JP2006296220A - Method for separating and refining dna or the like and separating and refining mechanism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simply and precisely obtain a target refined component from a biological sample in a short time by continuously making a biological sample liquid and various buffers flow. <P>SOLUTION: A target component is highly refined and separated by attaching a monolith structure having a physically solid silica skeleton to a disposable tube, etc., which is useable for centrifugal separator, making the biological sample flow, catching the sample in the monolith structure and eluting the sample. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a separation and purification method for DNA and the like and a separation and purification mechanism.

  Decoding the entire human gene sequence has been completed, and genomic research is shifting from conventional structural analysis to functional analysis such as elucidation of genes related to disease states and more specifically targeted drug discovery research .

  Along with this, bioinformatics (bioinformatics) technology for analyzing accumulated genetic information (database) with a computer has been rapidly developed and utilized. Therefore, in SNPs analysis (single nucleotide polymorphism analysis) and gene expression analysis, it is necessary to separate and purify Template DNA from biological samples, and there is a need for further miniaturization, higher performance, higher efficiency, and higher speed than before. It has been.

  Since a satisfactory method for efficiently separating and purifying a large amount of Template DNA from a small amount of biological sample has not yet been obtained, research on biological samples such as whole blood is currently limited. In addition, the number of white blood cells in a whole blood sample varies considerably among individuals due to many factors such as age, sex, immune status, and other storage conditions, and also affects the amount of DNA recovered. Due to this fluctuation, in order to obtain a sufficient amount of high-quality Template DNA from a whole blood sample having various white blood cell counts, a highly reproducible and adaptive purification technology for a minute amount of sample is required.

  As a method for separating and purifying Template DNA from a biological sample, various methods have been proposed after cell lysis and pretreatment for enzymatic degradation of the protein to lower its molecular weight. For example, an isolation method is shown in which DNA is bound by addition of a chaotropic substance on a glass fiber filter, followed by separation, washing and drying after pretreatment for reducing the molecular weight. (See Non-Patent Document 1)

  In addition, a method of adding glass powder to bind DNA to glass powder, centrifuging, collecting the glass powder, washing, eluting and isolating is shown (Patent Document 1, Non-Patent Document 2, Non-Patent Document 2, (See Patent Document 3 and Non-Patent Document 4).

  Also proposed 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 the nucleic acid is eluted. (See Patent Document 2).

  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 an agarose gel by electrophoresis or various extractions, but this method is time consuming and the resulting DNA is also very 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.

  However, none of these methods are optimized for high-efficiency purification from biological samples and purification of trace components, so high purification cannot be achieved and the purpose cannot be achieved, and satisfactory results are found to be difficult to use. Is not obtained.

JP 59-227744 A Japanese Patent No. 2680462 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)

  When purifying a biological sample, a container for handling the liquid is indispensable because the liquid sample is handled by pretreatment by enzymatic degradation of protein or precipitation purification by adding a solution. Naturally, the smaller the container used, the smaller the surface area of the container and the more convenient the handling of trace components. Considering ease of use, such as relocation, it is necessary to devise a method for attaching the separated body to the container. In that sense, a conventional purification type cartridge in which a disposable type container filled with silica gel beads or a filter woven with silica gel fibers or the like is attached to the container is suitable.

  In the particle type, the particle diameter, the surface area, the pore diameter, etc. can be freely selected, and there is an advantage that it can be used for various biological samples in which various matrices are mixed. However, a filter is needed to keep the particles in the cartridge. Since the filter portion cannot participate in purification, there is a drawback that purification efficiency is deteriorated. Further, the particle space cannot be made constant, and variations are likely to occur. There is also the possibility of particle elution. Therefore, it cannot be used because it cannot be used for stable high purification.

  On the other hand, instead of using particles, a cartridge in which a filter knitted with silica gel fibers is attached to a container has been proposed. However, in a filter in which silica gel fibers or the like are woven into a mesh shape, elasticity is generated, and the volume changes depending on how the liquid is contained. Further, the hardness is 20 Hk or less, and it is easy to deform and is inconvenient to handle. In particular, when using a centrifuge suitable for biological samples containing a large amount of matrix, the volume change becomes significant, and a large amount of matrix is mixed in biological samples, so the viscosity is high at first, and the viscosity decreases as purification proceeds. Tend to. Therefore, a separated body such as a fiber filter in which the liquid flowing space changes due to a change in resistance pressure is not suitable for a biological sample or the like whose viscosity changes.

  In order to purify a biological sample, a hard separator that does not cause a change in the space in which the liquid flows is required. What is important is the change in the space in which the liquid flows with respect to the pressure, but considering the handling, a separator with a hardness of 30 Hk or more is desired.

  Furthermore, in a biological sample that requires complicated pretreatment, a plurality of steps are required, and the separator is deteriorated during that time. If these steps are simply performed by passing only liquid, high purification cannot be expected with a conventional soft separator.

  Another important point is how to keep the separator on the cartridge. The method of retaining this solid separated body in a container or the like with a resilient resin ring or the like is not a big problem in general purification. However, in the case of a biological sample, the viscosity is high at the initial stage where the degree of purification is low, and attaching such a ring is not suitable because it reduces the liquid contact cross section through which the liquid passes. When handling trace components such as blood, it is necessary to carry out purification in a small amount of space, and it is not possible to make a part for attaching a ring.

  Therefore, a method that does not use a filter or a retaining ring is suitable for purification of a biological sample. Although it is not possible with a fiber filter or the like, if it is a hard separated body having a hardness of 30 Hk or more, it is easy to handle and can be easily attached to the cartridge by fusion or press fitting. The press-fitting can be easily done by attaching a stainless steel rod to the end of the drilling machine and pushing the separating body into a resin cartridge, but it is necessary to be able to withstand centrifugation, and a separating body with a hardness of 100 Hk or more is desired. .

  However, as a problem of the method of press-fitting a separated body such as a container, in the case of purification of a biological sample, a container that easily causes catalytic action such as glass or metal is often avoided, such as polyethylene and polypropylene. Resin is often used. These containers are susceptible to heat shrinkage and are sterilized by heating, even if they are pressed in, for purification of plasmid DNA from Escherichia coli, which is a biological sample that must be carefully sterilized at 120 ° C. It is possible that it will fall out.

  For this reason, a method in which a part of the cartridge resin is dissolved by vibration such as ultrasonic waves and the separator is fused to the partially used liquid is suitable. When the separator itself dissolves, it becomes meaningless as a separator, so a separator that does not change even under heating is required. A heat-resistant separator having a hardness of 30 Hk or more that is easy to handle is desired.

  As a separator satisfying all of these viewpoints, a monolith structure can be proposed as a purified separator of biological sample components.

  The monolith structure can be mainly produced by a sol-gel method. That is, a metal alkoxide such as tetramethoxysilane or a reactive organic monomer such as trimethoxysilane is used alone or in combination, and is partially hydrolyzed and polycondensed to form a colloidal oligomer (sol Production) and further hydrolyzed to promote polymerization and cross-linking to form a three-dimensional structure network (generation of gel). Since gelation is performed at 300 ° C. or higher, there is no denaturation even with heat of fusion.

  A monolithic solid phase can also be prepared 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.

  Since the phase separation is performed at 500 ° C. or higher, there is no denaturation even with heat of fusion.

  Various methods for producing these monolith structures have been reported as columns in HPLC, and have a pressure resistance of 20 Mpa or more and are known to be produced with good reproducibility.

  It is important for biological samples to be highly viscous and to be purified with good reproducibility without causing flow path changes even when pressure is applied. Naturally, the higher the pressure resistance, the faster the fluid can flow, and the purification time. Accelerates. A monolith structure having a pressure resistance of 1 Mpa or more can be recommended. This monolith structure is known to be formed in a rod shape, and can be used as a separator of the present invention by mechanically cutting it and cutting it into a disk shape.

  It has a hardness of 30Hk or more that is easy to handle and does not cause thermal denaturation, and is suitable for purification of biological samples. Furthermore, when this monolith structure is fired to 800 ° C. or higher, the skeleton becomes vitrified and the hardness becomes 100 Hk or higher, and a press-fitting method can be used.

  In addition, since the sol-gel method can be used to create a free space, a monolith structure can be directly formed in a container such as a cartridge from the beginning.

  For a small amount of biological sample, since it can be created in a small space at the tip of the cartridge chip, it is particularly suitable without causing a dead volume.

  All of these monolith structures have silanols, and conventionally known DNA purification methods can be used as they are.

  In particular, in the case of biological sample components, the viscosity varies depending on the target purified product, and control of the flow of the liquid is important. However, in the monolith structure, the through-pores through which the liquid flows can be freely controlled and generated. Suitable for purification.

  A through-pore of 10 μm or more is suitable for a highly viscous sample such as a blood sample. In addition, purification of DNA purified by gel electrophoresis can be performed with a through pore of less than 10 μm. In the monolith structure, the mesopores can be controlled at the same time other than the through-pore that is the passage of the liquid. In the case of purification of circular plasmid DNA from Escherichia coli, which is a biological sample, a monolith structure having no through pore is suitable.

  Since the monolith structure 1 suitable for the purification of the biological sample has a hardness of 40 Hk or more and high thermal stability, it is attached by fusing or press-fitting into various containers suitable for the purification of the biological sample. When targeting biological sample liquids such as blood with limited collection volume, ultrasonic fusion is easily performed on the tip of the pipette tip 2 as shown in FIG. Can do. Therefore, in combination with the centrifuge tube 3 of FIG. 1 (b) (FIG. 1 (c)), the biological sample liquid is passed through the monolith structure by centrifugal force and purified with various buffers, while purifying with a small amount of highly pure genome. Obtain DNA.

  Furthermore, when simultaneously preparing various samples, the monolith structure separators 42, 42, 42,... Can be freely placed in the holes 41, 41, etc. of the 96-well plate 4 (FIG. 2) and the 384-well plate, respectively. Can be installed by press-fitting or fusing. At this time, it is recommended that the monolith structure 42 be received by providing the step portion 44 in the liquid passage portion 43 such as the hole 41. Here, containers such as pipettes, cartridges, centrifuge tubes, and well plates are collectively referred to as disposal containers.

  The gist of the present invention is that the monolith structure is hard, so that the biological sample can be flowed into the monolith structure by force such as centrifugation by freely attaching it to the instrument according to the purpose by press-fitting or fusion. It is possible to purify, and it is not limited to the shape of these examples and the fusion method, but the above form is the best

  Moreover, in the monolith structure body by the sol-gel method, a monolith body body can be freely created within a space having a glass surface. Even without fusing or press-fitting, a monolith body can be formed first as shown in FIG. In this case as well, since the liquid is allowed to flow, pressure changes occur, pressure resistance is required, and it cannot be applied to the purification of biological samples unless the monolith structure is formed.

  For whole blood, put in a centrifuge tube, add lysis / adsorption solution, incubate at 70 ° C. for 15 minutes, mix by vortex to dissolve whole blood. After centrifugation, the supernatant is loaded onto a monolith column.

  Also, the E. coli culture solution is centrifuged, collected and discarded, and the precipitate is collected in a separate container. Next, the cells are suspended by mixing. Next, a lysis solution is added to lyse the cells. Further, neutralize by adding a neutralizing solution and mixing by inversion. Next, after centrifugation, the supernatant is loaded onto a monolith column.

  As described above, in the case of a biological sample, a complicated pretreatment process is required. After that, it becomes the actual purification process. In these steps, the diluted solution, lysate, and method to be added differ depending on the target biological sample such as blood, animal tissue, or E. coli culture solution. In addition, the number of purifications in centrifugal precipitation also varies. Naturally, it is better that the number of the pretreatment steps is small, but impurities exist in the actual purification step, and the liquid resistance increases. In the purification method using a solid monolith structure, there is no change in the flow path even if centrifugation is performed, and this can be handled sufficiently.

  1 ml of acetic acid was added to 2 ml of 7% polyethylene glycol aqueous solution, 1 ml of tetramethoxysilane was stirred and mixed, put into a polycarbonate tube and sealed at both ends, and gelled at 40 ° C.

  By changing the size of the polycarbonate tube, a monolith structure with a free diameter can be obtained.

  Substitute with 0.1 standard ammonia aqueous solution, age for several hours, replace with ethanol and dry, then overheat to 600 ° C., and obtain a silica monolith rod with a three-dimensional network structure with a through pore diameter of 30 μm and a mesopore diameter of 10 nm and an outer diameter of 0.4 mm. Obtained. This monolith rod was cut to a length of 1 mm to obtain a separated body having a hardness of 48 Hk.

  The monolith separated body was put in a commercially available pipette tip having a volume of 10 μl and pushed in for 0.2 seconds using an ultrasonic fusion device sonopet-150B, and the contact resin part was melted and fused by ultrasonic vibration. The shape is as shown in FIG.

  The photograph by an electron microscope is shown. It was proved that there was no gap and it could be fused. (FIG. 3) It was confirmed that there was no damage to the monolith structure due to fusion.

  For comparison, an appropriate amount of glass wool was packed at the end of the same pipette tip. In the case of the glass wool type, the tip of the fusion was crushed, and ultrasonic fusion was not possible.

  When 6 μl of Tris buffer solution was added to each of both pipette tips and centrifuged at 3000 rpm, 3 out of 6 glass wools were lost in the glass wool type. In the case of the fused ones, it was not possible to miss all six.

Similarly, when 10 μl of blood, which is a biological sample, was diluted to 1/5 and centrifuged at 3000 rpm, 6 out of 6 glass wool types lost glass wool.
None of the 6 monolith structures were missed.

  Furthermore, similarly, even if it centrifuged at 10,000 rpm, it did not come out and there was no fragment of the monolith structure in the eluate.

  Biological samples typified by blood are highly viscous and often require high centrifugation, but monolithic structures that are ultrasonically fused can be used up to high centrifugation and should not be crushed. Was confirmed.

  A 2% xylene solution (g / ml) of high-purity perhydropolysilazane (manufactured by Tonen Co., Ltd., high-purity product) is sucked up to the point of 1 mm with a 10 μl commercial pipette tip, discharged and discharged in air at 80 ° C. for 24 hours. Dried.

  This operation was repeated 5 times to obtain a pipette tip having a quartz film formed on the surface of the tip portion of 1 mm of the pipette tip, and steam sterilized at 120 ° C.

  1 mm of the tip of the pipette tip subjected to the above treatment was mixed with a uniform solution in which methyltrimethoxysilane, formamide as a coexisting substance, and 1 mol / l nitric acid aqueous solution as a catalyst were mixed at a molar ratio of 1: 2.5: 1.8. Inhaled and sealed the tip with sealing tape. Gelation was performed at 80 ° C. to prepare a monolith structure at the 1 mm portion of the tip of the pipette tip.

  A monolith structure having no mesopores with a through pore of 15 μm was obtained. Furthermore, mesopores can be created by combining silica phases as necessary, and the size can be controlled so as to be suitable for biological samples.

  In the circular DNA produced by E. coli, it is not necessary to create a mesopore, but this time, in order to make it a blood target, a silica phase was further formed to create a mesopore.

  A 5% trimethoxysilane nitric acid aqueous solution was sucked into a 1 mm portion and gelled at 80 ° C., whereby a silica phase was formed on the monolith surface, and mesopores were obtained as in Example 1.

  The monolith structure prepared at the tip of the pipette tip was centrifuged by the same method as in Example 1, but it was demonstrated that it could be used by flowing a biological sample without coming off.

  Six of those prepared in Example 1 were sterilized at 120 ° C. for 1 hour. Add 10 μl of fresh whole blood to the sample tube, add 20 μl of decomposition / adsorption buffer (8 M guanidine thiocyanate, 0.8 M potassium acetate), incubate for 15 minutes at 70 ° C., and mix by vortex to dissolve the whole blood. .

  In the conventional method, in order to lower the viscosity, RNase and proteolytic enzyme treatment is performed and the next step is performed. However, in this method, since a hard monolith structure is fused, a highly viscous biological sample is allowed to flow. Therefore, it is not necessary to use these decomposing enzymes. This degrading enzyme inhibits PCR, and it is important that it can be purified without using it.

  Next, the fused monolith solid phase column was set in a centrifuge tube, centrifuged at 10,000 rpm for 1 minute, passed through the monolith structure, and concentrated and collected into the monolith structure. Centrifugation was performed for 1 minute in a washing buffer (0.5 M potassium acetate, 60% ethanol), and the monolith structure was washed.

  Next, the monolith structure was set in a collection tube, 10 μl of elution buffer (Tris-HCI 10 mM, pH 8) was added, and centrifugation was performed at 10,000 rpm for 1 minute to elute purified genomic DNA through the monolith.

It was confirmed that A ₂₆₀ / A ₂₈₀ of each eluate was 1.7 or more, and a part of them was taken to confirm the result of gel electrophoresis. It was confirmed that the seven purification results were purified with good reproducibility (FIG. 4, lane 1: pH marker, lanes 2-7: PCR amplified product reproducibility).

  Furthermore, the gene sequence (408 bp) of human β-Globin was PCR amplified using the purified genomic DNA.

  It was confirmed that PCR could be performed with good reproducibility when purified using the method of the present invention even from a very small amount of blood sample of 5 μl (FIG. 5, lane 1: pHY marker, lanes 2-7: reproducibility of PCR amplification product).

  Moreover, sequence data could be confirmed with ABI Prism 3730xlGeneticAnalyzer (FIG. 6). It was found that even a short purification could be applied to actual biological sample analysis.

  The one prepared in Example 2 was sterilized at 120 ° C. for 1 hour. Add 3 μl, 6 μl, 9 μl, and 12 μl of frozen blood to the sample tube, add 2 volumes each of decomposition / adsorption buffer (6 M guanidine thiocyanate, 0.8 M potassium acetate), incubate at 70 ° C. for 15 minutes, and vortex The whole blood was dissolved by mixing.

  Next, the fused monolith solid phase column was set in a centrifuge tube, centrifuged at 10,000 rpm for 1 minute, passed through the monolith structure, and concentrated and collected into the monolith structure. Centrifugation was performed for 1 minute in a washing buffer (0.5 M potassium acetate, 50% ethanol), and the monolith structure was washed by washing.

  Next, it was set in a collection tube, 5 μl of elution buffer (RNA-free water) was added, and centrifugation was performed at 10,000 rpm for 1 minute to elute through the monolith structure.

It was confirmed that A ₂₆₀ / A ₂₈₀ of each eluate was 1.7 or more, and the result of gel electrophoresis was confirmed. It was confirmed that even a very small amount of 3 μl to 12 μl of frozen blood could be easily purified using the method of the present invention (FIG. 7 Lane 1: Whole blood 3 μl, Lane 2: Whole blood 6 μl, Lane 3: Whole blood 9 μl, Lane 4: whole blood 12 μl).

  In the same manner as in Example 1, the diameter of the polycarbonate tube was increased to produce a silica monolith rod having a diameter of 6 mm and cut to a thickness of 1.5 mm. As shown in FIG. 2, this was fused to each hole of the 96-well plate with an ultrasonic fuser.

  In the same manner as in Example 3, 50 μl of purified genomic DNA was eluted from 200 μl of fresh blood.

  As a result of gel electrophoresis, purified genomic DNA was obtained with good reproducibility as shown in FIG. 8 (M: molecular weight marker, 1-4: reproducibility by the method of the present invention).

  As a result of PCR amplification of the purified genomic DNA with 100 bp and 400 bp of human β-globin gene, it was confirmed that PCR was performed, and it was proved that the purification method of the present invention was highly purified. FIG. 9 (M: molecular weight marker, lane 1, 2: 100 bp PCR amplification product, lane 3, 4: 400 bp PCR amplification product).

  In the same manner as in Example 4, 200 μl of purified genomic DNA was eluted from 400 μl of frozen blood cells. As a result of gel electrophoresis, purified genomic DNA as shown in FIG. 10 (M: molecular weight marker, 1-4: reproducibility by the method of the present invention) was obtained.

  As a result of PCR amplification of the purified genomic DNA of human β-globin gene 740 bp, it was confirmed that PCR was performed with good reproducibility, and it was proved that the purification method of the present invention was highly purified (FIG. 11 (M : Molecular weight marker, 1-4: Reproducibility of PCR amplification product after purification by the method of the present invention)).

  1 ml of acetic acid was added to 2 ml of 7% polyethylene glycol aqueous solution and 1 ml of tetraethoxysilane was stirred and mixed, then put into a polycarbonate tube and sealed at both ends, and gelled at 40 ° C.

  After aging for several hours and substitution-drying with ethanol, the mixture was heated to 600 ° C. to obtain a silica monolith rod having a through-pore diameter of 10 μm and a non-porous three-dimensional network structure having no mesopores and an outer diameter of 7 mm. This monolith rod was cut into a length of 1 mm to obtain a separated body having a hardness of 42 Hk.

  Furthermore, in order to increase the hardness, sintering was performed at 1150 ° C. to obtain a monolith structure separator having a hardness of 120 Hk. A very hard monolithic body having a disk-like mesopore and a through-pore of 10 μm was obtained.

  Silica monolith is baked at a high temperature to form a strong monolith body while leaving a uniform through-pore skeleton with a three-dimensional network structure through which the liquid flows. Since it becomes stronger, press-fitting by physical force is also possible.

  A solid stainless steel rod having an outer diameter of 5.5 mm was attached to the tip of an upright drilling machine. A strong monolith structure 51 was placed in the spin type disposal cartridge 5, moved downward without rotating, and pushed down to a predetermined position to obtain a spin type disposal cartridge as shown in FIG.

  This solid monolith structure is particularly suitable for biological sample components that require complex processing. For example, purification of circular plasmid DNA from E. coli enters the purification step after cell suspension, degradation, and neutralization. However, depending on the previous process and sample condition, insoluble matter remains, and a sudden pressure fluctuation is likely to occur when the liquid is flowed by centrifugation. In the conventional method, the large pressure fluctuation sometimes causes deterioration of the separator during the process. Further, in order to improve the street, there is a demand for a purified product as thin as possible, but it is difficult to use a physically weak product. The fired monolith structure of the present invention can be made very hard and thin.

  By using a solid monolith structure, purification of a biological sample that requires such a complicated pretreatment can be easily performed by simply passing a liquid through the monolith structure by centrifugation or the like.

  Take 1 ml of E. coli culture solution, which is a biological sample, in a sample tube, centrifuge at 10,000 rpm for 2 seconds, collect the cells, and discard the supernatant. To the precipitate, 250 μl of Tris buffer containing RNase and 10 mM EDTA is added, and vortex mixing is performed to suspend the cells. Next, 250 μl of potassium hydroxide aqueous solution containing 1% sodium lauryl sulfate is added and mixed. Further, 350 μl of adsorption buffer (8M guanidine thiocyanic acid, 0.5M potassium acetate) is added and mixed to neutralize. Thereafter, the insoluble matter is precipitated after centrifugation at 15,000 rpm for 5 minutes.

  The monolith solid phase column of Example 7 was set in a centrifuge tube, and the supernatant was centrifuged at 10,000 rpm for 1 minute, and the plasmid DNA was concentrated and collected in the monolith through the monolith. A washing buffer (0.3 M potassium acetate, 65% ethanol) (500 μl) was added, the mixture was centrifuged at 10,000 rpm for 1 minute, and washed through the monolith. Once again, 800 μl of a washing buffer (0.5 M potassium acetate, 60% ethanol) was added, centrifuged at 10,000 rpm for 1 minute, and washed through the monolith. The monolith was then centrifuged at 10,000 rpm for 1 minute to remove the liquid in the monolith.

  Next, the sample was set in a collection tube, 200 μl of elution buffer (pH 8 aqueous solution) was added, and centrifugation was performed at 10,000 rpm for 1 minute to elute through the monolith.

  The purified plasmid DNA eluted was checked for purity by electrophoresis and compared with the conventional method.

  FIG. 13 shows the results of electrophoresis of purified circular DNA from E. coli plasmid DNA (circular DNA): pQE vectors 6 × Histag constructs. M is a molecular weight marker, 1 is a method of the present invention, and 2 is a conventional method. It can be seen that 4 kb of purified plasmid DNA was obtained at a higher recovery rate than the conventional method.

  The conventional method was carried out using QIAprep Spin Miniprep kit (Qiagen) using a silica gel membrane filter and using various buffers attached to the kit according to the attached protocol.

  The time required for purification using a soft conventional membrane filter took about 20 minutes, but was shortened to about 10 minutes in the method using a solid monolith structure.

  FIG. 14 shows the result of electrophoresis of purified circular DNA from Escherichia coli plasmid DNA (circular DNA): pUC119 vectors. M is a molecular weight marker, the conventional method is 1, and the method of the present invention is 2. It can be seen that the purified plasmid DNA of 3.7 kb is obtained with a higher degree of purification in the method of the present invention than in the conventional method.

  As described above, when the method of the present invention was used, it was proved that circular plasmid DNA could be highly purified from E. coli which is a biological sample with a high recovery rate.

(A) Side view of one embodiment of the present invention (b) Side view of one embodiment of the present invention (c) Use state explanatory drawing of the present invention The present invention-Example slope view (A) Main part electron microscope enlarged explanatory view of FIG. 1 (b) Enclosed part electron microscope enlarged explanatory view of FIG. 3 (a) Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention Example of the present invention Sequence data Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention Example of the present invention Electrophoretic diagram of the embodiment of the present invention Electrophoretic diagram of the embodiment of the present invention

Explanation of symbols

1 Monolith structure 2 Pipette 3 Tube 4 Well plate 5 Cartridge

Claims (6)

A method of separating and purifying DNA and the like by capturing and eluting a biological sample solution by passing it through a three-dimensional network monolithic separator having silanol groups attached to a container. The method for separating and purifying DNA or the like according to claim 1, wherein a separated body of a pressure-resistant monolith structure having a hardness of 30 Hk or more is used. The method for separating and purifying DNA or the like according to claim 1 or 2, wherein a separated body of a non-porous monolith structure cured by firing is used. A mechanism for separating and purifying DNA or the like, wherein a separation body of a monolith structure is formed and formed in a liquid passing part such as a disposal container. A separation mechanism of DNA or the like, wherein a monolith structure separated body is fused and fixed to a liquid passing part such as a disposable container. A mechanism for separating and purifying DNA or the like, wherein a monolith structure separated body is provided in each hole of a well plate.

Images