing 8% or more 15 N_pUC4-KIXX was generated.

[0079] (2) Proton irradiation of pUC4-KIXX plasmid Two types of plasmid samples were dropped onto a Si wafer substrate in an amount of 1 μL, dried, and then placed in a vacuum chamber. The proton beam was irradiated onto the sample. The proton beam irradiation was carried out using a 1MV tandem electrostatic accelerator at the Research Infrastructure Center. The conditions are the same as those in (2) of Example 2. 15 N_pUC4-KIXX plasmid of the sample 15 N( 1 H,α1γ) 12 The resonance curve of the C resonance nuclear reaction is shown below. 15 N-labeled Deo Xyribonucleotides 15 The resonance curve obtained for N-labeled dGTP is also shown. The proton energy was changed from 890 keV to 906 keV in 4 keV increments, and three types of The irradiation was carried out at three different doses. The three doses are shown in Table 3. [Table 3]

[0080] (3) Transformation of proton-irradiated plasmids 1×10 10 Add 50 μL of E. coli to 8.6 cells / mL of the proton-irradiated plasmid. The plasmid was then transferred to E. coli by electroporation. During the electroporation process, a DC electric field was applied at a voltage of 1500 V and an electrical resistance of 200 The current was applied for 3.5 to 3.7 ms at a load of 25 Ω and a capacitance of 25 μF. 1 mL of LB medium was added. After that, the cells were cultured for one hour at 37°C and 200 rpm. The plasmid is damaged by irradiation, the circular form is broken, and the linearized plasmid is then stored in the E. coli cells. Therefore, among the proton-irradiated plasmids, only those that maintained their circularity were replicated. Amplified and transformed.

[0081] (4) Evaluation of the extent of damage to drug resistance genes Three types of culture plates containing two drugs were prepared: (a) Ampicillin 100 μg / mL, (b) kanamycin 50 μg / mL, (c) ampicillin 100 μg / Each culture plate was cultured with 50 μg / mL of kanamycin and 50 μg / mL of proton-irradiated plates. 100 μL of E. coli transformed with the smid was plated on each plate, and the number of colonies formed was counted. As a control sample, the unirradiated plasmid was also cultured on three different culture plates containing the drug. Figures 14, 15, and 16 show the results of the three types of culture medium containing the drug. The vertical axis shows the number of colonies that appeared on the culture plate. The values ​​are normalized by the standard deviation (=1.0) and are the average values ​​of three measurements. The error is the difference between the three measurements. The upper and lower limits are shown. The number of colonies expressed at 10 μC in both figures was 0. The detection limit values ​​are plotted in the figure. The vertical axis shows the amount of the two types of plasmids that were irradiated with protons. This indicates the percentage of drug resistance genes that function normally without being damaged. Total irradiation dose 2.5 In μC, 15 The N_pUC4-KIXX plasmid was more damaged by radiation than the error. This result shows that 15 N( 1 H,α1γ) 12 C resonance nuclear reaction causes the ionization of protons In Figure 14, the total irradiation dose is roughly At levels above 5.0 μC, genes are thought to be nonspecifically damaged.

[0082] The above-described embodiments are intended to facilitate understanding of the present invention and are not intended to limit the present invention. The elements of the embodiment and their arrangement, materials, conditions, etc. The shape and size are not limited to those shown in the examples and can be changed as needed. In addition, the configurations shown in different embodiments may be partially substituted or combined with each other. It is Noh.

Claims

1. At least some of the DNA bases 15 The abundance of N exceeds the natural abundance ratio. Artificial genes.

2. 15 N unlabeled primer sequence; 15 N-labeled deoxyribonucleotides, The artificial gene according to claim 1.

3. The artificial gene according to claim 1 or 2; a vector linked to the artificial gene; An artificial gene comprising:

4. a plurality of biomolecule binding sites, and In this case, the gene sequence is mutated. The artificial gene according to claim 1 or 2.

5. The artificial gene according to claim 4 , wherein the biological molecule is a protein.

6. 15 N is an unlabeled artificial gene, having multiple biomolecule binding sites; At least some of the plurality of biomolecule binding sites have mutated gene sequences; In any of the plurality of biomolecule binding sites, 15 N-labeled biomolecules can be bound 、 Artificial genes.

7. The plurality of biomolecule binding sites are each selected from the group consisting of a plurality of biomolecule binding sites, each of which has a non-mutated gene sequence. 15 N label The artificial gene of claim 6, wherein the biomolecule is capable of binding.

8. The artificial gene of claim 7 , wherein the biological molecule is a protein.

9. The artificial gene according to any one of claims 6 to 8, capable of binding to any of the plurality of biomolecule binding sites 15 an N-labeled biomolecule; A kit comprising:

10. DNA 15 labeling with N; 15 Irradiating the DNA with a proton beam having an energy that causes a resonant nuclear reaction of N; A method for mutating a gene, comprising:

11. The DNA 15 Labeling with N is 15 N. The method according to claim 10.

12. The DNA 15 Labeling with N allows the vicinity of the DNA to be 15 N, The method of claim 10.

13. The DNA 15 Labeling with N allows the DNA to 15 N-labeled biomolecules are bound 13. The method of claim 10 or 12, comprising:

14. The method of claim 13 , wherein the biomolecule is a protein.

15. The DNA in living cells 15 15. The compound according to claim 10, wherein the compound is labeled with N. How to do it.

16. In the cellular components other than DNA in the living cells 15 N is maintained at its natural abundance ratio, and the D In N.A. 15 The method of claim 15, wherein the abundance ratio of N is increased above its natural abundance ratio.

17. The DNA in the living cell 15 Labeling with N 15 N-labeled deoxyribonucleic acid 15 or 16, comprising administering to said living cells an inhibitor of glutamine synthetase and a nucleotide.

16. The method according to claim 16.

18. The DNA in the living cell 15 Labeling with N 15 N-labeled deoxyribonucleic acid and administering to said living cells a nucleotide and an inhibitor of ribonucleotide reductase.

17. The method according to claim 15 or 16.

19. The DNA in the living cell 15 Labeling with N 15 N-unlabeled glutamine was added as above.

19. The method of claim 17 or 18, further comprising administering to a living cell.

20. 20. The method of claim 10, further comprising detecting the resonant nuclear reaction. How to post.

21. 21. The method of claim 20, wherein the detecting step measures a gamma ray dose of 4.43 MeV. How to do it.

22. and calculating the number of mutations that have occurred in the DNA based on the measured amount of γ-rays.

22. The method of claim 21 .

23. In the calculation, 15 It is generated by a resonant nuclear reaction that occurs in a standard sample with a known number of N atoms. The method of claim 22, wherein the dose of gamma radiation is referenced.

24. The aforementioned 15 11. The method of claim 10, further comprising changing the energy at which N undergoes a resonant nuclear reaction.

24. The method according to any one of claims 1 to 23.

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