JP2002345465A - Unit for purifying nucleic acid and method for purifying the nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a unit for purifying a nucleic acid, capable of separating the nucleic acid from a sample solution containing the nucleic acid by only using procedures of depressurizing and pressurizing the solution, and to provide a method for purifying the nucleic acid by using the unit. SOLUTION: The unit for purifying the nucleic acid comprises (a) a solid phase which is capable of adsorbing and desorbing the nucleic acid, (b) a container which receives the solid phase capable of adsorbing and desorbing the nucleic acid and has at least two openings, and (c) a pressure-difference generator which is connected to one of the openings of the container. The method for purifying the nucleic acid comprises using this unit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the field of molecular biology. In particular, the present invention relates to a unit for purifying nucleic acid and a method for purifying nucleic acid using the same.

[0002]

2. Description of the Related Art Nucleic acids are used in various fields and in various forms. For example, the area of recombinant nucleic acid technology requires the use of nucleic acids in the form of probes, genomic nucleic acids, and plasmid nucleic acids.

[0003] In the diagnostic field, nucleic acids are used in various ways. For example, nucleic acid probes are routinely used for the detection and diagnosis of human pathogens. Similarly, nucleic acids have been used to detect genetic disorders. Nucleic acids have also been used to detect food contaminants. In addition, nucleic acids are routinely used in locating, identifying and isolating nucleic acids of interest for a variety of reasons, from genetic mapping to cloning and recombinant expression.

[0004] In many cases, nucleic acids are available only in very small quantities, and isolation and purification procedures are complicated and time-consuming. This often time-consuming and complicated operation tends to lead to nucleic acid loss. The purification of nucleic acids from samples obtained from serum, urine and bacterial cultures also carries the risk of contamination and false positive results.

[0005] In a conventional purification method generally performed,
A centrifugation operation is included. For example, Tokuho 7-510
65, in the method disclosed in JP-T-2000-505295, etc., by contacting a nucleic acid-containing sample solution to a solid phase capable of binding nucleic acids, such as silica particles, to bind the nucleic acids to the solid phase; Next, the solid phase to which the nucleic acid is bound is separated by centrifugation.

However, the centrifugal separation operation has a significant problem. That is, the purification operation cannot be incorporated into a continuous process because of the necessity of centrifugation. Also,
Usually, since the sample is transferred, the chance of the sample being contaminated increases.

[0007] A purification method utilizing a chromatography method has also been disclosed (Japanese Patent Application Laid-Open No. 2000-9317).
1) In this method, the operation of sucking the liquid and discharging the liquid to the column is required using an independent liquid suction device, which complicates the operation.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to separate a nucleic acid from a sample solution containing a nucleic acid only by depressurizing and pressurizing operations, unlike a chromatographic method which does not require a centrifugation operation and which is complicated. An object of the present invention is to provide a nucleic acid purification unit and a method for purifying a nucleic acid using the unit.

[0009]

The object of the present invention is to provide (a)
A solid phase capable of adsorbing and desorbing nucleic acids, (b) a container containing the solid phase capable of adsorbing and desorbing nucleic acids, having at least two openings, and (c) binding to one opening of the container. Using the nucleic acid purification unit including the pressure difference generator, and the purification unit, suction and discharge operation of the sample solution containing nucleic acid by decompression and pressurization operation, and subsequent suction and discharge operation of the nucleic acid washing buffer solution The present invention has been achieved by a nucleic acid purification method including a suctioning and discharging operation of a liquid capable of desorbing a nucleic acid adsorbed on a solid phase capable of adsorbing and desorbing a nucleic acid subsequently. Here, in the present invention, the “nucleic acid” may be single-stranded or double-stranded, and there is no limitation on the molecular weight.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a solid phase capable of adsorbing and desorbing nucleic acids (hereinafter referred to as "solid phase"), silica gel, glass, alumina, zeolite, titanium dioxide, zirconium dioxide, metal oxide and / or A porous or non-porous inorganic material made of a mixed metal oxide or the like can be used. Of these, glass, particularly glass fiber filter paper, is preferred. Glass fiber filter papers include Whatman GF / A, G
F / B, GF / C, GF / D, products of Advantech, etc. can be used. At this time, the glass fiber filter paper can be used regardless of the density, retained particle diameter, thickness of glass fiber, and the like.

There is no particular limitation on the material of the container for containing the solid phase, as long as the solid phase can be stored and at least two openings can be provided, but plastic is preferred from the viewpoint of ease of production. For example, polystyrene, polymethacrylate, polyethylene, polypropylene,
It is preferable to use a transparent or opaque resin such as polyester, nylon and polycarbonate.

Hereinafter, the present invention will be described in detail by taking as an example an embodiment using glass fiber filter paper as a solid phase.

[0013] The container is usually produced in a form that is divided into a main body for accommodating the glass fiber filter paper and a lid, and each has at least one opening. One is an inlet and an outlet of a nucleic acid-containing sample solution, a nucleic acid washing buffer solution, and a liquid capable of desorbing nucleic acids adsorbed on a solid phase capable of adsorbing and desorbing nucleic acids (hereinafter referred to as “sample solution etc.”). The other is connected to a pressure difference generating device that can make the inside of the container into a reduced pressure or a pressurized state. Although the shape of the main body is not particularly limited, it is preferable to make the cross section circular in order to facilitate manufacture and facilitate the diffusion of the sample solution and the like over the entire surface of the glass fiber filter paper. It is also preferable to make the cross section square, in order not to generate cutting waste of the glass fiber filter paper.

The lid needs to be joined to the main body so that the inside of the container can be depressurized and pressurized by the pressure difference generating device. If this state can be achieved, the joining method can be arbitrarily selected. For example, use of an adhesive, screwing, fitting, screwing, fusion by ultrasonic heating and the like can be mentioned.

The inner volume of the container is determined only by the amount of the sample solution to be processed, but is usually represented by the volume of the glass fiber filter paper to be contained. That is, the thickness is about 1 mm and the diameter is about 2 m
It is preferable to have a size that accommodates about 1 to 6 glass fiber filter papers of m to 20 mm.

It is preferable that the end face of the glass fiber filter paper is brought into close contact with the inner wall surface of the container to such an extent that a sample solution or the like does not pass.

A space is provided below the glass fiber filter paper opposite to the opening used for the entrance of the sample solution or the like without making close contact with the inner wall of the container, and the sample solution or the like diffuses as uniformly as possible over the entire surface of the glass fiber filter paper. Make structure.

Preferably, a member having a substantially central hole is provided on the glass fiber filter paper facing the other opening, that is, the opening connected to the pressure difference generator. This member has the effect of holding down the glass fiber filter paper and efficiently discharging the sample solution, etc., and has a funnel-shaped or bowl-shaped shape with a slope such that the liquid collects in the central hole. Is preferred. Those skilled in the art can appropriately determine the size of the hole, the angle of the slope, and the thickness of the member in consideration of the amount of the sample solution or the like to be processed, the size of the container accommodating the glass fiber filter paper, and the like. It is preferable to provide a space between the member and the opening to store the overflowed sample solution and the like and prevent the sample solution from being sucked into the pressure difference generator. The size of this space can also be appropriately selected by those skilled in the art. In order to efficiently collect nucleic acids, it is preferable to aspirate a sample solution containing nucleic acids in an amount larger than that in which the entire glass fiber filter paper is soaked.

Further, in order to prevent the sample solution and the like from concentrating only on the portion directly below the opening to be sucked and to allow the sample solution and the like to pass through the glass fiber filter paper relatively uniformly, the glass fiber filter paper is used. It is preferable to provide a space between this member and this member. For this purpose, it is preferable to provide a plurality of protrusions from the member toward the glass fiber filter paper. The size and number of the projections can be appropriately selected by those skilled in the art, but it is preferable to keep the opening area of the glass fiber filter paper as large as possible while maintaining the space.

When three or more openings are provided in the container, it is necessary to temporarily close off the extra openings in order to enable the suction and discharge of the liquid accompanying the decompression and pressurization operations. Needless to say.

In the pressure difference generator, first, the pressure in the container containing the glass fiber filter paper is reduced, and the sample solution containing nucleic acids is sucked. Examples of the pressure difference generator include a pump capable of suction and pressurization such as a syringe, a pipettor, or a peristaltic pump. Of these, a syringe is suitable for manual operation, and a pump is suitable for automatic operation. Further, the pipetter has an advantage that one-handed operation can be easily performed.

Next, using the nucleic acid purification unit described above,
A method for purifying nucleic acids will be described. There is no limitation on the sample solution containing the nucleic acid that can be used in the present invention.For example, in the diagnostic field, a solution prepared from a body fluid such as whole blood, plasma, serum, urine, stool, semen, or saliva collected as a specimen Is the control.

First, these specimens are treated with a nucleic acid lysis buffer solution. The nucleic acid lysis buffer used in the present invention is an aqueous solution containing a main agent, a buffer, a surfactant, if necessary, and a reagent for dissolving a cell membrane such as protease K. As the main agent, guanidine chloride, guanidine isothiocyanate, guanidine thiocyanate, sodium perchlorate, sodium iodide, and the like can be used.
s, EDTA is Triton-X as a surfactant.
100 etc. can be used. Of these, guanidine chloride and Tris are preferred. The preferred concentration of the buffer is 1
0-100 mM, the preferred concentration of the surfactant is 0.1-
10%.

One opening of the nucleic acid purification unit according to the present invention is inserted into the thus prepared sample solution containing the nucleic acid. Next, the pressure inside the purification unit is reduced using a pressure difference generator connected to another opening, and the sample solution is sucked into the container. By this operation, the sample solution comes into contact with the solid phase, and the nucleic acid in the sample solution is adsorbed on the solid phase. At this time, it is preferable to aspirate an amount of the sample solution that comes into contact with almost the entire solid phase. However, if aspirated into the pressure difference generating device, the device will be contaminated.

After suctioning an appropriate amount of the sample solution, the inside of the container of the unit is pressurized using a pressure difference generator, and the sucked liquid is discharged. It is not necessary to provide an interval before this operation, and the discharge may be performed immediately after the suction.

Next, the nucleic acid washing buffer solution is sucked into the container by the same decompression-pressure operation as described above, and is discharged from the container to wash the inside of the container. This solution has a function of washing away the sample solution remaining in the container and also washing away impurities in the sample solution adsorbed on the glass fiber filter paper together with the nucleic acid.
Therefore, it is necessary to have a composition that does not desorb nucleic acids from glass fiber filter paper but desorb impurities. The nucleic acid washing buffer solution is composed of an aqueous solution containing a main agent, a buffer, and, if necessary, a surfactant. The main agent is about 10 to 90% (preferably about 50 to 90%) of methyl alcohol, ethyl alcohol, butyl alcohol, acetone or the like. (90%) aqueous solution as a buffer and a surfactant include the buffers and surfactants described above. Among them, a solution containing ethyl alcohol, Tris and Triton-X100 is preferable. Tris and Triton-X100
Are preferably 10 to 100 mM and 0.1 to 10%, respectively.

Next, a solution capable of desorbing the nucleic acid adsorbed on the glass fiber filter paper is introduced into the container by the same decompression-pressure operation as described above, and discharged from the container. Since the effluent contains the target nucleic acid, it is collected and provided for subsequent operations, for example, amplification of the nucleic acid by PCR (polymerase chain reaction).

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

[0029]

EXAMPLE 1 Nucleic acid purification unit FIG. 1 is a sectional view of a nucleic acid purification unit according to the present invention. However, the pressure difference generator is not shown. The container 1 containing the solid phase is composed of a main body 10 and a lid 20, and is made of transparent polystyrene. The main body 10 houses six GF / D (manufactured by Whatman) as glass fiber filter paper 30. Further, it has an opening 101 for sucking a sample solution or the like. The bottom surface 102 continuing from the opening is formed in a funnel shape, and a space 121 is provided between the bottom surface 102 and the glass fiber filter paper 30. In order to support the glass fiber filter paper 30 and keep the space 121, a frame 103 integrated with the bottom surface 102 is provided.

The main body has an inner diameter of 20.1 mm and a depth of 5.
9 mm, the length from the bottom surface 102 to the opening 101 is about 70
mm. Also, the built-in glass fiber filter paper 30
Has a diameter of 20.0 mm and a thickness of 0.91 mm.

In FIG. 1, a funnel-shaped holding member 13 is provided above the glass fiber filter paper. A hole 131 is provided at the center of the holding member 13, and a group of projections 132 is provided below the holding member 13.
22 are provided. Glass fiber filter paper 30 and body 10
Wall 1 so that the sample solution or the like is unlikely to leak from between the walls 104.
The diameter of the upper part of 04 is made larger than the diameter of the glass fiber filter paper, and the end of the pressing member 13 rides on the step 105.

The lid 20 is joined to the main body 10 by ultrasonic heating. At approximately the center of the lid 20, an opening 21 for connecting the pressure difference generator is provided. A space 123 for holding a sample solution or the like flowing out of the hole 131 is provided between the lid 20 and the pressing member 13. The volume of the space 123 is about 0.1 ml.

Example 2 Purification of Nucleic Acid Using the Nucleic Acid Purification Unit of Example 1 2 ml of human whole blood was collected using a commercially available blood collection tube. A nucleic acid lysis buffer solution 2 having the composition shown in Table 1 was added to the whole blood.
ml was added and incubated at 60 ° C. for 10 minutes.

Table 1 Composition of nucleic acid lysis buffer solution Guanidine chloride (manufactured by Life Technology) 382 g Tris (manufactured by Life Technology) 12.1 g Triton-X100 (manufactured by ICN) 10 g Protease K 20 μl Double distilled water 1000 ml

After the incubation, the opening 101 of the nucleic acid purification unit described in Example 1 was inserted into the solution, and the opening 21 was inserted.
Was connected to a Terumo 5 ml syringe (not shown) and aspirated. At this time, there is no need to limit the suction speed,
The liquid was visually adjusted so that it did not flow into the syringe. Then, the liquid was discharged by pressurizing using a syringe still connected to the opening 21. As described later, the nucleic acid was adsorbed on the glass fiber filter paper by this operation.

Immediately after the discharge, the opening 101 was inserted into the nucleic acid washing buffer solution having the composition shown in Table 2, and the same vacuum-
The inside of the unit was washed by a pressurizing operation.

Table 2 Composition of nucleic acid washing buffer solution 10 mM Tris-HCl 70% ethanol

This washing is an operation for washing the residue of the sample solution containing the impurities adsorbed on the glass fiber filter paper and the nucleic acid in the container, and has no effect on the nucleic acid adsorbed on the glass fiber filter paper. .

Immediately after the discharge of the washing liquid, the same suction-discharge operation was performed using 2 ml of purified distilled water, and the discharged liquid was collected. By this operation, the nucleic acid adsorbed on the glass fiber filter paper was desorbed into the purified distilled water and collected.

Example 3 Quantification of Recovered Amount of Nucleic Acid By measuring the absorbance of the solution recovered in Example 2,
The amount and purity of the nucleic acid contained in the solution were quantified. According to a method known to those skilled in the art, the amount of nucleic acid was quantified by the absorbance at a wavelength of 260 nm, and the purity was determined from the ratio of the absorbance at a wavelength of 260 nm and 280 nm. If the ratio is 1.8 or more, the purity is judged to be good.
Table 3 shows the measurement results.

Table 3 Measurement of Absorbance Sample Number Nucleic Acid Amount Absorbance Ratio (260 nm / 280 nm) 1 121 μg 1.865 2 108 μg 1.9323 3 (Buffy coat) 291 μg 1.899

Example 4 Amplification by PCR Using the solution recovered in Example 2, amplification of nucleic acid by PCR was performed. First, a liquid having the composition shown in Table 4 was prepared.

Table 4 Liquid composition Recovered liquid 2 μl Purified water 36.5 μl PCR buffer 10-fold dilution 5 μl 2.5 mM dNTP 4 μl Taq FP 0.5 μl Primer 2 μl (designed to amplify P53 exon 6)

After denaturing this solution at 94 ° C. for 30 seconds,
Annealing was performed at 5 ° C. for 30 seconds, and an extension reaction was performed at 72 ° C. for 1 minute. This step was repeated 30 times. Using the thus obtained solution, Human DNA manufactured by Clontech as a positive control, and purified water as a negative control, electrophoresis was carried out on a 1.5% agarose gel for 1 hour according to a standard method. The results are shown in FIG. In FIG. 2, the top row shows the results of the marker, the second row shows the results of the positive control, the third row shows the results of the electrophoresis of Sample 1, and the fourth row shows the results of the electrophoresis of Sample 2. The bottom row where nothing appears is the result of the negative control.

From the results of Examples 3 and 4, it can be seen that the recovered solution obtained in Example 2 contained a practical amount and purity of nucleic acid.

[0046]

According to the nucleic acid purification unit and the nucleic acid purification method of the present invention, a practical amount and purity of nucleic acid can be obtained from blood or the like by a simple operation of suctioning and discharging a sample solution or the like utilizing a pressure difference. it can. The method of the invention has the advantage, in particular, that it can be easily integrated during a continuous process.

[Brief description of the drawings]

FIG. 1 is an example of a nucleic acid purification unit according to the present invention. However, the pressure difference generator to be connected to the opening 21 is not shown.

FIG. 2 shows the nucleic acid recovered by the nucleic acid purification method of the present invention using the nucleic acid purification unit of the present invention, and
It is the result of electrophoresis of the sample amplified by R.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Container 10 ... Body 101 ... Opening 102 ... Bottom 103 ... Frame 104 ... Wall 105 ... Step 121 ... Space 122 ... Space 123 ... Space 13 ... Pressing member 131 ... Hole 132 ... Projection 20 ... Lid 21 ... Opening 30 ... Glass fiber filter paper

Claims (7)

[Claims] 1. A solid phase capable of adsorbing and desorbing a nucleic acid, (b) a container having at least two openings for containing a solid phase capable of adsorbing and desorbing the nucleic acid, and (c) A nucleic acid purification unit comprising: a pressure difference generator coupled to one opening of the container. 2. The nucleic acid purification unit according to claim 1, wherein the solid phase capable of adsorbing and desorbing the nucleic acid is a glass fiber filter paper. 3. The nucleic acid purification unit according to claim 1, wherein the pressure difference generator is detachably connected to one opening of the container. 4. The nucleic acid purification unit according to claim 1, wherein the pressure difference generator is a syringe. 5. The nucleic acid purification unit according to claim 1, wherein the pressure difference generator is a pipettor. 6. The nucleic acid purification unit according to claim 1, wherein the pressure difference generator is a peristaltic pump. 7. A nucleic acid purification method comprising the following steps using the nucleic acid purification unit according to claim 1. (a) inserting one opening of the nucleic acid purification unit into a sample solution containing a nucleic acid, (b) using the pressure difference generator coupled to the other opening of the nucleic acid purification unit to evacuate the inside of the container. A step of aspirating the sample solution containing the nucleic acid under reduced pressure and bringing the sample solution into contact with a solid phase capable of adsorbing and desorbing the nucleic acid, (c) the pressure difference generator coupled to another opening of the nucleic acid purification unit To the pressurized state in the container using,
Discharging the sample solution containing the sucked nucleic acid out of the container, (d) inserting one opening of the nucleic acid purification unit into a nucleic acid washing buffer solution, and (e) another one of the nucleic acid purification unit. (F) the step of aspirating the nucleic acid washing buffer solution while bringing the inside of the container into a reduced pressure state using the pressure difference generator coupled to an opening, and bringing the nucleic acid washing buffer solution into contact with a solid phase capable of adsorbing and desorbing the nucleic acid; The container is pressurized using the pressure difference generator coupled to another opening of the nucleic acid purification unit,
Discharging the sucked nucleic acid washing buffer solution out of the container; (g) allowing one of the openings of the nucleic acid purification unit to desorb the nucleic acid adsorbed on a solid phase capable of adsorbing and desorbing the nucleic acid. (H) using a pressure difference generator coupled to the other opening of the nucleic acid purification unit to reduce the pressure in the vessel to allow adsorption and desorption of the nucleic acid Suctioning a liquid capable of desorbing the nucleic acid adsorbed on the nucleic acid, and bringing the liquid into contact with a solid phase capable of adsorbing and desorbing the nucleic acid; and (i) generating the pressure difference coupled to another opening of the nucleic acid purification unit. Pressurizing the inside of the container using a device,
Discharging the liquid capable of desorbing the nucleic acid adsorbed on the solid phase capable of adsorbing and desorbing the sucked nucleic acid to the outside of the container.