Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • 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
  • C12Q1/6869—Methods for sequencing

Definitions

• the present invention relates to a method for chemically determining the nucleotide sequence of deoxyribonucleic acid. More specifically, the present invention relates to a method for base-selective modification of deoxyribonucleic acid labeled with radioactive phosphorus by a chemical reagent. In the process of cleaving the deoxyribonucleic acid strand at the position of the modified base, the chemically modified dexylribonucleic acid is removed from the reaction solution after the chemical modification reaction, and The present invention relates to a method for cleaving at the position of a modified base and an apparatus for carrying out the method.
• DNA deoxyribonucleic acid
• Maxam and Gilbert the chemical sequencing method developed by Maxam and Gilbert is one of the most popular methods. It is widely used because it has the advantages of relatively simple experimental procedures and the advantage that it is comparable to other determination methods in terms of speed and accuracy. The details of this method are described in Proc. Acad. Sci., Vol. 74, pp. 560, 1977, Protein Nucleic Acid Enzyme, Vol. 23, pg.
• a DNA sample is prepared, one end of which is labeled with a radioactive phosphorus, and the four bases (guanine, adenine, thymine, and cytosine) that form the DNA are prepared.
• G, A, ⁇ , and C, respectively are selectively modified with a chemical reagent, and then the DN chain is cleaved at the position of the modified base, and the obtained fragment is subjected to gel electrolysis.
• the fragments were separated and developed in the order of the molecular chain length of each fragment by base sequence by the swimming method, and then separated and separated on an X-ray film by autoradiography.
• a band is imaged, and then the base sequence of the first DNA sample is determined by identifying the base corresponding to the band on the X-ray film.
• the Maxam-Gilbaut method is widely used because of the above-mentioned advantages, but since the operations in each of the above steps are conventionally performed manually, many DN- ⁇ samples are used.
• the operation Hakiwa Umate annoyance Wei is a time consuming 0 Therefore operation It is desirable to improve work efficiency by mechanization.
• the present invention is intended to improve the efficiency of the work by mechanizing the operation of the second step of the above four steps, ie, the step of base-selectively modifying the labeled DNA and cutting the DNA strand at that position. It relates to methods and equipment.
• a sample solution containing labeled DNA is dispensed into four resin tubes, and each of the four bases, namely G, A, T, and C, is used for each.
• Specific chemical modifications (reagents and reaction conditions) were applied separately, ethanol was added, the mixture was cooled to -40 to -70, centrifuged, the supernatant was sucked off, discarded, and the residue remained. The ethanol was added to the precipitate again, and the precipitate was centrifuged.The supernatant was aspirated and discarded.The precipitate was washed repeatedly to remove the excess reagents used in the chemical modification from the precipitate.
• the DNA_chain was cleaved at the position of the modified base.
• a cutting sample sodium hydroxide or piperidine
• the present inventors have found that, among the above operations, particularly the operation of adding ethanol, cooling and centrifuging.
• the present invention separates a chemically modified DNA from a reaction solution after the completion of the DNA chemical modification reaction, and then adds a cleavage reagent thereto.
• the separation operation is performed by bringing the reaction solution after the chemical modification reaction into contact with an adsorbent. After the chemically modified DNA is adsorbed on the adsorbent, the adsorbent is washed with a washing liquid to remove the adhering chemical modification reagent, and then contacted with the eluate, thereby adsorbing the chemically modified adsorbent.
• This is a DNA cleavage treatment method characterized by eluting DNA and concentrating the eluate.
• the present invention is also an apparatus for performing the above method, the apparatus comprising a reactor accommodating the reaction solution, a transfer bomb, an adsorber for adsorbing and holding the chemically modified DNA, a three-way valve and And a concentrator, and these are interconnected by a feed pipe in the order described above.
• the adsorber for adsorbing and holding DNA from the solution after the completion of the chemical modification the facility for washing the adsorbed DNA and washing away the reagents used for the chemical modification
• the above-described method of the present invention can be carried out by operating the apparatus extremely simply and mechanically using equipment for elution and equipment for concentrating the eluate.
• FIG. 1 is an example of a system diagram of a preferred embodiment of the device of the present invention.
• Fig. 2 shows the DNA cleaved by the present invention. This is an example of a sketch of a moving radiograph. BEST MODE FOR CARRYING OUT THE INVENTION
• the radioactive-labeled DNA is placed in the reaction vessel la in the reactor 1 in Fig. 1, and various reagents are added.
• the reaction is carried out at a specified temperature for a specified time, and then the chemical modification reaction is performed.
• the passing liquid is discharged to the waste liquid tank 6 through the three-way valve 5 and is chemically modified: the DNA is adsorbed and retained by the adsorbent charged in the adsorber 2.
• the chemically modified DNA held by the adsorbent in the adsorber 2 is eluted and eluted into the concentration vessel 3a.
• the vacuum pump 12 and the vibrator 13 are operated, and the pressure reducing control valve 14 is opened to concentrate the eluate in the concentration vessel 3a under reduced pressure. Evaporate
• adsorbent used in the present invention either a positive ion or an anion exchanger can be used, and one having a polystyrene resin as a base or one having cellulose as a base
• a positive ion or an anion exchanger can be used, and one having a polystyrene resin as a base or one having cellulose as a base
• Dowe X -X2 Dowex-50W (both trade names, manufactured by Dow Chemical Company), AIEC-CM52, AIEC-DE52 (both trade names, manufactured by Petman)
• non-ionic porous resin for example,
• Amber 1 ite XAD-2 (trade name, manufactured by Roam And Haas), Diaion HP-10 (trade name, manufactured by Mitsubishi Kasei), Levacit OC1031 (trade name, Bayer) Can be used.
• fine particles treated in a reversed-phase distribution form for example, a packing material used in high-performance liquid ⁇ -macrographs, such as microbonder pack C18 and microbonder.
• microbonder pack C18 and microbonder.
• Micro Bonder Pack-Fenil both are trade names, manufactured by Ots Corporation are used.
• the above adsorbents are usually used by filling glass, stainless steel or polyethylene columns, for example, Step Pack C-18 (commercially available). Name, manufactured by Otters) Yes Is Bond.Elut C-18 (manufactured by Analytic Chem-International Co., Ltd.), which is commercially available after filling the column and is preferably used. I can do it.
• the adsorbent is an ion exchanger
• an aqueous solution of an acidic or basic substance is applied to the eluate for elution of the DNA, especially ammonia and piperazine.
• An aqueous solution of a volatile basic substance such as is preferred.
• volatile substances such as methanol, ethanol or acetate nitrile are used.
• a mixed solution of a water-soluble organic solvent and water is used.
• 38 10 (trade name, manufactured by Eppendorf) is preferably used.
• the transfer pump is used to send the sample solution in the reaction vessel to the adsorber via a Teflon or silicone tube, and has a low dead space.
• One pump can be used.
• the concentrator contains a synthetic resin or glass container that contains the DNA solution eluted from the adsorbent, and
• ATI03 ⁇ 4> It is desirable to have a seal that can be sealed as soon as possible and contact the exhaust system. In order to facilitate concentration under reduced pressure, it is more convenient to provide a mechanism for rotating, revolving, precessing, or vibrating the container.
• pure water is used when the adsorbent is a cation or anion exchanger, and non-ionic porous
• pure water or a water-soluble solution of 109 & below is used.
• the series of operations of the method of the present invention ie, the inversion of the apparatus of the present invention, can be easily performed automatically by using a multiple time constant oscillator. , And by arranging four sets of reaction vessels, adsorbers, and the like in parallel, it is possible to simultaneously perform four types of base-selective cleavage of the DNA.
• the device is a simple nickel-free device that does not require expensive chillers or high-speed centrifuges as compared with conventional methods, is a device that can be automated at low cost, and uses the device of the present invention. It can also be widely applied to chemical treatment of biochemicals such as enzymatic digestion of proteins or enrichment of trace dilute tJ, for example, pretreatment for analysis of cinnamon in rivers 7j ⁇ .
• each of the four Eppendorf tubes (2 pieces, manufactured by Eppendorf) is dispensed to about 500,000 counts, and the chemical modification reaction is performed.
• the reaction stop solution was added, and pure water (1 ⁇ ) was further added, and the mixture was set in the reactor 1 of the first S apparatus.
• Activate transfer pump 4 (tube pump, 1 / min) to allow the reaction solution to pass through adsorber 2 (adsorbent set-pack C-18, manufactured by Odarts), and then use pure water. Washed in 2.
• the eluate (40% aqueous ethanol solution) is passed through the adsorber 2 using the same transfer pump 4, and the eluate is opened by opening the three-way valve 5 to the concentrator 3 toward the concentrator 3 a Housed in The concentrator 3 was heated to 90 ⁇ , and the vibrator 13, the vacuum pump 12 and the pressure-reducing control valve 14 were operated to concentrate and dry.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Saccharide Compounds (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)

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• 1983
  • 1983-03-02 JP JP58032970A patent/JPS59161398A/ja active Granted
• 1984
  • 1984-03-01 EP EP84901004A patent/EP0143849B1/en not_active Expired
  • 1984-03-01 US US06/672,253 patent/US5106585A/en not_active Expired - Fee Related
  • 1984-03-01 GB GB08427461A patent/GB2146339B/en not_active Expired
  • 1984-03-01 DE DE19843490085 patent/DE3490085C2/de not_active Expired
  • 1984-03-01 WO PCT/JP1984/000074 patent/WO1984003512A1/ja not_active Ceased

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