WO2005073373A1 - Procédé de recueil d'un fragment endommagé d'adn - Google Patents

Procédé de recueil d'un fragment endommagé d'adn Download PDF

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WO2005073373A1
WO2005073373A1 PCT/JP2005/001085 JP2005001085W WO2005073373A1 WO 2005073373 A1 WO2005073373 A1 WO 2005073373A1 JP 2005001085 W JP2005001085 W JP 2005001085W WO 2005073373 A1 WO2005073373 A1 WO 2005073373A1
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dna
dna fragment
antibody
oxidative stress
damaged
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PCT/JP2005/001085
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Japanese (ja)
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Shinya Toyokuni
Shinya Akatsuka
Osamu Nikaido
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Shirankai Kyoto University Faculty Of Medicine Alumni Association Inc.
Nikken Seil Corporation
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Publication of WO2005073373A1 publication Critical patent/WO2005073373A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method for selective collection of damaged DNA, and more particularly, to a method for selectively selecting a DNA fragment corresponding to a DNA region damaged by the stress in a genome exposed to oxidative stress. And the use of DNA fragments collected in this way to pray for genomic damage.
  • Oxidative stress is a concept that means the amount of free radicals / active oxygen generated minus the erasing ability-defense ability * repair ability of injury, etc., for humans. It has been clarified to be related to lifestyle-related diseases such as arteriosclerosis and cancer.
  • Nucleic acids such as DNA are also target molecules for free radicals and active oxygen, but guanine, one of the purine bases among nucleic acid bases, is known to be easily damaged. Hydroxyradiation, singlet oxygen, or the modified base formed by photodynamic reaction include the hydroxylated 8-position of 2, -deoxyguanosine and 8-hydroxy-2'-deoxyguanosine ( Non-patent document 1). When 8-hydroxy-2'-deoxyguanosine is present in DNA, it can cause a transversion mutation (gene mutation) from GC (guanine-cytosine) to TA (thymine-adenine) during its replication. (Non-patent document 2) [Shibutani et al., Nature 349: 431, 1991].
  • 8-hydroxy-2'-deoxyguanosine has been used as a marker for DNA damage, which is a factor that causes senescence, cell mutation and canceration.
  • 8-hydroxydeoxyguanosine excreted in urine has been used as a marker that reflects the onset and disease state, such as the occurrence of cancer, management of diabetic patients, and monitoring after radiation exposure. The measurements were performed using thin-layer chromatography, high-performance liquid chromatography, electrochemical detection (HPLC-ECD), gas chromatography mass spectrometry, etc.
  • Kits for immunoassay using monoclonal antibodies against xyguanosine (8_ ⁇ HdG) Patent Document 1, Non-patent Document 3 have also been used. Antibodies to 8_ ⁇ HdG are frequently used to identify cells with a high content of 8-hydroxyguanine in the nucleus and play a crucial role in a wide range of fields such as medicine, pharmacy, and agriculture .
  • Patent Document 1 Patent No. 3091974
  • Non-Patent Document 1 Kasai, Mutat. Res. 387: 147, 1997
  • Non-patent document 2 Takatani et al., Nature 349: 431, 1991
  • Non-Patent Document 3 Toyokuni et al., Lab. Invest. 76: 365, 1997
  • the present invention specifies a region / site on a gene (DNA) susceptible to damage by stress. More specifically, to selectively collect DNA fragments containing one or more damaged nucleosides from a population of DNA fragments obtained from a sample. It is intended to provide a way to:
  • the present inventors succeeded in selectively collecting damaged DNA fragments by immunoprecipitation for the first time, and completed the present invention. That is, the present invention is as follows.
  • a method for collecting DNA fragments in a region containing DNA which has been damaged by oxidative stress comprising extracting DNA from a sample, fragmenting the DNA, and modifying the modified nucleoside or the modified nucleoside.
  • a method comprising incubating with a primary antibody specific for a polynucleotide containing a nucleoside, recovering a precipitated complex, and recovering a DNA fragment damaged by the complex.
  • modified nucleoside is selected from 8-hydroxy-2′-deoxyguanosine, cyclobutane-type pyrimidinin dimer, and acrolein-added 2′-deoxynucleoside.
  • the present invention it is possible for the first time to collect a DNA fragment containing a damaged nucleoside. Noh.
  • a means for identifying and analyzing a damaged or easily damaged region on the gene can be obtained.
  • protective means against genetic injury and by identifying sites (regions) that are susceptible to injury, if the site is a region specific to the type of cancer or organ, diagnosis of those sites can be made. It can contribute to future preventive treatment by elucidating the mechanism of onset of cancer and the like due to DNA damage, and understanding the genetic characteristics of each individual.
  • FIG. 1 shows the amount of DNA fragments collected from a methylene blue-treated genomic DNA sample using an antibody against 8-OHdG.
  • FIG. 3 shows the amount of DNA fragments collected from a DNA sample obtained from rat plasma using an antibody against 8-OHdG.
  • FIG. 4 shows the amount of DNA fragments collected from a UV-C-treated genomic DNA sample using an antibody against CPD.
  • FIG. 5 shows the amount of DNA fragments collected from DNA extracted from mouse kidney under oxidative stress load (6 hours after administration of Fe_NTA) using an antibody against acrolein-added 1′-deoxyadenosine.
  • modified nucleoside refers to a nucleoside damaged by oxidation stress caused by ultraviolet light, radiation, a chemical substance, or the like.
  • the 8'-position of 2'-deoxyguanosine is hydroxyl 8-hydroxy-2'-deoxyguanosine (hereinafter also referred to as “8_OHdG”), 8-nitroguanosine with 8-position guanosine nitrated, and cyclobutane-type pyrimidine dimer (hereinafter “CPD”). ), Nucleosides modified by reaction with acrolein, and the like.
  • Cyclobutane pyrimidine dimer is most frequently produced by ultraviolet light. This is one of the DNA damages that are caused.
  • acrolein is one of the unsaturated aldehydes formed by peroxidation of membrane lipids, and is widely detected in the environment and also found in cells under oxidative stress [Uchida et al. (Uchida et al.) , J. Biol. Chem. 273: 16058, 1998]. Since acrolein reacts with highly reactive nucleic acids to form adducts, it is known that nucleosides react with acrolein to form adducts under oxidative stress.
  • the present invention is directed to any nucleoside that is modified by reacting with acrolein under oxidative stress.
  • acrolein-added 2'-dexoxynucleoside is preferred for acrolein-added 2'-dexoxy.
  • Adenosine is particularly preferred.
  • an antibody specific to a modified nucleoside or a polynucleotide containing the modified nucleoside include polyclonal antibodies and monoclonal antibodies.
  • Antibodies to the modified deoxynucleoside can be prepared by a conventional method using a complex of a suitable carrier (for example, limpet hemocyanin (KLH)) and a synthetic modified deoxynucleoside as a hapten antigen as immunogen DNA. Can be manufactured. A polynucleotide (DNA fragment) containing the modified nucleoside is recognized by an antibody against the modified deoxynucleoside.
  • KLH limpet hemocyanin
  • an animal is immunized with the immunogen DNA prepared as described above.
  • the administration is performed by intravenously, subcutaneously or intraperitoneally administering an appropriate amount to a mammal (eg, rat, mouse, egret, human, etc.).
  • the interval between immunizations is not particularly limited, and is 1 to 10 times, preferably 4 to 5 times at intervals of several days to several weeks, preferably at intervals of 23 weeks.
  • the antibody titer is also measured 7 to 10 days after the final immunization, and blood is collected on the day showing the highest antibody titer to obtain antiserum.
  • the antibody titer can be measured by enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunohistochemical staining, and the like.
  • ammonium sulfate precipitation ion exchange chromatography Purification can be carried out by appropriately selecting a known method such as luffy, gel filtration, affinity chromatography and the like, or by combining them.
  • an animal is immunized with immunogenic DNA. Immunization is performed by administering a suitable amount to mammals (for example, rats, mice, etc.) intravenously, subcutaneously or intraperitoneally.
  • mammals for example, rats, mice, etc.
  • the interval between immunizations is not particularly limited, and the immunization is performed at least for four to five times at intervals of several days to several weeks, preferably at intervals of two to three weeks.
  • antibody-producing cells are collected. Spleen cells are preferred as antibody-producing cells.
  • Myeloma cells used for cell fusion between antibody-producing cells such as spleen cells and myeloma cells may be cells derived from animals such as mice and commonly available cell lines. . As a cell line to be used, it has drug selectivity, cannot survive in a HAT selection medium (including hypoxanthine, aminopterin and thymidine) in an unfused state, and can survive only in a state fused to antibody-producing cells. Are preferred.
  • specific examples of myeloma cells include mouse myeloma cell lines such as PSUI, P3X63-Ag, and X63Ag8.653.
  • Antibody-producing cells and myeloma cells are mixed at a predetermined ratio (e.g., 3: 1) in animal cell culture medium such as serum-free DMEM or RPMI-1640 medium, and then mixed in the presence of a cell fusion promoter. Alternatively, it is performed by an electric noise treatment (for example, electoral port correction).
  • the cells are cultured using, for example, a HAT medium containing hypoxanthine (100 / im), aminopterin (0.4 ⁇ ) and thymidine (16 ⁇ ), and the cells that grow can be obtained as hybridomas.
  • Screening for the presence of the target antibody in the culture supernatant of the proliferated hybridomas is carried out by enzyme-linked immunosorbent assay (EIA) or the like. Cloning of the fused cells that have finally confirmed antibody production is performed by limiting dilution, etc., and finally, hybridomas, which are monoclonal antibody-producing cells, are established.
  • a usual cell culture method or the like can be employed.
  • a mouse is immunized using a complex of synthetic 8_OHdG and KLH as an immunogen, a hybridoma is prepared, and the obtained hybridoma is used.
  • a mouse monoclonal antibody against 8-hydroxy-2, -deoxyguanosine (clone N45.1 (Niken Zile), hereinafter referred to as “ ⁇ 45.1”) was prepared by preparing ascites fluid and fractionating and purifying it by ammonium sulfate salting out. (Name).
  • TD II-2 [Mori et al., Photochem Photobiol 54: 225, 1991] is known as a monoclonal antibody against clobtan-type pyrimidine dimer (CPD).
  • CPD clobtan-type pyrimidine dimer
  • 8_nitroguanosine have been provided (Clone # N02G52, Dojin).
  • monoclonal antibodies against the reaction product of acrolein with 2'-deoxyadenosine can be prepared by methods known in the literature [Kawai et al. (Kawai et al.), J. Biol. Chem. 278:
  • the DNA fragment having oxidative stress injury is specifically recognized by the mouse monoclonal antibody N45.1 against 8_OHdG. Therefore, they are precipitated as antigen-antibody complexes.
  • the immunoprecipitation method is well known in the art as an experimental technique for separating and purifying an antigen protein corresponding to a specific antibody.
  • a DNA fragment containing 8-hydroxydeoxyguanosine can be separated from a miscellaneous population of DNA fragments by immunoprecipitation using N45.1 under the conditions described below.
  • the amount of the injured DNA fragment containing the modified deoxynucleoside collected in this manner was determined by the sample used for preparing the original DNA fragment population as the starting material. The degree of oxidative stress is reflected, and the oxidative stress level of a living body can be appropriately evaluated by the method of the present invention.
  • each of the DNA fragments collected by the method of the present invention can be cloned using an appropriate vector-host system.
  • Such vectors are capable of replicating in a host. Is not particularly limited so long as it is, for example, plasmid DNA, phage DNA, etc.
  • plasmid DNA examples include a plasmid derived from Escherichia coli (eg, pBR322), a plasmid derived from Bacillus subtilis (eg, pUB110), a plasmid derived from yeast (eg, YEp13), and the like.
  • I phage eg, gtlO
  • animal viruses such as retrovirus, adenovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.
  • the purified DNA fragment is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multiple cloning site of the vector DNA, and ligated to the vector. Is adopted.
  • a transformant can be obtained by introducing the above-described recombinant vector into a host suitable for expressing a DNA fragment.
  • the host is not particularly limited.
  • bacteria of the genus Escherichia such as Escherichia coli
  • bacteria of the genus Bacillus such as Bacillus subtilis
  • yeasts such as Saccharomyces cerevisiae
  • COS cells And animal cells such as CHO cells, and insect cells such as Sf9 and Sf21.
  • a method for introducing a recombinant vector into a host is also known in the art, and examples thereof include a method using calcium ions, an electoporation method, a calcium phosphate method, and a lipofection method.
  • Culture of the transformant is performed according to a usual method used for culturing a host, and a medium and culture conditions suitable for each host are also known.
  • a microbial host is suitable for the present invention.
  • the medium contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism to efficiently culture the transformant.
  • Either a natural medium or a synthetic medium may be used as long as the medium can be used.
  • Cultivation is usually carried out at 37 ° C for 812 hours under aerobic conditions such as shaking culture or aeration and stirring culture.
  • the cloning in the present invention can also be carried out using a commercially available cloning kit.
  • a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) is available. is there.
  • the nucleotide sequence of each cloned collected DNA fragment is decoded to obtain sequence information, and the genome database is queried to determine the position of the chromosome corresponding to the damaged DNA fragment in the whole genome sequence. be able to.
  • the injured DNA fragment is mapped onto the chromosome as a single point.
  • mapping for example, there is a method of creating a distribution map based on the positional coordinate information on the chromosome obtained from the genome database of Celera.
  • the following steps employ a commonly used method.
  • a very small amount of the target substance is contained in the DNA (generally 100% of the amount of the sample DNA as a starting material) (About 1 / 1000th of the damaged DNA), so the special treatment described below was adopted and the first successful collection of the damaged DNA was achieved.
  • any sample containing DNA such as cultured cells, tissue cells of experimental animals, and blood collected from the human body
  • Preparation of DNA from specimens can be carried out in the usual way. It is necessary to select conditions under which DNA (deoxynucleoside) is not artificially modified in the process. For example, in order to suppress the production of 8-hydroxydeoxyguanosine to a very small amount, a deproteinization treatment using an organic solvent such as phenol cannot be applied as a DNA purification operation. Therefore, it is effective to add a chaotropic agent (such as sodium iodide) and suppress the co-precipitation phenomenon of DNA and protein, thereby omitting the phenol treatment.
  • a chaotropic agent such as sodium iodide
  • Specimen strength Fragment the obtained DNA by restriction enzyme treatment.
  • Select the type of restriction enzyme to be used so that the average length of the DNA fragment after enzyme digestion is about 1,000 bases.
  • Such restriction enzymes vary depending on the target (animal, plant, tissue, etc.).
  • Haelll recognition base sequence: GG ICC
  • the time of the restriction enzyme reaction is kept to a necessary minimum (1 hour) in order to reduce the generation of modified DNA (eg, 8-OHdG) as an artifact.
  • the solvent, temperature, etc. for the restriction enzyme reaction differ depending on the restriction enzyme used. To completely digest the sample DNA in one hour, it is desirable to add as many restriction enzymes as possible to the reaction system.
  • the immunoprecipitation reaction can be performed by the following method.
  • conditions were selected under which the antibody stably forms a complex with the antigen and precipitates.
  • a fragment containing 8_OHdG is separated from a heterogeneous population of DNA fragments.
  • PBS phosphate buffered saline, pH 7.4
  • the amount of antibody to be used depends on the amount of DNA and the amount of 8-OH dG contained therein (expected value), but it is added so that the antibody molecule is in excess with respect to the DNA molecule.
  • the number of antibody molecules is adjusted to be about 50 times or more, preferably 300 times or more, and more preferably 625 times or more with respect to one DNA fragment (one molecule).
  • the upper limit of the antibody molecule for the DNA fragment can be appropriately determined by those skilled in the art according to the purpose and the reaction system to be used. Generally, when a sample obtained by fragmenting genomic DNA extracted from mammalian tissue cells under physiological conditions into an average lkb length is used as a sample, the weight ratio of antibody to fragmented DNA is 0.08: 1, preferably 0.48: 1, most preferably about 1: 1.
  • DNA and antibody are combined in a test tube in a buffer, for example PBS, at a concentration of 0.01-0.05 / ⁇ ⁇ / ⁇ 1, preferably 0.01-0.02 zig / ⁇ , and this is rotated on a vertical surface. And mix each component. This mixing operation is performed at a low temperature (4 ° C) and continues for 1 to 3 hours.
  • bead particles having a molecule having an affinity for mouse immunoglobulin G bound to the surface thereof are added to the mixture obtained in (1).
  • carrier particles include Sepharose and agarose beads bound to Protein A or Protein G, and antibodies derived from other animals against mouse immunoglobulin G.
  • Combined magnetic beads eg, Dynabeads
  • proteins such as antibodies and restriction enzymes remain together with DNA.
  • the protease Proteinase K
  • the protease is allowed to act at 37 ° C for 1 hour in a TE buffer.
  • phenol-chloroform extraction and ethanol precipitation are performed, and the damaged DNA is purified and concentrated.
  • the following method is used to determine the amount of the damaged DNA fragment containing the modified deoxynucleoside (eg, 8_OHdG) collected by the method of the present invention.
  • the amount of DNA fragments damaged by oxidative stress is small, and the amount of DNA fragments collected by the method of the present invention is extremely small (about 100 minutes of the amount of sample DNA as a starting material). About 1 / 1000th of 1).
  • the residual level of 8-hydroxyhydroxyguanosine in the genomic DNA of mammalian cells is on the order of one per million guanine. Therefore, the spectrophotometer measurement method usually used for DNA quantification cannot be applied because the concentration is below the detection limit concentration.
  • the present inventors have succeeded in detecting and quantifying a trace amount of DNA by using a bromide reagent.
  • the quantification procedure is as follows.
  • the standard DNA solution of known concentration and the collected damaged DNA fragment solution are mixed with appropriate amounts of bromide solution.
  • the mixed solution of DNA-brominated medium is placed as droplets on an ultraviolet irradiation device, and the intensity of the fluorescence by ultraviolet irradiation is compared between the standard solution and the sample solution to determine the target damaged DNA content.
  • the damaged DNA collected in an appropriate vector-host system is cloned, for example, by the method described above, and the fragment population is separated as separate clones.
  • the base sequence (part of) is determined for each clone. Based on this sequence, a publicly available genome database can be searched to obtain detailed genomic information on the chromosome, a site having a sequence that matches the DNA fragment, and its surroundings. According to this analysis method, it is possible to comprehensively identify the remaining site of DNA damage due to oxidative stress over the entire genome.
  • the present invention is based on the combination of immunoprecipitation and a monoclonal antibody against a modified deoxynucleoside (e.g., 8-OHdG). And a method for selectively isolating and identifying the same.
  • the “optimal condition” can be determined based on the following points.
  • the amount of the antibody is adjusted as described above so as to efficiently recognize the damaged DNA fragment contained in the fragmented DNA population and form a complex.
  • Thorough washing was performed to completely remove non-target fragments non-specifically attached to or in proximity to the beads without antigen-antibody reaction. A total of eight washes were performed using four types of buffers with different surfactant compositions.
  • DNA is quantified using a spectrophotometer, utilizing the absorption characteristics of nucleic acids.However, since the amount of damaged DNA is lower than the detection sensitivity of a normal spectrophotometer, it is mixed with a bromide reagent. It is quantified by the fluorescence signal intensity of the mixed droplet.
  • Genomic DNA was prepared from kidney tissue of mice (16 weeks old, male C57BLZ6). DNA extraction from tissue cells was performed using the sodium iodide method (Wang et al., Nucleic Acids Res. 22: 1774, 1994), a commercial reagent kit (Wako Pure Chemical Industries; DNA Extractor WB Kit). It was implemented according to the attached instructions. During the extraction process, keep the sample at a low temperature as much as possible to suppress the new generation of 8-hydroxyguanine (ie, 8-hydroxy-2'-deoxyguanosine as a modified deoxynucleoside) during the extraction process. (On ice) and light-shielded. The buffers were saturated with argon gas, and 0.1M ImM desferrioxamine was added to inactivate the catalytic iron.
  • the mouse genomic DNA obtained in the above (1) was digested with the restriction enzyme Haelll at 37 ° C in 10 mM Tris-HCl (H7.5), 10 mM MgC12, 1 mM dithiothreitol and 50 mM NaCl, and the fragment was digested. It has become.
  • the reaction system was adjusted so that the enzyme could be completely cleaved in one hour.
  • the genomic DNA extracted was introduced artificially into the sample, and samples having different contents of 8-hydroxydeoxyguanosine were prepared.
  • guanine in the DNA is reduced to 8-hydroxydeoxy. It can be artificially changed to ciguanosine. Residual levels of 8-hydroxydeoxyguanosine in the genomic DNA of mammalian cells are usually on the order of one per million guanine.
  • Table 1 8_OHdG content of genomic DNA treated with methylene blue and light
  • the amount of PBS to be initially added was adjusted so that the total volume of the mixed system was 900 ⁇ l. This tube was stirred for 3 hours using a rotary shaker in a cold room (4 ° C).
  • Magnetic beads (Dynabeads M-280) conjugated with a secondary antibody (She-marked anti-Mouse IgG) manufactured by Dynal were used as a carrier for sedimentation.
  • a magnetic bead suspension (PBS containing 0.1% of plasma albumin) was used.
  • (Medium) (100 / il) was added to the mixed system, and the stirring operation was continued for another 3 hours by using a rotary shaker at a low temperature.After completion of the binding reaction, the beads were washed sequentially with four kinds of buffers.
  • composition of each buffer is as follows:
  • Solubilization buffer 140 mM NaCl
  • Solubilization buffer 500mM NaCl
  • 0.1% sodium deoxycholate 0.1% sodium deoxycholate, ImM EDTA, 50mM Hepes-KOH (pH7.5), 500mM NaCl, 1% TritonX-100
  • washing was carried out twice in each buffer in the order of 1 to 4 described above. After washing, the beads were suspended in 80 ⁇ l of an elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl (pH 8.0)). The collected DNA fragments were eluted by heat treatment at 65 ° C for 15 minutes. The beads were then separated from the solution components using a magnet. The operation of adding the elution buffer ⁇ heating ⁇ separation was repeated once.
  • an elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl (pH 8.0)
  • Fig. 1 shows the results when the initial DNA fragments to be subjected to immunoprecipitation were treated with 0, 5, 10, 50 ⁇ M methylene blue to cause artificial damage.
  • the vertical axis of the figure represents the amount of damaged DNA fragments collected by the method of the present invention from DNA samples in which the content of 8-—dG was increased stepwise. The results indicate that the amount of damaged DNA fragments collected depends on the amount of 8-hydroxydeoxyguanosine in the starting DNA sample.
  • Fe-NTA Iron nitrite triacetic acid
  • oxidative stress agent Iron nitrite triacetic acid
  • DNA extraction from kidney tissue and its fragmentation, and subsequent collection and purification of 8-hydroxyguanine-containing fragments were performed as described in Example 1.
  • the DNA cloning kit used in the following step (3) before purifying the collected DNA fragment, remove the 5'-terminal phosphate group from the DNA fragment Added a fostase treatment. That is, the collected DNA fragments were treated with a small intestine alkaline phosphatase (Takara Bio) at 50 ° C. for 30 minutes.
  • a commercial kit for blunt-end PCR product cloning (Invitrogen; Zero Blunt TOPO PCR Cloning Kit for Sequencing) was used.
  • the collected DNA fragment was inserted into a plasmid vector (pCR4Blunt-TOPO) according to the instructions attached to the kit, E. coli was transformed, and the resulting transformant was cultured on an agar plate. This was used as the library of the collected fragment clone.
  • Escherichia coli colonies were sequentially picked up from the agar plate of the above (3), and the nucleotide sequence of the DNA fragment clone was comprehensively determined using an autosequencer (ABI PRISM377).
  • FIG. 2 shows the mapping result of the remaining 8-hydroxyguanine under the oxidative stress load state.
  • the points indicated by black squares in the figure indicate the origin of each collected fragment on the chromosome. ing.
  • the DNA content in plasma is extremely low as compared with the kidney tissue used in Examples 1 and 2.
  • the amount of DNA obtained from blood (5-7 ml) collected from a single rat is shown in Table 2 below.
  • the plasma DNA obtained in the above (1) was fragmented in the same manner as described in Example 1, and the DNA fragment containing 8-hydroxydeoxyguanosine was collected and purified by immunoprecipitation.
  • the initial amount of DNA to be subjected to immunoprecipitation was 1.5 / ig.
  • 1.5 ⁇ of DNA was used from the combined extracted DNA of three animals. This initial amount of DNA is about one-third of the amount in Example 1.
  • FIG. 3 shows the results of collecting fragments containing 8-hydroxydeoxyguanosine from DNA in rat plasma by the method of the present invention.
  • Ultraviolet irradiation is also considered to be one of the oxidative stresses, and cyclobutane-type pyrimidine dimer (CPD) is one of the most frequently generated DNA damage by ultraviolet light
  • TDM-2 [Mori et al, Photochem Photobiol 54: 225, 1991] is a monoclonal antibody against CPD.
  • the DNA extracted from the tissue was irradiated with UVC (254 nm) to prepare a sample in which CPD was artificially introduced into the DNA fragment.
  • UVC 254 nm
  • SPECTROLINKER XL-100 UV crosslinker
  • Example 2 In immunoprecipitation with Example 1 the procedure, it was used TDM- 2, 20 ⁇ ⁇ as the primary antibody.
  • FIG. 4 shows the results of collecting CPD-containing DNA fragments by the method of the present invention. These results indicate that the amount of collected DNA fragments depends on the amount of UV irradiation.
  • Oxidative damage also includes peroxidation of lipids.
  • Acrolein is one of the unsaturated aldehydes formed by the peroxidation of membrane lipids, and is also found in cells under oxidative stress. (Uchida et al., Supra). Acrolein reacts with nucleic acids to form adducts.
  • mAb21 is a monoclonal antibody to the reaction of acrolein with 2'-deoxyadenosine
  • Each of 12-week-old and 17-week-old male C57BLZ6 mice was administered Fe-NTA in an amount equivalent to 3 mg of iron per kg of mouse body weight. Mice were sacrificed and extracted 6 hours after administration for S storage sample. Samples were also taken from untreated mice of the same age for controls. As in Example 1, genomic DNA was extracted from kidney tissue using a sodium iodide kit.
  • Kidney genomic DNA was fragmented in the same manner as described in Example 1. Further, according to the procedure of Example 1, the acrolein-added fragment was collected and purified by immunoprecipitation. However, as a primary antibody, 2 ⁇ g of a monoclonal antibody (mAb21) against a reaction product of acrolein and 2′-deoxyadenosine was used.
  • mAb21 monoclonal antibody against a reaction product of acrolein and 2′-deoxyadenosine
  • a genomic location (or gene) that is susceptible to damage is searched using the sequence information of the damaged DNA. It becomes possible.
  • a gene that is susceptible to injury is a candidate for a gene that is susceptible to mutation, and such information provides, for example, insight into the earliest stages of carcinogenesis. Therefore, in the end, information that is essential for establishing cancer prevention measures It can be used as basic information for deriving.

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Abstract

Il est prévu de fournir un procédé destiné à recueillir sélectivement un fragment d'ADN dans une région endommagée par un stress oxydatif, provenant d'un échantillon contenant de l'ADN, caractérisé en ce qu'il comprend l'extraction de l'ADN de l'échantillon, la fragmentation de l'ADN, l'incubation avec un anticorps primaire spécifique sur un nucléoside modifié, ayant été modifié par le dommage, ou un polynucléotide contenant le nucléoside modifié comme décrit ci-dessus, le recueil d'un complexe ainsi précipité et ensuite le recueil d'un fragment endommagé de l'ADN provenant du complexe.
PCT/JP2005/001085 2004-01-29 2005-01-27 Procédé de recueil d'un fragment endommagé d'adn WO2005073373A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2010084998A1 (fr) * 2009-01-26 2010-07-29 Kyushu University, National University Corporation Procédé de prédiction de l'efficacité d'un médicament
CN103212066A (zh) * 2013-03-14 2013-07-24 曹毅 一种环丁烷嘧啶二聚体光修复酶脂质体的制备方法
CN108530539A (zh) * 2018-03-20 2018-09-14 北京博雅捷康生物科技有限公司 一组识别dna受紫外线照射造成的嘧啶二聚体的抗体

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010084998A1 (fr) * 2009-01-26 2010-07-29 Kyushu University, National University Corporation Procédé de prédiction de l'efficacité d'un médicament
CN103212066A (zh) * 2013-03-14 2013-07-24 曹毅 一种环丁烷嘧啶二聚体光修复酶脂质体的制备方法
CN108530539A (zh) * 2018-03-20 2018-09-14 北京博雅捷康生物科技有限公司 一组识别dna受紫外线照射造成的嘧啶二聚体的抗体
CN108530539B (zh) * 2018-03-20 2021-08-13 北京博雅捷康生物科技有限公司 一组识别dna受紫外线照射造成的嘧啶二聚体的抗体

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