JP4080462B2 - Nucleic acid recovery method - Google Patents

Description

  The present invention relates to a method for recovering nucleic acid from a substance containing nucleic acid. More specifically, an automatic recovery device for nucleic acid components present as endogenous or exogenous genes from body fluid components for genetic diagnosis by nucleic acid testing, or genetically modified E. coli for nucleic acid sequencing The present invention relates to a method for recovering nucleic acid suitable for an apparatus for automatically recovering plasmid DNA.

  Advances in molecular biology have developed a number of gene-related technologies, and these technologies have isolated and identified many diseased genes. As a result, even in the medical field, molecular biology techniques have been incorporated into diagnosis or examination methods, making it possible to make diagnoses that were not possible in the past, and greatly reducing the number of examination days.

  This rapid progress largely depends on the nucleic acid amplification method, particularly the PCR method (Non-patent Document 1).

  Since the PCR method can amplify nucleic acids in solution in a sequence-specific manner, for example, a virus that is present in a very small amount in serum is indirectly detected by amplifying the nucleic acid that is the gene of the virus. Can prove the existence of the virus.

  However, there are some problems when this PCR method is used for daily examinations in clinical settings. Among these, it is pointed out that the difficulty of extraction and purification steps of nucleic acids in pretreatment is a problem, and that the extraction and purification steps of these nucleic acids are important (Non-patent Document 2).

  This is caused by the influence of an inhibitor that could not be removed during nucleic acid purification. Known inhibitors include hemoglobin in blood, surfactants used in the extraction process, and the like.

  Moreover, regarding an extraction process, complicated operation and the great effort by an expert are required. Therefore, it has become an obstacle when introducing a new genetic test into a hospital laboratory, and the automation of this process is eagerly desired.

  In addition, plasmid DNA is frequently used as a material for genetic recombination in institutions conducting molecular biological research, but from the point of labor saving, etc., as in the laboratory, It is desired to automate the process of extracting and purifying nucleic acids.

  In addition to the method using the aqueous phase / organic phase separation described above, other methods for recovering the nucleic acid can be obtained from an agarose gel by utilizing the property of nucleic acid binding to the glass surface in the presence of a chaotropic substance. A method for recovering DNA (Non-patent Document 3) or a method for recovering plasmid DNA from E. coli (Non-patent Document 4) has been reported.

  Furthermore, Patent Document 1 describes a method for simplifying the operation for the purpose of recovering nucleic acids from biological materials.

Conventionally, in order to efficiently perform an operation for washing a solid phase at room temperature, it is recommended to use ethanol having a concentration of at least 75% (Non-Patent Document 5).
JP-A-2-289596 polymerase chain reaction: polymerase chain reaction, Saiki et al. , Science, 239, 487-491 (1988). Oshima et al., JJCLA, 22 (2), 145-150 (1997) B. Vogelstein and D.W. Gillespie, Proc. Natl. Acad. Sci. USA, 76 (2), 615-619 (1979) M.M. A. Marko, R.A. Chipperfield and H.M. C. Birnboim, Anal. Biochem. 121, 382-387 (1982) C. W. Chen and C.I. A. Thomas, Jr. , Anal. Biochem. , 101, 339-341 (1980)

  By the way, as a known method for recovering nucleic acid from a biological sample containing nucleic acid in a highly purified state containing no inhibitor, a surfactant is allowed to act on the biological sample in the presence of a proteolytic enzyme, A method is known in which nucleic acid is freed, mixed with phenol (and chloroform), separated into aqueous and organic phases by a centrifuge several times, and then recovered from the aqueous phase in the form of a precipitate with alcohol. It has been.

  However, this preparation method has a problem associated with using an organic solvent such as phenol which is a toxic substance in the process. That is, an organic solvent such as phenol may dissolve the plastic part of the device used for nucleic acid recovery, and may deteriorate the device.

  Furthermore, after using an organic solvent such as phenol, the treatment necessary for its disposal is complicated.

  In the method described in Patent Document 1, a biological sample is mixed with a sufficiently large amount of a chaotropic substance (for example, guanidine salt) and silica particles, and nucleic acid can be rapidly recovered by binding the released nucleic acid to a solid phase.

  However, in order to obtain a nucleic acid in a purified state, it is necessary to remove the guanidine salt from the solid phase to which the nucleic acid is bound while maintaining the state bound to the solid phase. It was not shown.

  In the technique described in Non-Patent Document 5, since ethanol is a volatile component, a lid opening / closing mechanism and a cooling mechanism for preventing evaporation are required when the apparatus is built, and the apparatus is increased in size and reliability is reduced. Invite.

  Further, cleaning with 70% ethanol or acetone is required. For this reason, an operation for removing acetone by drying is required. However, since 70% or more of ethanol and acetone are volatile, when mounted on an automatic device, as described above, a highly airtight container and lid opening / closing mechanism or / and volatilization is not possible. A cooling mechanism to prevent is needed.

  In addition, the use of acetone may dissolve the plastic part of the device used for nucleic acid recovery, as described above, due to its property as a strong organic solvent, and may deteriorate the device.

  Therefore, the materials used for the apparatus, container, and dispenser to be used are significantly limited. Moreover, since it is an acute toxic substance and a flammable substance, it is necessary to sufficiently prevent the release of acetone into the environment accompanying the evaporation operation.

  An object of the present invention is to realize a method and an apparatus for recovering nucleic acid that can be recovered from a nucleic acid-containing material quickly and easily without reducing the yield of highly purified nucleic acid.

  Another object of the present invention is to realize a nucleic acid recovery method suitable for an apparatus capable of automatically recovering a nucleic acid component present in a biological sample.

  Furthermore, another object of the present invention is to realize a nucleic acid recovery method suitable for an apparatus capable of automatically recovering plasmid DNA from E. coli.

In order to achieve the above object, the present invention is configured as follows.
(1) A method for recovering nucleic acid from a nucleic acid-containing material, wherein a mixed solution containing a chaotropic substance and a nucleic acid-containing material is brought into contact with a silicon oxide-containing solid phase to separate the silicon oxide-containing solid phase from the mixed solution. The silicon oxide-containing solid phase is washed with a solution containing a chaotropic substance, and then washed with an aqueous solution containing acetate and no alcohol, and nucleic acids are eluted from the silicon oxide-containing solid phase.

( 2 ) Preferably, in (1) above, the nucleic acid recovery method is characterized in that the acetate is potassium acetate.
(3) Preferably, in the above (2), the concentration of the potassium acetate is 0.2 to 1 mol / liter.

(4) Preferably, in (1) above, the acetate salt is sodium acetate.
  (5) Preferably, in the above (4), the concentration of the sodium acetate is 0.2 to 1 mol / liter.

(6) Preferably, in the above (1) , the mixed solution and the silicon oxide-containing solid phase are stirred at room temperature, and the mixed solution and the silicon oxide-containing solid phase are brought into contact with each other.

(7) Preferably, in the above (1) , the silicon oxide-containing solid phase is glass particles, silicon oxide powder, quartz filter paper, quartz wool, crushed materials thereof, or diatomaceous earth.

(8) Preferably, in the above (1) , the nucleic acid does not elute from the silicon oxide-containing solid phase during washing.

(9) Preferably, in the above (1) , the silicon oxide-containing solid phase is a particle having a particle size of about 1 to 100 μm.

  ADVANTAGE OF THE INVENTION According to this invention, the collection method of the nucleic acid which can collect | recover the nucleic acid from the material containing a nucleic acid rapidly, easily and highly purified, without reducing a yield is realizable. That is, a nucleic acid having a purity suitable for genetic testing or gene analysis can be collected quickly and easily without using a dangerous substance.

  In addition, it is possible to realize a nucleic acid recovery method suitable for an apparatus capable of automatically recovering a nucleic acid component present in a biological sample.

  Furthermore, a nucleic acid recovery method suitable for an apparatus that can automatically recover plasmid DNA from E. coli can be realized.

(Embodiment 1)
Recovery of nucleic acid when a washing step using a 1 mol / liter salt aqueous solution was employed According to the flowchart of FIG. 1, nucleic acid was recovered from an aqueous solution containing nucleic acid. As a nucleic acid-containing material, commercially available purified pBR322 DNA was diluted with TE buffer (pH 7.4).

  In this case, since the nucleic acid is already in the free state in the nucleic acid-containing material, the first step, which is a step for promoting the release of the nucleic acid from the nucleic acid-containing material, was skipped. In the first step, the free material usually used includes a surfactant and an enzyme. As means for promoting the release of nucleic acid from the nucleic acid-containing material, there are means for applying heat, means for applying ultrasonic waves, and the like.

  The second step is a step of mixing the released nucleic acid and a substance that promotes the binding of the nucleic acid to the solid phase. In a reaction vessel of 1.5 ml capacity, 6 mol / 900 μL of a liter GuHCl solution was added and stirred.

  The third step is a step of bringing the mixed solution into contact with the nucleic acid-binding solid phase, adding 100 mg of glass particles as the nucleic acid-binding solid phase, and stirring the solution with a rotary mixer at room temperature. Liquid contact was made.

  The fourth step is a step of separating the solid phase to which the nucleic acid is bound from the liquid. For this purpose, a centrifuge is used to obtain a solid phase precipitate by centrifugation at 15000 g for 1 minute, and the supernatant is discarded. .

  In the fifth step, the solid phase to which the nucleic acid was bound was washed with a salt-containing solution. The solid phase was suspended in a 1 mol / liter salt aqueous solution, and the supernatant was discarded.

  The sixth step is a step of eluting the nucleic acid from the solid phase to which the nucleic acid is bound, adding TE buffer (pH 8.0) to the solid phase, elution by heating at 60 ° C. for 1 minute, 15000 g, The supernatant was obtained by centrifugation for 1 minute. Electrophoresis was performed using 0.8% agarose gel as a sample, and after ethidium bromide staining, the intensity of each band was quantified by densitograph, and the recovery rate was based on the amount of nucleic acid in the first step. Was calculated.

  The results are shown in Table 1. Nucleic acids can be effectively retained during washing by using chloride and acetate.

  As described above, Embodiment 1 of the present invention is a first step for promoting the release of nucleic acid from a nucleic acid-containing material, and a second step of mixing a released nucleic acid and a substance that promotes binding of the nucleic acid to a solid phase. A third step of bringing the mixed solution into contact with the nucleic acid-binding solid phase, a fourth step of separating the solid phase and the liquid, and a fifth step of washing the solid phase to which the nucleic acid is bound with a solution containing a salt, And a sixth step of eluting the nucleic acid from the solid phase.

  Therefore, according to Embodiment 1 of the present invention, it is possible to realize a method for recovering nucleic acid that can recover a nucleic acid containing a nucleic acid quickly, easily, and highly purified without reducing the yield. . That is, a nucleic acid having a purity suitable for genetic testing or gene analysis can be collected quickly and easily without using a dangerous substance.

  In addition, it is possible to realize a nucleic acid recovery method suitable for an apparatus capable of automatically recovering a nucleic acid component present in a biological sample.

  Furthermore, a nucleic acid recovery method suitable for an apparatus that can automatically recover plasmid DNA from E. coli can be realized.

(Embodiment 2)
Recovery of nucleic acid when a washing step using a solution containing sodium chloride, potassium chloride, sodium acetate, and potassium acetate is adopted According to the flowchart of FIG. 2, nucleic acid was recovered from an aqueous solution containing nucleic acid. In the flowchart of FIG. 2, the first, second, third, fourth and sixth steps are the same as the flowchart of FIG. 1, and the fifth step of FIG. It is divided into a process and a 5b process.

  In the second embodiment, as in the first embodiment, as a nucleic acid-containing material, commercially available purified pBR322 DNA was diluted with a TE buffer (pH 7.4).

  The steps from the first step to the fourth step are the same as those in FIG.

  Step 5a is a step in which the solid phase to which the nucleic acid is bound is washed with a salt-containing solution, and the non-nucleic acid component is washed from the solid phase. The solid phase is washed with 1000 μl of a 6 mol / liter GuHCl (guanidine hydrochloride) solution. The solid phase was washed by suspending, obtaining a solid phase precipitate by centrifugation at 15000 g for 1 minute, and discarding the supernatant.

  Step 5b is a step of washing the solid phase to which the nucleic acid has been bound with a salt-containing solution, and washing substances that promote the binding of the nucleic acid to the solid phase of the nucleic acid, with different concentrations of NaCl, KCl, NaOAc The solid phase was suspended with an aqueous KOAc solution, and the supernatant was discarded.

  In the sixth step, TE buffer solution (pH 8.0) is added to the solid phase, elution is carried out by heating at 60 ° C. for 1 minute, and a supernatant is obtained by centrifugation at 15000 g for 1 minute. Then, electrophoresis was performed using 0.8% agarose gel as a support, and after staining with ethidium bromide, the intensity of each band was digitized by densitograph, and the recovery rate was calculated based on the amount of nucleic acid in the first step.

  The results are shown in FIG. With NaCl and KOAc, a recovery rate of 50% or more is obtained at a concentration of 500 mmol / liter or more, and with Kcl, a recovery rate of 50% or more is obtained at a concentration of 200 mmol / liter or more. I understand that.

  As described above, also in the second embodiment of the present invention, the same effect as in the first embodiment described above can be obtained.

(Embodiment 3)
Recovery of nucleic acid when a washing process using a solution containing sodium acetate and sodium chloride is employed According to the flowchart of FIG. 3, nucleic acid was recovered from an aqueous solution containing nucleic acid. In the flowchart of FIG. 3, the first, second, third, fourth, fifth, and sixth steps are the same as those in the flowchart of FIG. Therefore, the description from the first step to the fifth step is omitted.

  In step 5b, the solid phase was suspended with KOAc, NaCl and 40% ethanol aqueous solution having different concentrations, and the supernatant was discarded. In the sixth step, TE buffer (pH 8.0) was added to the solid phase, elution was performed by heating at 60 ° C. for 1 minute, and a supernatant was obtained by centrifugation at 15000 g for 1 minute. The seventh step is a step of removing alcohol, and ethanol was removed by heating and heating at 60 ° C. to obtain a purified nucleic acid aqueous solution.

  Using this portion as a sample, electrophoresis is performed using 0.8% agarose gel as a support, and after ethidium bromide staining, the intensity of each band is digitized using a densitograph, and the recovery rate is calculated based on the amount of nucleic acid in the first step. did.

  The results are shown in FIG. By combining with 40% ethanol, nucleic acid can be effectively recovered at a KOAc of 10 mmol / liter or more, or a NaCl concentration of 25 mmol / liter or more.

As described above, also in the third embodiment of the present invention, the same effect as in the first embodiment described above can be obtained.
(Embodiment 4)
Recovery of Nucleic Acid When Ethanol Concentration Changed When Using Ethalol, Sodium Acetate, and Sodium Chloride Solution in Washing Step According to the flowchart of FIG. 3, nucleic acid was recovered from an aqueous solution containing nucleic acid. In the flowchart of FIG. 3, the first, second, third, fourth, fifth, and sixth steps are the same as those in the flowchart of FIG. Therefore, the description from the 1st process to the 5a process is omitted.

  Step 5b is a step of washing the solid phase bound to the nucleic acid with a mixed solution of a salt-containing solution and alcohol to wash a substance that promotes binding of the nucleic acid to the solid phase of the nucleic acid. In this step 5b, the solid phase was suspended with 25 mmol / liter, 50 mmol / liter, or 100 mmol / liter of an aqueous solution of ethanol having a different concentration from NaCl, and the supernatant was discarded.

  In the sixth step, TE buffer (pH 8.0) was added to the solid phase, elution was performed by heating at 60 ° C. for 1 minute, and a supernatant was obtained by centrifugation at 15000 g for 1 minute. The seventh step was a step of removing alcohol, and ethanol was removed by heating at 60 ° C. to obtain a purified nucleic acid aqueous solution.

  Using this portion as a sample, electrophoresis is performed using 0.8% agarose gel as a support, and after ethidium bromide staining, the intensity of each band is digitized using a densitograph, and the recovery rate is calculated based on the amount of nucleic acid in the first step. did.

  The results are shown in FIG. By combining 50 mmol / liter or more of KOAc or 100 mmol / liter or more of NaCl and ethanol, it is possible to effectively suppress a reduction in the recovery rate when the ethanol concentration fluctuates.

As described above, also in the fourth embodiment of the present invention, the same effect as in the first embodiment described above can be obtained. In this Embodiment 4, ethanol is used, but it is an aqueous solution of 25 mmol / liter, 50 mmol / liter, or 100 mmol / liter NaCl and ethanol having different concentrations. Such a problem does not occur.
(Embodiment 5)
Recovery of DNA added to serum Nucleic acid added to serum was recovered according to the flowchart of FIG.

  A sample obtained by adding commercially available purified pBR322 DNA to human serum was used as a sample. Although the nucleic acid is already free, sodium dodecyl sulfate was added in the first step to prevent nuclease.

  Since the second process to the fifth process are the same as those in the fourth embodiment, the description thereof is omitted.

  In step 5b, the solid phase was suspended in a 50% aqueous ethanol solution containing 50 mmol / liter of KOAc, and the supernatant was discarded.

  In the sixth step, TE buffer (pH 8.0) was added to the solid phase, elution was performed by heating at 60 ° C. for 1 minute, and a supernatant was obtained by centrifugation at 15000 g for 1 minute. In the seventh step, ethanol was removed by heating at 60 ° C. to obtain a purified nucleic acid aqueous solution.

  Using this portion as a sample, electrophoresis is performed using 0.8% agarose gel as a support, and after ethidium bromide staining, the intensity of each band is digitized using a densitograph, and the recovery rate is calculated based on the amount of nucleic acid in the first step. did. The recovery rate was 70%, and A260 / A280 was 1.90. Moreover, the time for processing the first to seventh steps was about 30 minutes.

  In the fifth embodiment, the same effect as in the first embodiment can be obtained. Although ethanol is used in the fifth embodiment, since it is a 50% ethanol aqueous solution containing 50 mmol / liter of KOAc, there is no problem as in the prior art.

(Embodiment 6)
Recovery of plasmid DNA from cultured recombinant E. coli Plasmid DNA was recovered from the cultured recombinant E. coli according to the flowchart of FIG. Escherichia coli into which pBR322 DNA was genetically engineered was used as a sample for the E. coli HB101 strain. A product obtained by culturing E. coli in 1 ml of LB medium overnight at 37 ° C. was collected by centrifugation, and a sample suspended in 100 μl of 0.15 mol / l NaCl was used as a sample.

  In the first step, 8 mg / ml in 50 mmol / liter glucose, 10 mmol / liter EDTA, 25 mmol / liter Tris-HCl (pH 8.0) was used to destroy peptidoglycan in the cell wall of E. coli. A solution containing lysozyme was added and mixed.

  Further, 0.2 mol / liter NaOH, 1% SDS solution was added to free the nucleic acid. To this solution was added 5 mol / liter potassium acetate.

  In the second step, 1000 μL of a 6 mol / L GuHCl solution was added to the nucleic acid-containing aqueous solution and stirred.

  In the third step, 150 mg of glass particles were added as the nucleic acid-binding solid phase, and the solid phase and the liquid were brought into contact with each other by stirring the solution with a rotary mixer at room temperature.

  In the fourth step, a centrifuge was used to separate the solid phase to which the nucleic acid was bound from the liquid, and a solid phase precipitate was obtained by centrifugation at 15000 g for 1 minute, and the supernatant was discarded.

  In step 5a, the solid phase was washed by suspending the solid phase with 1000 μl of a 6 mol / liter GuHCl solution, obtaining a solid phase precipitate by centrifugation at 15000 g for 1 minute, and discarding the supernatant. .

  In step 5b, the solid phase was suspended in a 50% aqueous ethanol solution containing 50 mmol / liter of KOAc, and the supernatant was discarded.

  In the sixth step, TE buffer (pH 8.0) was added to the solid phase, elution was performed by heating at 60 ° C. for 1 minute, and a supernatant was obtained by centrifugation at 15000 g for 1 minute. In the seventh step, ethanol was removed by heating at 60 ° C. to obtain a purified nucleic acid aqueous solution. Electrophoresis using 0.8% agarose gel as a support, using this portion as a support, and obtaining an electrophoresis band of nucleic acid by ethidium bromide staining, and confirming the bands of plasmid DNA and rRNA from the transferred amount of marker It was done.

  In this state, A260 / A280 was 1.96. Moreover, the yield of the obtained plasmid DNA was 4.5 micrograms by calculation from the A260 light absorbency after a ribonuclease process and an ethanol precipitation process. The time for processing the first to seventh strokes was about 45 minutes.

  Also in the sixth embodiment, the same effect as in the first embodiment described above can be obtained. In this Embodiment 6, ethanol is used, but since it is a 50% ethanol aqueous solution containing 50 mmol / liter of KOAc, there is no problem as in the prior art.

(Embodiment 7)
Recovery of DNA added to serum by nucleic acid recovery apparatus which is an automatic apparatus FIG. 9 shows a planar layout of a nucleic acid recovery apparatus (automatic apparatus) which is Embodiment 7 of the present invention.

  In FIG. 9, the stirring device 101 can move in the circumferential direction and the vertical direction by mechanical control, and can move between the particle suction port 107 and the washing place 108. The pipettor A102 and the pipettor B103 can move in the circumferential direction and the vertical direction by machine control, and have a nozzle 118 and a nozzle 119 for installing disposable tips, respectively.

  The reaction container transfer mechanism 104 can transfer the reaction container 120 to positions A, B, and C by mechanical control, and permanent magnets 122 are installed on both sides of the position C according to the size of the reaction container 120. ing. The purified product storage container transfer mechanism 105 can transfer the purified product storage container 121 to the positions D and E by machine control.

  According to the flowchart of FIG. 3, the nucleic acid added to the serum was recovered by the apparatus shown in FIG.

  A sample obtained by adding commercially available purified product pBR322 DNA to human serum was dispensed into a reaction container 120 in a predetermined amount and placed at position A. Although the nucleic acid is already free, SDS was added in the first step to prevent nuclease.

  In the second step, the reaction vessel 120 is moved to the position B by the reaction vessel transfer mechanism 104, and the pipettor A118 is attached to the tip at the tip 102 at the tip attachment position 106 by rotating in the circumferential direction. Move to the mouth 109 and aspirate 900 μL of 8 mol / L GuHCl solution for 100 μL of aqueous nucleic acid solution.

  Further, the pipetter A118 was moved to the position B, GuHCl was discharged into the reaction vessel 120, and further, suction and discharge operations were performed a plurality of times to perform stirring. Thereafter, the pipette A102 was moved to the tip disposal position 116, and the tip was discarded from the nozzle 118.

  In the third step, a magnetic particle solution is used as the nucleic acid-binding solid phase, the contents are stirred from the magnetic particle suction part 107 by the stirring mechanism 101, and the tip newly attached to the nozzle 118 of the pipettor A102 at the tip mounting position 106. To suck a predetermined amount. Thereafter, the pipette A102 was moved to the position B, the chip contents were discharged to the reaction vessel 120, and further, suction and discharge were performed and stirring was performed. The stirring mechanism 101 moved to the cleaning unit 108 to perform cleaning, and the pipetter A102 moved to the tip discarding position 116, and the tip was discarded from the nozzle 118.

  In the fourth step, the reaction vessel 120 is moved from the position B to the position C by the reaction vessel transfer mechanism 104 in order to separate the solid phase and the liquid to which the nucleic acid is bound, and the permanent magnet 122 moves the inside of the reaction vessel 120. Magnetic particles are captured on the side wall of the container. Then, the pipetter B103 is moved to the tip attachment position 113 by rotation, the tip is attached to the nozzle 119, and then moved to the position C to suck the liquid phase in the reaction vessel 120. Furthermore, it moved to the solution disposal position 114, discarded the solution in the chip, moved to the chip disposal position 115, and discarded the chip from the nozzle 119.

  In step 5a, the reaction vessel 120 is moved from the position C to the position B by the reaction vessel transfer mechanism 104, the pipette A102 is moved to the tip attachment position 106, and the tip is attached to the nozzle 118. After that, it moves to the GuHCl suction port 111, sucks a predetermined amount, moves to the position B, and discharges GuHCl into the reaction vessel 120. After stirring by this suction / discharge operation, it moved to the tip disposal position 116 and the tip was discarded from the nozzle 118.

  Further, the reaction container 120 is moved from the position B to the position C by the reaction container transfer mechanism 104, and the magnetic particles in the reaction container 120 are captured on the container side wall by the permanent magnet 122. Then, the pipettor B103 is moved to the tip attachment position 113 by rotation, and after the tip is attached to the nozzle 119, it is moved to the position C, and the liquid phase in the reaction vessel 120 is sucked. Next, it moved to the solution disposal position 114, discarded the solution in the chip, and further moved to the chip disposal position 115 to discard the chip from the nozzle 119.

  In the step 5b, the reaction vessel 120 is moved from the position C to the position B by the reaction vessel transfer mechanism 104. Next, the pipette A102 is moved to the tip attachment position 106, and after the tip is attached to the nozzle 118, it is moved to the 50% ethanol aqueous solution inlet 110 containing 50 mmol / liter KOAc, and a predetermined amount is sucked. After that, it moves to the position B, discharges a 50% ethanol aqueous solution containing 50 mmol / liter KOAc into the reaction vessel 120, and after stirring by suction / discharge operation, moves to the tip disposal position 116 and from the nozzle 118 to the tip. Was discarded.

  Further, the reaction container 120 is moved from the position B to the position C by the reaction container transfer mechanism 104, and the magnetic particles in the reaction container 120 are captured on the container side wall by the permanent magnet 122. Next, the pipettor B103 is moved to the tip attachment position 113 by rotation, and after the tip is attached to the nozzle 119, it is moved to the position C, the liquid phase in the reaction vessel 120 is sucked and moved to the solution disposal position 114. Discard the solution in the chip. Furthermore, it moved to the chip disposal position 115 and the chip was discarded from the nozzle 119. This process was further repeated a predetermined number of times.

  In the sixth step, the reaction vessel 120 is moved from the position C to the position B by the reaction vessel transfer mechanism 104, the pipette A102 is moved to the tip attachment position 106, and the tip is attached to the nozzle 118. After moving to the buffer solution (pH 8.0) suction port 109 and sucking a predetermined amount, it moves to the position B. Then, the TE buffer solution heated to 60 ° C. was discharged into the reaction vessel 120, stirred by suction / discharge operation, moved to the chip disposal position 116, and the chip was discarded from the nozzle 118.

  Further, the reaction vessel 120 is moved from the position B to the position C by the reaction vessel transfer mechanism 104, the magnetic particles in the reaction vessel 120 are captured on the side wall of the vessel by the permanent magnet 122, and the pipettor B103 is moved to the tip attachment position 113 by rotation. To do. Next, after attaching a chip to the nozzle 119, the nozzle moves to the position C, sucks the liquid phase in the reaction vessel 120, and then moves to the position D. Then, the solution was discharged into the refined product storage container 121, and this process was repeated a predetermined number of times.

  In the seventh step, the purified product storage container 121 was moved to the position E on the heat block 112 at 60 ° C. by the purified product storage container transfer mechanism 105 and heated for a predetermined time to obtain a purified nucleic acid aqueous solution.

Using this portion as a sample, electrophoresis was performed using 0.8% agarose gel as a support, and after ethidium bromide staining, the intensity of each band was quantified by densitograph, and the recovery rate was calculated based on the amount of nucleic acid in the first step. Calculated. As a result, the recovery rate was 65%. Moreover, the time for processing the first to seventh steps was about 30 minutes.
As described above, according to Embodiment 7 of the present invention, it is possible to realize a nucleic acid recovery apparatus that recovers nucleic acid quickly and easily from a material containing nucleic acid without reducing the yield. . In Embodiment 7, ethanol is used. However, since it is a 50% ethanol aqueous solution containing 50 mmol / liter KOAc, there is no problem as in the prior art.

  Next, a comparative example for comparing the present invention with the prior art will be described.

(Comparative example)
In the same manner as in the conventional example, the nucleic acid was recovered from the aqueous solution containing the nucleic acid according to the flowchart of FIG. As a nucleic acid-containing material, commercially available purified pBR322 DNA was diluted with TE buffer (pH 7.4).

  In this case, since the nucleic acid is already in the free state in the nucleic acid-containing material, the first step, which is a step for promoting the release of the nucleic acid from the nucleic acid-containing material, was skipped. The second step is a step of mixing the released nucleic acid and a substance that promotes the binding of the nucleic acid to the solid phase, and in a 1.5 ml capacity reaction vessel, 6 mol / 900 μL of a liter GuHCl solution was added and stirred.

  The third step is a step in which the mixed solution and the nucleic acid-binding solid phase are brought into contact with each other. 100 mg of glass particles are added as the nucleic acid-binding solid phase, and the solution is stirred with a rotary mixer at room temperature. Liquid contact was made.

  The fourth step is a step of separating the solid phase to which the nucleic acid is bound from the liquid, and using a centrifuge to separate the solid phase to which the nucleic acid is bound from the liquid, centrifuge at 15000 g for 1 minute. To obtain a solid phase precipitate and discard the supernatant.

  The fifth step is a step of washing the solid phase to which the nucleic acid has been bound with an aqueous ethanol solution, suspending the solid phase with various aqueous ethanol solutions having different concentrations, and obtaining a solid phase precipitate by centrifugation at 15000 g for 1 minute. The solid phase was washed by the operation of discarding the liquid.

  The sixth step is a step of releasing nucleic acid from the solid phase to which the nucleic acid is bound. TE buffer (pH 8.0) is added to the solid phase, and elution is carried out by heating at 60 ° C. for 1 minute. The supernatant was obtained by centrifugation for 1 minute. Then, this part was used as a sample, electrophoresis was performed using 0.8% agarose gel as a support, and ethidium bromide staining was performed. Thereafter, the intensity of each band was digitized using a densitograph, and the recovery rate was calculated based on the amount of nucleic acid in the first step.

  The results are shown in FIG. It was confirmed that the recovery rate decreased at an ethanol concentration of around 80%. In other words, the recovery rate of nucleic acid is the highest at an ethanol concentration of 80%, but a high concentration of ethanol must be used. As described above, 70% or more of ethanol is volatile, so an automatic device When mounted on top, a highly airtight container and lid opening / closing mechanism and / or a cooling mechanism for preventing volatilization are required, which complicates the apparatus configuration.

On the other hand, the above-described Embodiments 1 to 7 of the present invention are a nucleic acid recovery method and apparatus that can recover a nucleic acid-containing material quickly and easily without reducing the yield of highly purified nucleic acid. Can be realized.
The present invention can be applied to any material containing nucleic acid, but in particular, clinical specimens such as whole blood, serum, sputum, urine, or biological samples such as cultured cells and cultured bacteria, Or the nucleic acid etc. of the state hold | maintained at the gel after electrophoresis are suitable.

  In addition, the method of the present invention is also effective for materials containing reaction products such as DNA amplification enzymes and nucleic acids in an unpurified state. The nucleic acid referred to here is double-stranded, single-stranded, or partially double-stranded or DNA or RNA having a single-stranded structure.

  Further, in the first step of the present invention, the method for promoting the release of nucleic acid from the nucleic acid-containing material is a physical method using a mortar, ultrasonic wave, microwave, or the like, or a chemical method using a surfactant, a denaturant, or the like. Or a biochemical method using a proteolytic enzyme, a method combining them, or the like can be used.

  In the second step of the present invention, substances that promote the binding of nucleic acid to the solid phase include NaI (sodium iodide), KI (potassium iodide), NaClO4, NaSCN (sodium thiocyanate), GuSCN (thiocyanate). Acid guanidine), etc. are suitable, but since these substances have a peak at around 260 nm where the nucleic acid absorbs, they are generally used when mixed in a purified nucleic acid solution. This may have an adverse effect on the result of assaying the purity and amount of nucleic acid using a spectrophotometer.

  In addition, NaSCN and GuSCN containing thiocyanic acid generate lethal HCN (hydrogen cyanide) by reacting with an acid, so that attention is required in handling the waste liquid.

  Therefore, in the present invention, it is desirable to use GuHCl (guanidine hydrochloride) as a substance that promotes the binding of nucleic acids to the solid phase. GuHCl is readily available and has a significantly lower adverse effect on the absorbance at 260 nm than the chaotropic agent. Also, there is no potential danger of generating lethal gas.

  In the present invention, in order to specifically bind the nucleic acid component to the solid phase, the final concentration of GuHCl is desirably 4 to 6 mol / liter.

  The nucleic acid-binding solid phase in the third step of the present invention should be used as long as it contains silicon oxide such as glass particles, silica powder, quartz filter paper, quartz wool, crushed materials thereof, diatomaceous earth, and the like. However, it is possible to improve the binding efficiency or shorten the binding time by increasing the contact probability between the nucleic acid and the solid phase in the mixed solution of nucleic acid and chaotropic agent. Is desirable.

  In the present invention, a particle size of about 1 to 100 μm is suitable. In order to increase the contact probability between the nucleic acid and the solid phase, it is desirable to perform a stirring operation according to the specific gravity of the particles to be used.

  Further, as a means for separating the solid phase and the liquid in the fourth step of the present invention, separation can be performed by centrifugation or filtration. However, in the automation and instrumentation, an immunoanalyzer utilizing an antigen-antibody reaction The known B / F (bond form / free form) separation technique generally used in the above can be applied.

  In the fifth step of the present invention, the means for washing the solid phase to which the nucleic acid is bound with a solution containing a salt maintains the binding of the nucleic acid to the solid phase with respect to the solid phase after the completion of the fourth step. Washing can be performed by mixing a solution containing a salt for washing the solid phase and separating the solid phase and the liquid as in the fourth step.

  The fifth step promotes the binding of the non-nucleic acid component nonspecifically bound to the solid phase from the solid phase 5a and the binding of the nucleic acid to the solid phase from the solid phase. Divided into step 5b of washing the material. At this time, it is important that the liquid used for washing the solid phase in the steps 5a and 5b does not elute the nucleic acid bound to the solid phase, which will increase the yield of nucleic acid finally obtained. affect.

  In the present invention, ethanol having a concentration of 75% or more is not used as a cleaning liquid. However, as the cleaning liquid used in the step 5a, GuHCl is suitable as in the fourth step. . This is because GuHCl has a denaturing agent action, and nonspecifically adsorbed substances can be removed from the solid phase when used at a concentration of 4 to 6 mol / liter.

  The washing liquid used in step 5b is preferably a salt that does not adversely affect the use of the nucleic acid after elution and can prepare an aqueous solution having a medium concentration. This is because the nucleic acid that is in a non-solution state on the surface of the solid phase becomes difficult to dissolve in an aqueous solution having a high salt concentration. For example, NaCl (sodium chloride) is added at a concentration of 500 mM or more. It is desirable to use it. Alternatively, a mixed solution of an aqueous solution containing a salt and an alcohol can be used in the step 5b.

  In this case, it is possible to make the concentration even lower than when the salt is used alone, and it is tolerant to changes in the alcohol concentration due to volatilization of the alcohol component. In the present invention, it is effective to use a 50% ethanol aqueous solution containing 50 mmol / liter or more of NaCl.

  In addition, when a solution containing alcohol is used in Step 5b, a seventh step for removing a trace amount of alcohol components that may be mixed in the purified nucleic acid solution is added after Step 6. It will be necessary. This seventh step can be achieved by heating the solution.

  The means for eluting the nucleic acid from the solid phase in the sixth step of the present invention is achieved by mixing an aqueous solution having a low salt concentration equal to or higher than the volume of the solid phase or water with the washed solid phase. As a result of this operation, the nucleic acid moves from the solid phase to the aqueous phase. Thus, a purified nucleic acid aqueous solution can be obtained by separating the solid phase and the liquid as in the fourth step.

  The low salt concentration aqueous solution or water used in the present invention is preferably in a sterilized state, or it is desired to use a product subjected to DEPC treatment if necessary.

  In addition, the low salt concentration aqueous solution or water used here is preferably used in a state heated to 55 to 60 ° C. in order to increase the yield of the nucleic acid to be obtained. Even when the container used at the time is heated to the same temperature, the same effect can be obtained.

  In the present invention, it is desirable to perform the sixth step at least twice in order to increase the yield of nucleic acid.

  Moreover, although the washing | cleaning of a 5th process is performed with the washing | cleaning liquid containing an acetate or a chloride, acetate can be made into the potassium acetate aqueous solution which has a density | concentration of 0.5 mol / l or more. The chloride may be sodium chloride having a concentration of 0.5 mol / liter or more, or potassium chloride having a concentration of 0.2 mol / liter or more.

  Further, in the step 5b, the solution for washing can be a solution containing 40% ethanol and 10 mmol / liter or more of potassium acetate.

  Further, in the step 5b, the solution for washing can be a solution containing 40% ethanol and 25 mmol / liter or more of sodium chloride.

  The nucleic acid-binding solid phase used in the third step can be a substance containing silicon dioxide.

It is an operation | movement flowchart for performing the collection | recovery of the nucleic acid in Embodiment 1 of this invention. It is an operation | movement flowchart for performing collection | recovery of the nucleic acid including the washing | cleaning process by the solution containing sodium chloride etc. in Embodiment 2 of this invention. 6 is an operation flowchart for collecting a nucleic acid including a washing step in Embodiments 3, 4, 5, and 6 of the present invention. It is an operation | movement flowchart for performing the collection method of the nucleic acid which is the collection method of the nucleic acid of a prior art, and includes the washing | cleaning process only by ethanol aqueous solution. It is a graph showing the result of the washing | cleaning process by ethanol in a prior art. It is a graph showing the result of the washing | cleaning process by Embodiment 2 of this invention. It is a graph showing the result of the washing | cleaning process by Embodiment 3 of this invention. It is a graph showing the result of the washing | cleaning process by Embodiment 4 of this invention. It is a plane layout figure of the collection | recovery apparatus of the nucleic acid which is Embodiment 7 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Stirrer 102, 103 Pipetter 104 Reaction container transfer mechanism 105 Refined product storage container transfer mechanism 106, 113 Tip attachment position 107 Magnetic particle suction part 108 Cleaning place 109, 111 GuHCl suction port 110 50% ethanol aqueous solution suction port 112 Heat block 114 Solution disposal position 115, 116 Chip disposal position 118, 119 Nozzle 120 Reaction container 121 Fine product storage container 122 Permanent magnet

Claims (9)

A method for recovering nucleic acid from a nucleic acid-containing material,
Contacting a liquid mixture containing a chaotropic substance and a nucleic acid-containing material with a silicon oxide-containing solid phase;
Separating the silicon oxide-containing solid phase and the mixture;
The silicon oxide-containing solid phase is washed with a solution containing a chaotropic substance, and then washed with an aqueous solution containing acetate and no alcohol .
A method for recovering nucleic acid, comprising eluting nucleic acid from a solid phase containing silicon oxide.   The nucleic acid recovery method according to claim 1, wherein the acetate is potassium acetate.   The nucleic acid recovery method according to claim 2, wherein the potassium acetate concentration is 0.2 to 1 mol / liter.   2. The nucleic acid recovery method according to claim 1, wherein the acetate is sodium acetate.   5. The nucleic acid recovery method according to claim 4, wherein the concentration of sodium acetate is 0.2 to 1 mol / liter.   The nucleic acid recovery method according to claim 1, wherein the mixed solution and the silicon oxide-containing solid phase are stirred at room temperature, and the mixed solution and the silicon oxide-containing solid phase are brought into contact with each other.   The nucleic acid recovery method according to claim 1, wherein the silicon oxide-containing solid phase is glass particles, silicon oxide powder, quartz filter paper, quartz wool, crushed materials thereof, or diatomaceous earth. Method.   The nucleic acid recovery method according to claim 1, wherein the nucleic acid is not eluted from the silicon oxide-containing solid phase during washing.   The nucleic acid recovery method according to claim 1, wherein the silicon oxide-containing solid phase is a particle having a particle size of about 1 to 100 µm.

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