JP2005295869A - Method for cleaving nucleic acid molecule and apparatus therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for physically and efficiently cleaving a nucleic acid molecule in order to artificially manipulate especially a DNA or an RNA carrying genetic information. <P>SOLUTION: A hollow circular cylinder made of a metal in which at least the tip part of an outer peripheral surface and an inner peripheral surface of a capillary are coated with insulating ceramics having ≥10<SP>5</SP>Ω×m electric resistance is used as the capillary when the nucleic acid molecule is passed through the inside thereof and cleaved. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and apparatus for effectively cleaving nucleic acids roughly classified into DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). In particular, the present invention relates to an apparatus for physically and efficiently cleaving nucleic acid molecules in order to artificially manipulate DNA carrying genetic information.

  In recent years, the progress of molecular biology related to genetic information, mainly DNA, has been remarkable, and new products have been actively developed using DNA recombination technology. As shown in the schematic diagram of FIG. 1 (see, for example, the life science concept “Molecular Biology” by Haruo Koseki et al., Chemical Dojin, p. 28), the thickness is about 20 mm (2.3 nm in the figure). It is a chain polymer that is very long (for example, about 17 μm in lambda phage which is a kind of bacterial virus), but it is not easy to cleave it to a physically suitable size. Absent.

At present, such DNA cleavage is generally performed using an enzyme as described in Non-Patent Document 1, for example, but the operation is complicated and requires a long time. In order to cleave at the position, there is a problem that an appropriate enzyme must be selected.
Sambrook, J., EFFritsch, and TJManiatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  Here, when DNA can be cleaved without using an enzyme, it becomes easy to bind or recombine the target DNA. Therefore, production of a new recombinant gene or artificial synthesis of a gene control system It is expected that this will be possible, but no technology has yet been developed that fully responds to that expectation.

  Therefore, the present invention has an object to propose a method for physically and efficiently cleaving a nucleic acid molecule in order to artificially manipulate DNA or RNA carrying genetic information, together with the apparatus. .

  In view of the above-mentioned present situation, the inventors have made many trial experiments because there can be expected a dramatic progress in research in this direction if there is a technique that enables physical cleavage of DNA.

  As a result, when DNA is cut, DNA molecules can be effectively cut by adopting an instrument made of a material completely different from the conventional one, specifically, a capillary tube (injection needle) coated with insulating ceramics. The development of the method and apparatus is realized.

That is, the gist configuration of the present invention is as follows.
(1) When nucleic acid molecules are cut through the inside of a thin tube, the thin tube has an insulating property with an electrical resistance of 10 ⁵ Ω · m or more on at least the tip and the inner surface of the metal hollow cylinder. A method for cleaving nucleic acid molecules, comprising using ceramics.

(2) The method for cleaving a nucleic acid molecule according to (1), wherein the passage speed of the nucleic acid molecule in the capillary is a constant speed within a range of 0.1 to 100 mm / min.

(3) The method for cleaving a nucleic acid molecule according to (1) or (2) above, wherein the nucleic acid molecule passes through the capillary once or more.

(4) An apparatus for cutting nucleic acid molecules through the inside of a thin tube, wherein the thin tube has an electrical resistance of at least 10 ⁵ Ω · m at least on the outer peripheral surface and inner peripheral surface of a metal hollow cylinder An apparatus for cleaving nucleic acid molecules, which is coated with an insulating ceramic.

  By using the apparatus of the present invention, it is possible to effectively cleave DNA arranged in a long main chain. In addition to the field of genes, long-chain polymers (for example, sugar chains and polypeptide chains) Application to the field of basic research on physical properties and shearing force is also expected.

The present invention will be specifically described below. Although the present invention will be described with reference to DNA, the present invention is naturally applicable to RNA as well as DNA.
First, the inventors have invented a puncture needle that is completely different from the puncture needle of the current conductor metal (stainless steel: electric resistance 10 ⁶ to 10 ⁸ Ω · m), and proposed in Japanese Patent Laid-Open No. 2003-310759. did. In other words, using a high plasma atmosphere, we developed a thin-film ceramic-coated puncture needle that has excellent insulating properties (with an electrical resistance of 10 ⁹ Ω · m or more) in a high vacuum. We developed a human-friendly medical ceramic-coated puncture needle that can be used effectively during renal biopsy.

  Next, the inventors have found that when DNA is collected, DNA can be cut very efficiently by passing through a ceramic-coated thin tube (hollow needle) having an infinite electrical resistance. Patent application was filed in Japanese Patent Application No. 2004-91193.

Based on the above technical development, the present invention has further found a condition for enabling DNA cleavage with high reproducibility and capable of continuously collecting cut pieces, and has also developed an apparatus that contributes to the realization thereof. .
First, details of this apparatus will be described with reference to FIG.

FIG. 2 is an example of a vertical type schematically showing the DNA cutting apparatus of the present invention. The apparatus of the present invention can be installed in a horizontal type.
That is, reference numeral 1 in FIG. 2 is a variable-speed step rotation motor that rotates in the direction of the arrow 2, for example, and converts the rotational movement here into vertical reciprocation 4 through the column 3, for example, rubber The made piston 5 can be moved up and down in the cylinder 6. Furthermore, the base end of the thin tube 8 is connected to the bottom surface of the cylinder 6 via the support 7, and the tip of the thin tube 8 is immersed in the DNA solution 9.

  In the apparatus having the above configuration, the step rotation motor 1 is driven to raise the piston 5 on the bottom surface of the cylinder 6, whereby the DNA solution 9 is guided from the tip of the narrow tube 8 through the narrow tube 8 into the cylinder 6. When the DNA solution 9 passes through the narrow tube 8, the DNA is cut by a so-called shearing force. By passing the DNA through the capillary 8 at least once, the DNA is cut.

Next, when the piston 5 descends over the top dead center, the solution 9a containing the cut DNA is returned again to the lower DNA solution 9 tank. Subsequently, when the step rotation motor 1 is driven and the piston 5 is raised, the DNA solution 9 passes through the narrow tube 8 again from the tip of the thin tube 8, and the DNA is cut again.
By repeating the above operation once or a plurality of times, DNA cleavage is reliably achieved.

Here, at least the tip portion and the inner circumferential surface of the outer peripheral surface of the thin tube 8, that is, the portion in contact with the DNA solution 9 may be coated with insulating ceramics having an electric resistance of 10 ⁵ Ω · m or more. It is essential. This is because the influence of shearing force on the collected DNA is small in a ceramic having an electric resistance of less than 10 ⁵ Ω · m. The ceramic film does not necessarily have to be applied to the entire surface of the thin tube, and it is sufficient that at least the above-mentioned region that comes into contact with the DNA solution is used.
In the puncture needle research, when the sample extracted from the liver of an actual rat was prepared, when the resistance range of the ceramic membrane covering the puncture needle was 10 ⁵ Ω · m or more, the degree of adverse effects on surrounding cells was small. Is known.

  Specifically, as shown in FIG. 3 (a), at least a tip portion of the outer peripheral surface and an inner peripheral surface of a thin tube 10 made of a stainless steel cylinder are thinly insulated using a dry plating method. The ceramic film 11 having excellent properties is plasma coated. In particular, it is preferable to coat the ceramic film by using a magnetron sputtering method that can uniformly form a dense and excellent adhesive ceramic film.

As the ceramic film 11, for example, Al ₂ O ₃ , AlN, SiO _2, BN, or the like can be used in addition to SiNx having excellent insulating properties and preferably infinite electric resistance.

As such a ceramic film, the ceramic film of the type already disclosed by the inventors in Japanese Patent Application No. 2004-91193 can be suitably used.
That is, a ceramic thin film made of at least one selected from the group consisting of nitrides, carbides and oxides of Al, B and Si having an insulation resistance of 10 ⁵ Ω · m or more, or diamond-like carbon (DLC: Diamond, Like, and Carbon) are suitable for use.

  On the other hand, the cylindrical thin tube used in the present invention is not particularly limited as long as it is a metal material in order to form ceramics at least at the tip portion and the inner peripheral surface of the outer peripheral surface, but is preferably stainless steel. This is because the surface of stainless steel does not rust and precision processing is easy, and ferritic stainless steel is particularly suitable.

  For example, when the base of the thin tube is made of stainless steel, the outer diameter is 0.02 to 2.5 mm and the inner diameter is obtained by precision casting after continuously casting a stainless steel material and performing hot rolling, cold rolling, and bright annealing. Processed into various injection needle shapes depending on the purpose, such as 0.01 to 2.0 mm and length: 10 to 250 mm. In addition, what is necessary is just to perform the process process in this case according to a prior art.

Next, the surface of the obtained cylindrical capillary is cleaned by ultrasonic cleaning, electrolytic polishing, etc., and then the above-mentioned ceramic film is formed. As such ceramic, the electric resistance is 10 ⁵ Ω. • It is important to use an insulating ceramic of m or more.

  The coating thickness of the ceramic film is preferably 0.05 to 5.0 μm. This is because if the ceramic film thickness is less than 0.05 μm, it is difficult to ensure sufficient insulation, while if the ceramic film thickness exceeds 5.0 μm, it is difficult to ensure adhesion between the ceramic film and the matrix. This is because the coating increases the cost.

  Further, as described above, as described above, the application of the magnetron sputtering method capable of high ionization and high-speed film formation is optimal as a coating method of the ceramic film as described above, but other RF (Radio Frequency), hollow A known PVD coating method such as a cathode discharge method or an arc discharge method can also be used.

Incidentally, the preferable coating conditions of the ceramic film by the magnetron sputtering method are as follows.
For example, when a ferrosilicon target is used to perform SiNx coating, the input power is 3 to 10 kW, the degree of vacuum is 0.8 × 10 ^{4 to} 3 × 10 ³ Torr, the Ar gas flow rate is 50 to 500 sccm and N ₂ Gas flow rate: 50-500sccm is the optimum condition.

  By adopting the above apparatus configuration, a series of operations for DNA cutting can be performed efficiently, and by using this apparatus, it has become possible to realize cutting of many types of DNA with extremely high reproducibility. In addition, it is possible to cope with the cutting of more kinds of DNA by exchanging the thin tube with one of a different size.

  In addition, the apparatus shown in FIG. 2 uses a single thin tube coated with a ceramic film. However, in order to collect a larger amount of cut DNA, the ceramic film shown in FIG. It is effective to use a plurality of such tubes, for example, three as shown in FIG. 3B or seven as shown in FIG.

  Furthermore, when DNA passes through the narrow tube, the passing speed is preferably a constant speed within a range of 0.1 to 100 mm / min. This is because it has been experimentally known that the inner diameter and passage speed of the tubule greatly affect DNA cleavage, and are important conditions for obtaining a uniform and highly reproducible DNA fragment fragment.

As a DNA solution used for DNA cleavage, for example, a DNA solution having a small number of molecules (λc1857 phage DNA: 4 × 10 ⁶ molecules / ml) is suitable in order to detect DNA cleavage with high accuracy. That is, as the concentration increases, the shearing force is less likely to be applied.

DNA was cleaved using the apparatus of the present invention shown in FIG. In this apparatus, the specification of the narrow tube 8 is as follows.
Pipe dimensions: 0.83mm outer diameter, 0.58mm inner diameter and 40mm length
Tube base material: Stainless steel Ceramic film: SiN _x ceramic film Ceramic film thickness: 1.0μm thickness Ceramic film electric resistance: ∞

That is, a DNA solution having a small number of molecules [λc1857 phage DNA: 4 × 10 ⁶ molecules / ml] is used as the DNA solution 9 of the apparatus shown in FIG. 2, and the piston 5 is moved at a speed of 50 mm / min. By inhaling, the DNA solution 9 was passed through the narrow tube 8 at a passing speed of 50 mm / min to cut the DNA. Then, after extracting the solution 9a after cleaving the DNA and adding this solution to the packaging system of lambda phage to make bacterial virus particles in the test, the number of active bacterial virus DNA molecules in the solution is determined. It was measured by a plating method using a soft agar medium.
The number of viral DNA molecules extracted from the experiment at this time was 55.

  For comparison, the number of active bacterial virus DNA molecules in the solution was measured when the device was not used, that is, when DNA was cleaved with a control sample before treatment and a device using a stainless steel injection needle. In the device equipped with a stainless steel injection needle, approximately 15 times more active molecules (uncut molecules) remained as compared with the case of this device.

  Thus, the fact that the number of bacterial viruses in the solution after the cleaving treatment is reduced by the cleaving treatment by the apparatus of the present invention means that the DNA is cleaved more efficiently. The actually treated DNA molecules are analyzed by agarose gel electrophoresis to confirm that they are cleaved. Usually, DNA cleavage uses a solution having a large number of molecules, but this apparatus is characterized by being capable of efficient cleavage at a low concentration. Incidentally, when the number of molecules increases, the viscosity increases, and it works to cancel the shearing force, so that it is considered that the cutting efficiency deteriorates.

  According to the present invention, DNA can be cleaved much more easily than conventional cleaving methods using enzymes, so that medical and pharmaceutical research, development of animal and plant varieties using genetic recombination techniques, and other biochemistry. It can be widely used in fields.

It is a schematic diagram which shows the structure of DNA. It is a figure which shows the DNA cutting device. It is a figure which shows the method of changing the cutting amount in order to cut | disconnect DNA.

Explanation of symbols

1 step rotation motor 3 column 5 piston 6 cylinder 7 support 8 capillary 9 DNA solution

Claims (4)

When cutting nucleic acid molecules through the inside of a thin tube, the thin tube is coated with an insulating ceramic having an electrical resistance of 10 ⁵ Ω · m or more on at least the tip and the inner peripheral surface of the metal hollow cylinder. A method for cleaving a nucleic acid molecule, comprising using   2. The method for cleaving nucleic acid molecules according to claim 1, wherein the passing speed of the nucleic acid molecules in the tubule is a constant speed in the range of 0.1 to 100 mm / min.   3. The method for cleaving a nucleic acid molecule according to claim 1, wherein the nucleic acid molecule passes through the capillary tube at least once. An apparatus for cutting nucleic acid molecules through the inside of a capillary tube, the capillary tube having an electrical resistance of at least 10 ⁵ Ω · m or more at least on the outer peripheral surface and the inner peripheral surface of a metal hollow cylinder A nucleic acid molecule cutting apparatus characterized by being coated with ceramics.

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