JP2010233579A - Separating and purification mechanism for dna or the like - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for highly efficiently isolating and refining a nucleic acid, especially a fragment DNA in high reproducibility without elution with a highly concentrated salt to obtain a high-purity fragment by simple mechanism and technique with no need of elution refining. <P>SOLUTION: There is provided the mechanism, which aims at purifying a nucleic acid, in particular a fragmental DNA with the use of a monolith structure, wherein the monolith structure is made of glass or silica, namely, a porous integral structure and having small pores penetrating thorough from the top to the bottom and the penetrating pores being in a size fitting for the nucleic acid size of from 35 bp (mer) to 100 Kbp (mer). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a mechanism for separating and purifying DNA and the like.

  Until now, it is well known that nucleic acid mixture is adsorbed to glass and silica gel particles, glass, quartz wool, silica, glass membrane, polymer, etc. in the presence of chaotropic salts. It has been. Biological raw materials such as cultured cells / tissues, body fluids such as blood / serum / urine / feces, bacteria such as bacteria / human tuberculosis, viruses such as HIV / hepatitis B / hepatitis C, etc. The plasmid DNA, genomic DNA, chromosomal DNA, RNA, mitochondrial DNA, fragment DNA, etc. can be separated and purified.

Fragment DNA purification is a very frequently used technique in molecular biological research, and is performed prior to applications such as PCR, cloning, sequencing, restriction enzyme digestion, and other enzymatic actions.
For example, there is a method for isolating DNA from recombinant M13 phage. An isolation method in which M13 phage DNA is bound by addition of a chaotropic substance on a glass fiber filter, followed by separation, washing and drying is known as Nucleic Acids Research Vol. .15 5507-5516 (1987) (Non-Patent Document 1).

  A method of adding glass powder to bind DNA to glass powder, centrifuging, collecting the glass powder, washing, eluting and isolating is Pvoc. Natl. Acad. Sci. USA Vol. 76, 615-619. (1979) (Non-Patent Document 2). Similar methods are disclosed in JP-A-59-227744 (Patent Document 1), Analytical Biochemistry Vol. 121.382-387. (1982) (Non-Patent Document 3), Molecular cloning: A Laboratory Manual 188-190. 1982) (Non-Patent Document 4) and the like.

Patent No. 2680462 is a method in which a nucleic acid-binding solid phase containing a complex biological starting material, a chaotropic substance and silica or a derivative thereof is mixed, the solid phase to which the nucleic acid is bound is separated from the liquid, washed, and nucleic acid is eluted Proposed in the Gazette.
Although the physical mechanism for adsorbing DNA and RNA in the presence of a chaotropic reagent is not clarified in detail, it is considered that a cation exchange reaction occurs between a negatively charged carrier and a nucleic acid. Therefore, the efficiency of purification can be considered as equal to the efficiency of contact between the carrier surface and the biological sample.

  Regardless of which carrier is used, the carrier to be adsorbed is held in a container (cartridge, chip, etc.), the biological sample is passed through the container, the nucleic acid is adsorbed to the carrier with the adsorption buffer solution, and then the nucleic acid is washed with the washing solution. A general procedure is to expel contaminants other than the components out of the cartridge, and then pass the eluate to take out the nucleic acid components together with the solution.

  In addition, fragment DNA is often purified from agarose gels by electrophoresis or various extractions, but this method is time consuming and the resulting DNA is extremely dilute and contains salts and organic solvents. Therefore, it is necessary to further desalinate or concentrate by ethanol precipitation. In addition, it is very difficult to separate molecules having similar molecular weights by conventional methods such as gel filtration purification technology.

  Since these separation methods using a carrier require the use of a high concentration of salts, a phenomenon causing degradation or denaturation of DNA on the particle surface of glass or silica gel has been confirmed. In the presence of a high concentration of chaotropic salt, this adsorption occurs approximately quantitatively, but the adsorbed DNA is eluted in the presence of a salt buffer. The processing of DNA fragments is limited to a range of 100 base pairs (bp) to 10,000 base pairs (bp), DNA fragments of 100 base pairs (bp) or less, or 100 base pairs (bp) or more of 100, It was impossible to perform quantitative separation and purification of DNA up to 000 base pairs (bp).

  In the preparation method based on glass powder as a carrier, it is conceivable that the beads are made smaller and the amount is increased in order to increase the contact efficiency between the surface and the nucleic acid. However, the pressure at the time of passing the liquid rises, and the operability is remarkably lowered, the gap between beads is reduced, and the nucleic acid molecule has a high molecular weight, so that the problem that the efficiency cannot be reduced can not enter the gap. . Further, if the length of the container is increased for the purpose of improving the separation, the amount of the eluting solvent is increased in addition to the increase in pressure, so that the concentration efficiency is lowered, and it is far from simple processing. In addition, if beads or wool is used, the fragments and particles enter the eluate in a small amount, which causes a problem for later applications. In addition, if the filling method when filling the container is not constant, the separation time and pattern change, which causes the problem of poor separation stability.

The method of using a membrane or a filter as a carrier has an advantage that it can be easily processed, but it is difficult to produce by controlling the pores suitable for separation, and is not practical.
In addition, the method using a polymer is not simple enough to react specifically with nucleic acids depending on the nature of the polymer, and there are parts that affect other parts, making the separation system complicated and simple. Purification with a simple protocol is not possible.
In any case, it is impossible to purify highly pure fragment DNA. In addition, there are drawbacks such as difficulty in handling the powdered silica resin and suspension and the possibility of hindering subsequent application reactions.

As in the invention of Patent Document 2, the concept of directly isolating nucleic acid from a complicated starting material without pretreatment involves so-called “digestion” and “purification” simultaneously. For this reason, extreme reaction conditions are required, and there is a negative effect that the molecular weight range of the applicable nucleic acid is narrowed.
A nucleic acid mixture is adsorbed on a porous, non-porous inorganic substrate such as silica gel and glass from an aqueous solution containing a high concentration of salt (ionic strength) and containing an organic acid such as aliphatic alcohol or polyethylene glycol, A method for obtaining a nucleic acid by washing and then elution with a solution containing a lower concentration of salt (ionic strength) has been proposed in Japanese Patent Publication No. 8-5011321 (Patent Document 2).

  However, in this method, a nucleic acid mixture is adsorbed on an inorganic substance from an adsorption aqueous solution containing a high concentration salt, and the nucleic acid is eluted with a solution containing a salt at a low concentration. Is contained in a large agarose piece, it is necessary to process using multiple columns, and the eluted fragment DNA is pooled, and the resulting fragment DNA contains salts and organic solvents. An operation step such as desalting is required, and the purified DNA may be lost during ethanol precipitation.

JP 59-227744 A JP-T-8-5011321

Nucleic Acids Research Vol.15 5507-5516 (1987) Pvoc. Natl. Acad. Sci. USA Vol. 76, 615-619. (1979) Analytical Biochemistry Vol.121.382-387. (1982) Molecular cloning: A Laboratory Manual 188-190. (1982)

  Therefore, in the present invention, the above-mentioned points of the prior art are improved, adsorption and elution separation can be performed very easily and easily, elution with a high concentration of salt is not performed, elution purification is not required, and nucleic acid can be used. I would like to propose a highly reproducible method in which the separation and purification of medium-fragment DNA etc. is remarkably efficient.

  An integrated Molinos structure having a continuous through hole from one end to the other end and a through hole corresponding to a nucleic acid size of 35 bp (mer) to 100 Kbp (mer). The monolith structure is an inorganic substance such as glass or silica, or a hybrid containing an organic substance in an inorganic substance, characterized in that each of the nucleic acids corresponding to the through-holes can be retained by passing the contained solution. The porous body having macropores penetrating from the upper surface to the lower surface is used, and the porous body of the monolith structure is characterized by having micropores inside the macropores. The porous body of the monolith structure is characterized by having 1 to 100 μm macropores and 0 to 100 nm micropores, and is formed by a monolith structure on a column tube. A monolithic solid-phase column is formed by arranging a disk, and a monolithic solid-phase column formed by detachably mounting a substrate composed of a monolithic structure on a cylindrical body that is open at the top and bottom It is characterized by doing.

  According to the present invention, it is possible to quantitatively separate and efficiently purify a DNA fragment of 100 bp (mer) or less and a wide range of DNA fragments from 10000 bp (mer) to 100 Kbp (mer). Nucleic acid can be adsorbed and separated without using sensitive reaction conditions, so there is no need for dissolution or adsorption with high-concentration salt, no need for concentration and desalting operations, and DNA fragment purification, especially during DNA purification, is easy. It is. Further, it can be eluted with a salt-free solution or sterile water, and a high-purity fragment DNA can be easily obtained.

Comparison diagram of the first embodiment according to the present invention with the conventional method Comparison diagram of the first embodiment according to the present invention with the conventional method Evaluation chart by HPLC of Example 1 of the present invention Example of DNA fragment purification separation diagram of Example 2 according to the present invention Example of DNA fragment purification separation diagram of Example 4 according to the present invention Example of DNA fragment purification separation of Example 5 according to the present invention Separation diagram of single-stranded PCR amplification product of Example 6 according to the present invention Comparison of purification by sodium and potassium of Example 7 according to the present invention Example of the present invention Column tube explanation slope view Same as above Same as collection tube Same as above Monolith solid phase column

  The present inventors have found that when an efficient monolith structure is used for nucleic acid purification and the buffer conditions are adjusted, it is not necessary to use a buffer with a high salt concentration, which has been common knowledge so far. In addition to the EDTA tris hydrochloride added for nucleic acid preservation, water is used alone, and the binding is released from the adsorbed carrier and eluted. When adsorbing nucleic acid, an adsorption solvent is isopropanol that acts as a separation solvent and a salt compound that supplies ions thought to react with a silanol group, and guanidine hydrochloride that is a chaotropic salt for dissolving an agarose gel. At this time, if an alkali metal salt that easily becomes a cation is present, it is considered that a dehydrogenation reaction easily occurs, whereby the cation causes a cation crosslinking reaction with the phosphate portion of the nucleic acid and adsorbs the nucleic acid. Examples of the alkali metal include lithium, sodium, potassium, rubidium, cesium, and francium. Among them, potassium salts that have a low electronegativity and easily become cations and undergo a cation crosslinking reaction are likely to react, and finally elute together with nucleic acid components in an ionic state, but are useful because they do not interfere with subsequent applications. It is. In addition, since sodium salt interferes with subsequent applications, it cannot be used unless desalting is performed, and is not very useful. In other words, with this apparatus and method, there is no need for desalting operation or alcohol precipitation, and the resulting purified solution can be directly used for subsequent operations (PCR, cloning, sequencing, enzymatic operation). become. Simplification of the operation procedure is a very important matter for preventing nucleic acid damage.

  The object of the present invention is to purify nucleic acids by using a monolith structure silica or glass with high purification efficiency and using a simpler buffer without affecting subsequent application operations such as cloning. It is to provide a method that can.

The monolith structure refers to an integrated porous body having open-structured macropores communicating from the upper end to the lower end, and many macropores having further micropores are synthesized.
The monolith structure can be mainly produced by a sol-gel method. That is, a metal alkoxide is partially hydrolyzed to produce a reactive monomer, and this monomer is polycondensed to form a colloidal oligomer (formation of a sol), and further hydrolyzed to promote polymerization and cross-linking, thereby providing a three-dimensional It is synthesized by creating a structure (generation of gel).

  When various organic monomers are added during this reaction, an organic / inorganic hybrid monolith can be easily prepared. Therefore, chemical characteristics can be added depending on the type of organic monomer to be added. For example, the water absorption of the aqueous sample component can be improved by adding an organic monomer having a hydrophilic group. In addition, by adding organic monomers with functional groups that exhibit selective chemical action, it can be used for adsorption of characteristic components that become impurities in purification, and the impurities remain in the solid phase, thereby increasing the efficiency of nucleic acid purification. Will be able to. In addition, by adding a polymer having a high elastic modulus during the sol-gel process, the monolith structure can be made elastic. Furthermore, basically, it is possible to increase the chemical stability of the monolith structure by mixing organic and inorganic substances.

  This means that the organic / inorganic hybrid monolith structure produced by the sol-gel method constitutes a chemical surface according to the purpose depending on the kind of the added organic monomer, and improves the chemical stability. This means that a property can be added, and the monolith properties effective in DNA pretreatment can be freely improved depending on the purpose. Furthermore, the monolith prepared from the sol-gel method is suitable in that the metal content is small as the solid phase for DNA of the present invention. General silica gel or the like is made from sodium silicate or the like, and a large amount of metal remains. Certainly, some are also made from purified sodium silicate and high-purity silica gels from sol-gel methods, but they are expensive and not suitable for the single use disposable. Even if it can be made inexpensively, it is batch-type synthesis at the time of particle preparation, and there is a high possibility that metal concentration will occur from the synthesis atmosphere.

  In the synthesis of the monolith structure in the sol-gel method, it is prepared by a continuous process, and there is no metal contamination. Conventionally, silica gel has been devised such as washing with hydrochloric acid or nitric acid or the like, and EDTA has been added to reduce the influence of metal, but the solid phase of the present invention is not necessary at all.

  Alternatively, a monolithic solid phase can be created by glass phase separation. Basically, it has the same effectiveness as the synthesis of the monolith structure from the sol-gel method, but it can make macropores larger than the sol-gel monolith, so it is effective when making secondary micropores on the inner surface. It is. Further, the glass phase separation has the advantage that it has higher alkali resistance than its composition and can be regenerated by alkali cleaning.

Hereinafter, the present invention will be described in detail with reference to the embodiments shown in the drawings.
In the present invention, the most basic constitution is to purify a fragment DNA from agarose gel, PCR reaction product, restriction enzyme-treated DNA, single-stranded DNA, RNA, etc., in the presence of chaotropic salt in glass or silica. Adsorption, especially monolithic structure formed of glass or silica with excellent separation ability, that is, an integrated porous body having an open structure with pores communicating from the upper end to the lower end. There is to make it.
Adsorption of fragment DNA on glass or silica in the presence of chaotropic salt has been proposed and practiced as described above.

  However, all conventional methods used fillers. For this reason, there is a variation in the filling of the filler, it is not uniform, the particles remain in the DNA solution after passing through the filler, the contact area with the solution is small and the reaction efficiency is poor, the liquid passing pressure However, there are still some drawbacks such as being difficult to handle.

In the present invention, in the presence of a chaotropic salt, a part adsorbed on glass or silica gel particles is extracted from an agarose gel or PCR reaction solution, and there is a part in common with the conventional technique.
This conventional technique quantitatively causes adsorption in the presence of a high concentration of chaotropic salt, and elution of the adsorbed nucleic acid is performed at a lower salt concentration.

  However, the method disclosed in JP-A-8-5011321 is a method in which a nucleic acid is fractionated in one operation step without a preliminary purification step. This necessitates extreme reaction conditions with a high-concentration salt buffer, narrowing the applicable molecular weight range of nucleic acids. Furthermore, since purified DNA is extremely dilute and contains salts and organic solvents, it is necessary to further precipitate ethanol and concentrate it.

The present invention improves these disadvantages, and does not adsorb or elute with a high concentration of salt, but elutes with water. This results in a very high concentration sample that does not require further purification.
One object of the present invention is to purify a DNA fragment from an agarose gel, a PCR amplification reaction DNA solution, a fragment DNA from an enzyme reaction solution, and the like.

  The present invention can extract and purify a DNA fragment of 35 bp to 100 Kbp from a standard agarose gel or a low boiling point agarose gel prepared with Tris acetic acid (TAE) buffer or Tris boric acid (TBE) buffer, and recover 60 to 80%. You can get a rate. In addition to extraction from a gel, a PCR product of 35 bp to 100 kbp can be directly purified from a PCR amplification reaction solution, and a recovery rate of 80 to 95% can be obtained. Since the obtained fragment DNA does not contain salt or organic solvent, there is no need for ethanol precipitation, desalting or concentration. This product is a monolith-based system, with a maximum DNA binding capacity of 5-8 μg and the isolated fragment DNA can be recovered in as little as 5 minutes.

  In the case of purification from gel, the target DNA band is cut out from the gel after electrophoresis and dissolved in the presence of guanidine thiocyanic acid (dissolution, adsorption buffer). In the case of purification after PCR amplification, an adsorption buffer is added directly to the amplification reaction solution. The dissolved gel piece is passed through a monolith solid phase column using a microcentrifuge or a suction device. At that time, the target fragment DNA binds to the surface of the silica monolith or glass monolith, the bound fragment DNA fragment is washed with a washing buffer, and then the DNA is eluted with water (elution buffer).

The following is used as this buffer.
A buffer (dissolution, adsorption buffer)
B buffer (washing buffer)
C buffer (elution buffer or sterile water)
This will be described in more detail. A1 buffer for PCR reaction solution (guanidine hydrochloride 1-8M, potassium acetate 0.1-1M, 2-propanol 1-70%)
A2 buffer for agarose gel (guanidine thiocyanic acid 1-8M, potassium acetate 0.1-1M, 2-propanol 1-70%)
B buffer (potassium acetate less than 0.1-1M, ethanol 1-80%)
C buffer (pH = 8 to 8.5 Tris-HCI 10 mM, EDTA 1 mM, or sterile DNA, RNA free water)

In the A1 buffer, the guanidine hydrochloride is preferably guanidine thiocyanic acid, more preferably 1 to 8M. As for 2-propanol, 40% or more is efficient.
The above conditions also apply to the A2 buffer.
In elution buffer C, elution with water is possible, but in order to prevent contamination by germs, use RNase-free water that has been treated with ultrafiltration membrane or diethyl pyrocarnate. Is recommended. In addition, when the purified DNA is stored, in order to prevent contamination with bacteria, water to which EDTA buffer has been previously added may be used as the elution buffer solution C. Even when viewed from the separation mechanism, the purification efficiency does not change with or without the EDTA buffer.

  In the present invention, by using a monolith structure for the substrate, the reaction between the phosphate group and the silanol group of the nucleic acid can proceed efficiently. In addition, it is possible to prevent a carry-over problem of silica that may be hindered in so-called application reaction because the silica particles are purified while adhering to a sample that is inevitable when a granular filler is used. Furthermore, the pressure at the time of separation is kept low, and unlike the case where a filler is used, there is an advantage that a sealant is unnecessary.

  This monolith structure can be produced, for example, by a so-called sol-gel method in which an inorganic porous material such as silica gel is purified from a low molecular weight compound sol that can be polymerized to finally obtain an aggregate or polymer gel. . This method generally has a central pore diameter of 1 to 100 μm, but several nanometers are possible due to subsequent technological advances.

In addition, a monolith structure having pores in a silica skeleton having two types of pores can be prepared by utilizing spinodal decomposition in a sol-gel process.
In addition, a porous body having a structure in which a porous body having micropores having an open structure is filled inside macropores penetrating from the upper surface to the lower surface can be produced. Incidentally, this porous body has a macropore diameter of 1 to 100 μm and a micropore of 0 to 100 nm.

In addition, as long as it is formed by containing glass and silica, it is needless to say that an appropriate monolith structure can be used.
In this monolith structure, the through-holes of the porous body can be formed as desired depending on the manufacturing method thereof, so that the monolith structure is appropriately selected and formed. However, this selection is determined by the raw material used, for example, agarose gel, PCR reaction solution, buffer, and the purpose.

  A use form of the base body constituted by this monolith structure will be described. A cylindrical column tube 1 is formed, and a hermetically sealed lid 3 is detachably provided on the upper end opening portion 2. A narrow-diameter outlet 4 is formed at the lower end, and an intermediate-diameter portion 6 is formed by providing a step 5 at the top of the outlet 4. A disk 7 as a base formed of a monolith structure can be placed or fitted to the step 5. The disk 7 may have a disk shape having a desired thickness, for example, about 0.1 to 10 mm, or a conical shape if desired. The column tube 1 and the disk 7 constitute a monolith solid phase column 9. Reference numeral 8 denotes a collection tube having a diameter that allows the column tube 1 to be inserted, and an upper end edge of the column tube 1 is configured to be locked at an upper portion.

The column tube 1 and the collection tube 8 are made of polypropylene, but organic polymers that do not affect nucleic acids such as polyethylene, polyethylene terephthalate, polystyrene and inorganic compounds such as glass and silica have good visibility and have some strength. Can be used if there is.
Further, although the disk 7 is mentioned as the substrate of the monolith structure, the present invention is not limited to this, and it can be used in a mold that allows the solution to pass freely, such as a dish or cylinder.

  The molecular size of DNA is said to be about 3.4 nm per 10 base pairs. For example, in a DNA of 35 bp to 300 bp, a macropore of about 10 nm to 100 n, in a DNA of 300 bp to 3 Kbp, a macropore of about 100 nm to 1 μm, and in a DNA of 30 Kbp to 300 Kbp, about 1 μm to 10 μm In the case of DNA having a macropore of 30 Kbp to 300 Kbp, it is considered that if the macropore has a size of about 10 μm to 100 μm, the DNA molecule can pass through without being damaged or broken. .

  Further, in order to enhance the interaction with impurities or the like and to purify more efficiently, addition of micropores is performed. From experience of various tests and experiments, it has been confirmed that if the micropores are around several tens of nanometers, the compound has a molecular weight of 100,000, and if it is around 10 nm, it interacts with a compound of several hundred to several tens of thousands of molecular weight. In addition, it has been found that a compound having a higher molecular weight has a good number of micro pores that are said to promote the destruction of the molecule, that is, 0 nm is preferable.

  Several types of monolithic monolith structures having micropores added to macropores can be prepared and used for different purposes such as DNA types and impurity removal. Incidentally, in order to create several types of monolithic monolithic structures, a method of creating macropores in advance and then forming micropores is more convenient for synthesis.

In the purification currently required, separation and purification from low molecules such as primers and agarose gel degradation products are the main, and even a single monolith structure can be sufficiently separated.
If a monolith structure having macropores of 1 to 100 μm, preferably about 20 μm and micropores of 0 to 100 nm, preferably about 10 nm, is used, 35 bp (mer) to 100 Kbp (as in the examples) mer) a wide range of DNA can be sufficiently purified.

A. There are the following methods for generating a PCR product using a monolith solid phase column.
(1) Microcentrifugation This protocol is designed for the purpose of purifying double-stranded DNA fragments from PCR reaction solutions. If one monolith solid phase column and a purification buffer are used, a fragment of 35 bp to 100 Kbp can be obtained by centrifugation. Can be separated from primers, nucleotides, polymerases, salts and the like.

100 μl of buffer A1 (adsorption buffer) is added to 10 μl of the PCR reaction solution. Wash buffer B with approximately 300 μl (wash buffer).
All centrifugation operations are performed at 10,000 rpm with a common desktop microcentrifuge.

  1. Add 10 volumes of Buffer A1 to the PCR reaction mixture and mix. There is no need to remove mineral oil. For example, 500 μl buffer A1 is added to 50 μl PCR reaction solution (amount not containing oil).

  2. The monolith solid phase column 9 is inserted into the collection tube 8 and the adjusted sample is applied to the monolith solid phase column. In order to obtain a high recovery rate, the sample liquid is added to the monolith solid phase column 9 without leaving it.

  3. After the monolith solid phase column 9 was centrifuged at 10,000 rpm for 30 seconds, the monolith solid phase column 9 was removed, and the liquid in the collection tube 8 was removed. The monolith solid phase column 9 is reinserted into the collection tube.

4). 500 μl of buffer B (washing buffer) was added, and the monolith solid phase column 9 was centrifuged at 10,000 rpm for 30 seconds. Centrifuge for 1 minute at 10,000 rpm.
After discarding the liquid in the collection tube 8, it is necessary to perform a re-centrifugation operation in order to completely remove residual ethanol derived from the buffer.

5). Transfer the monolith solid phase column 9 to a new 1.5 ml centrifuge sampling tube, add 10-50 μl of buffer C (elution buffer) to the center of the monolith surface, incubate the monolith solid phase column 9 for 1 minute at room temperature, Centrifuge at 000 rpm for 1 minute. The DNA eluted in the centrifuge tube is purified DNA and stored at −20 ° C. Or, use it as it is for later operations.
Elution buffer C is added to the central part of the monolith surface so that the DNA bound to the monolith is completely eluted. When 10 μl of elution buffer C is used, the volume of the eluate is 9 μl.
The elution efficiency is maximum when the pH is between 8 and 8.5. When using a sterilized water system for elution, it is better to confirm that the pH is within this range.

(2) Suction manifold method The monolith solid phase column can be operated by a suction manifold including a general luer adapter. This protocol is designed for the purpose of purifying double-stranded DNA fragments from PCR reaction solutions. If one monolith solid phase column and a purification buffer are used, a fragment of 35 bp to 100 Kbp can be separated from primers, nucleotides, polymerases, salts, and the like by a sample processing operation using a suction device.

100 μl of buffer A1 (adsorption buffer) is added to 10 μl of the PCR reaction solution. Wash buffer B with approximately 300 μl (wash buffer).
The suction switch is turned off at each operation step so that constant and stable suction is performed.

  1. Add 10 volumes of buffer A1 to the PCR reaction and mix. There is no need to remove mineral oil. For example, 500 μl buffer A1 is added to 50 μl PCR reaction solution (amount not containing oil).

2. Prepare a suction manifold and monolith solid phase column.
Connect the luer adapter suction manifold to the suction device.

  3. Equipped with a vacuum adapter attached to the port on the suction manifold. The monolith solid phase column 9 is inserted into the vacuum adapter.

4). The adjusted PCR sample is applied to the monolith solid phase column 9 with a pipette and aspirated to bind the DNA. Aspirate the solution completely through the monolith solid phase column 9. Stop suction after the sample has passed through the column.
In order to obtain a high recovery rate, the sample liquid is added to the monolith solid phase column 9 without leaving it. The maximum addition volume is 800 μl, and if the sample volume is more than 800 μl, add in several batches.

  5. Buffer B (washing buffer) 500 μl is added to the monolith solid phase column 9 and sucked until the liquid passes through the monolith solid phase column.

6). The monolith solid phase column 9 is removed from the manifold and transferred to the collection tube 8. Centrifuge for 1 minute at 10,000 rpm.
Centrifugation is necessary to completely remove residual ethanol from the buffer. (The liquid is sucked until it passes through the monolith solid phase column and dried. This is a necessary means for completely removing the washing buffer remaining in the monolith solid phase column 9.)

7). Transfer the monolith solid phase column 9 to a new 1.5 ml centrifuge sampling tube, add 10-50 μl of buffer C (elution buffer) to the center of the monolith surface, incubate the column for 1 min at room temperature, 10,000 rpm, 1 min Centrifuge. The eluted DNA in the centrifuge tube is purified DNA and stored at −20 ° C. Or, use it as it is for later operations.
Elution buffer C is added to the central part of the monolith surface so that the DNA bound to the monolith is completely eluted. When 10 μl of elution buffer C is used, the volume of the eluate is 9 μl.
The elution efficiency is maximum when the pH is between 8 and 8.5. When using a sterilized water system for elution, it is better to confirm that the pH is within this range.

B. There are the following methods for purifying an agarose gel using the monolith solid phase column 9.
(1) Microcentrifugation This protocol is designed for the purpose of purifying DNA fragments from standard or low-temperature melting agarose gels (using TE or TEB buffer). If one monolith column and purification buffer are used, centrifugation is performed. Thus, a fragment of 35 bp to 100 Kbp can be separated from primers, nucleotides, polymerases, salts and the like. Up to 1000 mg of agarose can be processed per monolith column.
Add 10 μl of buffer A (lysis, adsorption buffer) to 10 mg of agarose gel. Wash buffer B with approximately 500 μl (wash buffer).
All centrifugation operations are performed at 10,000 rpm with a common desktop microcentrifuge.

  1. Cut out the band of interest with a clean razor or scalpel and place it in a 1.5 ml centrifuge tube. Remove excess gel to minimize gel slice size.

2. Add 100 μl of buffer A2 (lysis, adsorption buffer) to 100 mg of gel slice.
To 100 mg of gel, 100 μl of buffer A2 is added. When an agarose gel having a concentration of 2% or more is used, 600 μl of buffer B is added. Since the amount of gel that can be processed by one monolith column is 1,000 mg, when the gel amount exceeds 1,000 mg, two or more monolith columns are used.

  3. Incubate at 60 ° C. for 5 minutes or until gel slice is completely dissolved. During the incubation, vortex the tube twice to mix the solution. Dissolve the agarose completely. When a gel of 2% or more is used, the recovery rate increases as the incubation time is increased.

The following operations are performed according to the above A.1. Purification of PCR reaction solution using a monolith solid phase column (1) Since this is the same as the microcentrifugation method, the description is omitted.
(2) Suction manifold method Monolithic columns can be operated with a suction manifold that includes a luer adapter. This protocol was designed to purify DNA fragments from standard or cold-melted agarose gels (using TE or TBE buffer). If one monolith column and a purification buffer are used, a 35 bp to 100 Kbp fragment can be separated from primers, nucleotides, polymerases, salts, and the like by sample processing operations using a suction device.

Add 10 μl of buffer A2 (lysis, adsorption buffer) to 10 mg of agarose gel. Wash buffer B with approximately 500 μl (wash buffer).
Elution is carried out by centrifugation at a desktop microcentrifuge at 10,000 rpm.
The suction switch is turned off at each operation step so that constant and stable suction is performed.

  1. Cut out the band of interest with a clean razor or scalpel and place it in a 1.5 ml centrifuge tube. Remove excess gel to minimize gel slice size.

2. Add 100 μl of buffer A2 (lysis, adsorption buffer) to 100 mg of gel slice.
To 100 mg gel, 100 μl of buffer A2 is added. When an agarose gel having a concentration of 2% or more is used, 600 μl of buffer A1 is added. Since the amount of gel that can be processed with one monolith column is 600 mg, when the amount of gel exceeds 600 mg, two or more monolith columns are used.

  3. Incubate at 60 ° C. for 5 minutes or until gel slice is completely dissolved. During the incubation, vortex the tube twice to mix the solution. Dissolve the agarose completely. When a gel of 2% or more is used, the recovery rate increases as the incubation time is increased.

The following operations are performed according to the above A.1. Purification of PCR reaction solution using a monolith solid phase column (2) Since this is the same as the suction manifold method, it will be omitted.
C. There are the following methods for purifying an enzyme reaction solution using a monolith solid phase column.
(1) Microcentrifugation This protocol was designed for the purpose of purifying double-stranded DNA fragments from enzyme reaction solutions such as restriction enzyme digestion and labeling reactions. If one monolith column and a purification buffer are used, a fragment of 35 bp to 100 Kbp can be separated from enzymes, primers, nucleotides, salts and the like by centrifugation.

Add 30 μl of buffer A1 (adsorption buffer) to 10 μl of enzyme reaction solution. Wash buffer B with approximately 300 μl (wash buffer).
All centrifugation operations are performed at 10,000 rpm with a common desktop microcentrifuge.

1. Add 3 volumes of Buffer A1 to the enzyme reaction mixture and mix. The maximum volume of the enzyme reaction solution that can be processed by the monolith column is 100 μl.
For example, 300 μl buffer A1 is added to 100 μl enzyme reaction solution.

The following operations are performed according to the above A.1. Purification of PCR reaction solution using a monolith solid phase column (1) Since this is the same as the microcentrifugation method, the description is omitted.
(2) Suction manifold method Monolithic columns can be operated with a suction manifold that includes a luer adapter. This protocol was designed for the purpose of purifying double-stranded DNA fragments from enzyme reaction solutions such as restriction enzyme digestion and labeling reactions. If one monolith column and a purification buffer are used, a fragment of 35 bp to 35 Kbp can be separated from enzymes, primers, nucleotides, salts, and the like by a sample processing device using a suction device.

For example, 30 μl of buffer A1 (adsorption buffer) is added to 10 μl of the enzyme reaction solution. Wash buffer B with approximately 300 μl (wash buffer).
All centrifugation operations are performed at 10,000 rpm with a common desktop microcentrifuge.
The suction switch is turned off at each operation step so that constant and stable suction is performed.

1. Add 3 volumes of Buffer A1 to the enzyme reaction mixture and mix. The maximum volume of the enzyme reaction solution that can be processed by the monolith column is 100 μl.
For example, 300 μl buffer A1 is added to 100 μl enzyme reaction solution.

The following operations are performed according to the above A.1. Purification of PCR reaction solution using a monolith solid phase column (2) Since this is the same as the suction manifold method, it will be omitted.
The advantage that the adsorption and elution separation of the present invention can be carried out very easily, elution with a high concentration of salt is not required, and the nucleic acid can be purified very efficiently is based on the point that the nucleic acid component is adsorbed on the monolith structure.

  In the conventional type method, silica gel particles, glass particles, and those obtained by filtering them are used. In all of them, the space through which the liquid passes passes through the particle surface, hits the particle, generates turbulent flow, and becomes non-uniform flow. Therefore, it is impossible to touch the surface uniformly. Since the monolith structure is a monolithic structure and has continuous pores inside, it is an image passing through the inside of the particle. That is, all the liquids are in uniform contact. Also, compared to particles, the skeleton is small and the flow is uniform without turbulent flow after the liquid hits.

That is, in the conventional solid phase type, turbulent flow occurs in the particles, the contact with the surface becomes non-uniform, and the low-molecular-side DNA is not adsorbed and comes off.
In order to prevent the omission, the conventional type facilitates the reaction by increasing the chaotropic salt concentration. However, in this case, salt precipitation occurs and a limit occurs, so that there is a limit in collecting low molecular weight DNA.

In the monolith type of the present invention, a uniform liquid flow is guaranteed, and adsorption of lower molecular weight DNA is possible.
Of course, the same thing happens in the cleaning process. In the conventional method, a turbulent flow of the cleaning liquid is generated, and it is difficult to clean the gel surface. As described in Example 1, even after the first washing, primers and the like which are not targeted by the conventional method remain considerably, but hardly remain in the method of the present invention.
The same is true for turbulent flow even with a filter in which particles are embedded in the fiber or the fiber itself.

As a conventional example, an inorganic base material is arranged near the outlet of a cylindrical hollow body having an inlet and an outlet, and the inorganic base material is sandwiched and held between tightly pressed polyethylene frit. There is an example. (Patent Document 3)
In this case, the part contributing to the separation is the inorganic base material part, and the upper and lower frit are used to hold the inorganic body in the hollow body.
No matter how hard it is pushed in, a space is created between the frit and the inorganic material, and the liquid remains in the space. It is difficult to eject or replace the liquid in the space. In particular, in the depressurization method as described above, if a gas phase portion is partially formed, the portion preferentially flows, and the liquid cannot be uniformly discharged. In the sample addition and cleaning process, it is difficult to replace the liquid.

Moreover, in the last elution, a liquid remains and collection | recovery worsens.
In the monolith structure of the present invention, the macropores through which the liquid flows become a continuum, and the liquid changes uniformly in the direction of flow. That is, the liquid replacement efficiency is significantly increased. As in Example 1, this conventional type is considered to be one of the causes that a lot of sample components remain even in the second elution. In the continuum monolith structure of the present invention, since the liquid replacement efficiency is high, it can be seen that one elution is sufficient and the second elution is hardly left.

Further, in the conventional type, the space changes depending on the pushing condition, and the lot-to-lot variation at the time of pushing into the hollow body easily occurs. In the present invention, the monolith structure is an integral structure, and there is no variation at the time of pushing into the hollow body.
Although it is expensive as a commercial product and does not actually exist, the frit and the inorganic material can be further crushed and integrated, but this method also forms different layers at the crushed interface portion. Again, the liquid flow is hindered compared to a monolithic structure with uniform continuous pores.

Furthermore, in the present invention, if an inorganic material such as silica gel and an organic material such as polyethylene as a frit material are mixed in a sol-gel process, a hybrid homogeneous phase having the properties of silica and polyethylene can be produced.
In the case where the inorganic substance is in the form of particles, the upper and lower frits are indispensable to stop the problem, and the problem of substitution efficiency as described above arises.

  Even when silica gel thin film embedded with silica fiber or Kel particles (for example, 3M Emporedisk (registered trademark)) is used as an inorganic material, it is still not physically hard and is subjected to rapid decompression or high-speed centrifugation. Therefore, part of the fibers and particles may be eluted due to deformation. Even with a slight deformation, the space volume will change, causing a variation. In the monolith structure, since the separation is performed by the continuous pores inside the hard skeleton, it is not deformed even by pressure fluctuations, so that reproducibility is also obtained.

For example, even if a hard fiber membrane or silica gel thin film that does not need to be stopped with a frit can be formed, turbulent flow occurs due to the flow of the liquid flowing on the fiber or gel surface, and uniform separation cannot be obtained. The separation cannot be expected as in the case of using the monolith structure.
By using a monolith structure that does not generate turbulent flow, it is possible to achieve adsorption of low-molecular DNA and a high cleaning effect for the first time.

  Furthermore, the flow path in the conventional type particle and the micropore in the monolith structure of the present invention are different in that they are in contact with the liquid flow. In the particle type or the like, there is no uniformity of pressure in the liquid resistance between the side where the liquid enters and the side where the liquid escapes, and the contact of the liquid into the pores is different. In a pressure system such as HPLC, a uniform pressure can be obtained, and the influence thereof is reduced. However, in the pressure reduction system used in the field of the present invention, the inlet side is normal pressure, and the outlet side is negative pressure. The inside and outside of the pores in the individual becomes uneven. That is, even in the same component, depending on the component molecule, there are those that enter the inside of the pore and those that do not enter, and the total width at the time of elution increases. Therefore, it is difficult to eliminate only the components that are desired to be removed at the time of washing, and the low-molecular-weight primer remains in the finally eluted components as in the conventional example shown in Example 1. In the monolith structure of the present invention, since there are micropores on the surface of the macropores through which the liquid flows, uniform penetration occurs. Therefore, it is possible to easily remove the primer which is a low molecular impurity.

  In the conventional method, in the case of large DNA, it becomes difficult to enter into the fine hole, and further, the viscosity of the liquid containing the component increases, so that the contact becomes non-uniform, and two kinds of phenomena occur simultaneously. It is thought that there are many parts that are not adsorbed. In addition, turbulent flow causes physical damage to the polymer DNA and increases the possibility of destruction.

  Basically, these phenomena can be reduced by increasing the concentration of chaotropic salt, but they are not removed even during washing, but are eluted during elution. That is, a large amount of salt enters the sample components after purification. This is a major problem for later use.

In the method of the present invention using a monolith structure, the salt concentration can be lowered, and the above problems can be solved, which is very effective. Furthermore, it becomes more effective when used together with a potassium salt having a more cation exchange action. Potassium salt has a strong cation exchange action, contributing to the adsorption of nucleic acids to the surface. Therefore, if it remains on the surface of the substrate during elution, the target purified DNA will not be eluted. Cleaning is impossible. In the case of the particle type, turbulent flow is generated even during cleaning, and the penetration into the pores is non-uniform, so that a portion where the potassium salt remains on the substrate at a high concentration inevitably occurs. As a countermeasure, a buffer capable of removing potassium, that is, another salt may be added to the eluate, but this is also not suitable for the purpose of later application.
In the monolith structure, a uniform liquid flow and uniform penetration into the pores are possible, so that the potassium salt acts effectively.

  For purification of the PCR reaction solution (fragment DNA), 50 μl of the PCR amplification reaction product is mixed with 300 μl of buffer A1 (1 M guanidine hydrochloride, 0.2 M potassium acetate, 50% 2-propanol). The silica monolith solid phase column 9 is inserted into the collection tube 8, and the mixture is injected into the silica monolith solid phase column 9 and centrifuged in a 1.5 ml centrifuge tube. The silica monolith solid phase column 9 is washed with B buffer (0.2 M potassium acetate, 50% ethanol) so as not to contain salt.

  For elution, 20 μl of elution buffer C (EDTA 4 mM, Tris-HCI 10 mM, pH 8 or sterile DNA, RNA free water) is centrifuged through a centrifuge column in another 1.5 ml centrifuge tube. The PCR product (fragment DNA) purified in this way does not contain primers, dNTPs, polymerases and salts, and can be used directly in subsequent operations. (See FIGS. 1 and 2) In FIG. 1, M: molecular weight marker, (1) sample purified by conventional method, (2) sample purified by the present invention.

(1) is an evaluation by electrophoresis on a sample (400 bp) purified by the method of conventional Patent Document 3. (2) is the method of the present invention. In (1), a large amount of the low molecular side (lower) part remains, but in (2), almost no part remains, and high purification efficiency is obtained. Further, in FIG. 2, 1-1 and 1-2 are performed twice by the conventional method, and the remaining is seen, and 2-1 and 2-2 are those of the present invention. In the case of the present invention, almost no residue remains at the first time, and it can be seen that high purification efficiency is obtained. FIG. 3 is an evaluation using HPLC. HPLC conditions are as follows: HPLC conditions Column: CIM DEAE
Eluent:
A: 20 mM Tris-HCl Ph7.4
B: A + 1M NaCl
A / B = 50 / 50- (10MIN) -0/100 Flow rate: 3ml / min
Detection: UV260nm

  The HPLC evaluation data of the unpurified PCR solution in FIG. 3 is the top chromatogram, and there is no change in the pattern compared to the chromatogram of the purification by the conventional Patent Document 3 method (bottom), It can be seen that dNTPs and primers are hardly removed. In the method of the present invention, it can be seen that the two peaks of dNTPs and primer are largely removed as in the second chromatography, and the target nucleic acid is highly purified. (See Figure 3)

For purification of DNA fragments from an agarose gel, PCR amplification products are electrophoresed using a standard or low melting point agarose gel (using TE or TBE buffer) and the DNA is separated in an agarose gel (TE or TBE 0.5%). . A DNA fragment to be isolated with a clean razor or scalpel is excised from the gel and placed in a 1.5 ml centrifuge tube. Mix in 300 μl of buffer A2 (2M guanidine thiocyanate, 0.4M potassium acetate, 30% 2-propanol) and incubate at 60 ° C. for 5 minutes or until gel slice is completely dissolved.
According to Example 1, the lysate is inserted into the collection tube 8 and the mixture is injected into the monolith solid phase column 9 and centrifuged in a 1.5 ml centrifuge tube. The monolith solid phase column 9 is washed free from salt by treatment with buffer B (0.2 M potassium acetate, 50% ethanol).

  For elution, 20 μl of buffer C (EDTA 1 mM, Tris-HCI 10 mM, pH 8 or sterile DNA, RNA free water) is centrifuged through a centrifuge column in another 1.5 ml centrifuge tube. 4, FIG. 4-1 is evaluated by electrophoresis at 35 bp, FIG. 4-2 is evaluated by electrophoresis at 100 to 500 bp, FIG. 4-3 is evaluated by 10,000 bp, and FIG. 4-4 is evaluated by electrophoresis at 35,000 bp.

  In the method of the present invention, a low molecular weight of 35 bp to about 100 Kbp are recovered, and it can be seen that a wide range of DNA can be purified even from an agarose gel. Similar results were obtained with water containing no EDTA buffer as the elution buffer.

  100 μg of DNA after the restriction enzyme reaction is treated with the restriction enzyme. This DNA restriction reaction solution was mixed with 1,300 μl of buffer A according to Example 1, and the subsequent treatment was carried out in the same manner as in Example 1. After elution, the purified DNA obtained did not contain restriction enzymes and salts, and the ratio of absorbance measurement ratio (mm) 260/280 was as good as 1.8.

For purification of a 35 bp PCR amplification product having a small molecular size, 10 μl of the PCR amplification reaction product was mixed with 1,100 μl of buffer A according to Example 1, and the subsequent processing was carried out in the same manner as in Example 1. In the figure, M: molecular weight marker, 1: sample before purification, 2: sample purified by the present invention.
Here, even a small DNA of 100 bp or less than 100 bp could be purified from a PCR reaction solution of 35 bp DNA.

For purification of PCR amplification products having a large molecular size (from 100 bp to 100,000 bp), 20 μl of the PCR amplification reaction solution was mixed with 1,200 μl of buffer A according to Example 1, and the subsequent processing was carried out in the same manner as in Example 1. (See FIG. 6) In the figure, M: molecular weight marker, 1: sample before purification, 2: sample purified by the present invention, 3: sample purified by the conventional method.
Even DNA from 1000 bp to 100 Kbp could be purified from the PCR reaction solution.

For purification of the single-stranded DNA solution, 20 μl of the solution was mixed with 1,200 μl of buffer A according to Example 1, and the subsequent treatment was performed in the same manner as in Example 1. (See FIG. 7) In the figure, M: molecular weight marker, 1: sample before purification, 2: sample purified by the present invention.
The single-stranded DNA 35mer could not be recovered by the conventional method (3), but in the present invention (2) it was recovered both times with good reproducibility.

Comparison of purification with sodium and potassium (retention mechanism) (see FIG. 8) In the figure, M: molecular weight marker, 1: glass monolith, sample after purification with potassium, 2: silica monolith, sample after purification with potassium, 3: silica monolith, sodium After purification by sample,
A purification comparison was made between sodium, which has been frequently used, and potassium, which has the effect of the present invention. With sodium, almost no retention was obtained, and with potassium, high purification efficiency was obtained.
In addition, similarly to the silica monolith, the glass monolith also obtained higher production efficiency with potassium.

In order to make the present invention effective, it is meaningful to carry out the following method.
A method for separating and purifying DNA, comprising: adsorbing a nucleic acid corresponding to each through-hole of an integrated monolith structure by interposing an alkali metal salt in a solution containing a nucleic acid, washing with a washing solution, and then elution.
A method for separating and purifying DNA, wherein the alkali metal salt is potassium acetate.
A method for separation and purification, wherein a dissolution adsorption buffer containing less than 0.1 to 1 M of potassium acetate is used.
A method for separating and purifying DNA, comprising performing dissolution and adsorption using a dissolution and adsorption buffer containing a guanidine salt or a potassium salt such as potassium acetate.
A method for separating and purifying DNA, wherein elution is performed with water containing Tris-HCl and EDTA.
A method for separating and purifying DNA, wherein the dissolution, adsorption, separation and washing operations are carried out using a single monolith solid phase column.
A kit consisting of a dissolution adsorption buffer, water, and a separation and purification mechanism.

  As described above, the mechanism for separating and producing DNA and the like according to the present invention is used very frequently in molecular biological research. C. Quantitative separation of a wide range of DNA from 35 bp (mer) to less than 100 Kbp (mer), especially useful for purification of fragment DNA prior to R, cloning, sequence sync, restriction enzyme digestion, and other enzyme action applications It can be used for purification of a wide range of nucleic acids.

Claims (6)

  A monolithic Molinos structure that has a continuous through hole from one end to the other end, and has a through hole having a size corresponding to the size of the nucleic acid, and allows the solution containing the nucleic acid to be separated to pass through. And a mechanism for separating and purifying DNA or the like, characterized in that each of the nucleic acids corresponding to the through-holes can be retained by The monolith structure is an inorganic material such as glass or silica, or a hybrid material containing an organic material in an inorganic material, and uses a porous material having macropores penetrating from the upper surface to the lower surface. 1. A separation and purification mechanism for DNA or the like according to 1. The mechanism for separation and purification of DNA or the like according to claim 1 or 2, wherein the porous body of the monolith structure has micropores inside the macropores. The mechanism for separation and purification of DNA or the like according to any one of claims 1 to 3, wherein the porous body of the monolith structure has macropores of 1 to 100 µm and micropores of 0 to 100 nm. The mechanism for separating and purifying DNA or the like according to any one of claims 1 to 4, wherein a monolith solid phase column is constituted by disposing a disk formed of a monolith structure in a column tube. 6. A monolith solid phase column formed by detachably mounting a base body constituted by a monolith structure on a cylindrical body whose upper and lower sides are open is used. A mechanism for separation and purification of DNA.

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