JP2001083050A - Nucleic acid recovery method and reagent kit - Google Patents

Abstract

(57) [Problem] To provide a method for recovering a nucleic acid containing a nucleic acid rapidly, easily, and highly purified from a material containing the nucleic acid without reducing the yield, and particularly to a nucleic acid component present in a biological sample. And a method suitable for an automatic recovery apparatus for plasmid DNA from Escherichia coli. A first step for promoting release of a nucleic acid from a nucleic acid-containing material, a second step of mixing a substance which promotes binding of the released nucleic acid and the nucleic acid to a solid phase, a mixed solution and a nucleic acid binding solid phase, Third step of contacting the nucleic acid under acidic conditions, a fourth step of separating the solid phase and the liquid, a fifth step of washing the solid phase to which the nucleic acid is bound with an acidic solution containing a salt, A sixth step of eluting from According to the present invention, a nucleic acid having a purity suitable for a genetic test or a genetic analysis can be obtained quickly, easily, and without using dangerous substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 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 a genetically modified E. coli for nucleic acid base sequence determination The present invention relates to a method for recovering a nucleic acid suitable for use in an apparatus for automatically recovering plasmid DNA from the same.

[0002]

2. Description of the Related Art Advances in molecular biology have led to the development of a number of gene-related technologies, and the isolation and identification of many disease-causing genes by these technologies. as a result,
In the field of medical treatment, molecular biology techniques have been introduced into diagnosis or testing methods, enabling diagnosis that has been impossible in the past, and significantly shortening the number of testing days.

[0003] This rapid progress is mainly due to nucleic acid amplification methods, especially PCR (polymerase chain reaction, Saiki et al., Science, 239, 48).
7-491 (1988)). Since the PCR method is capable of sequence-specifically amplifying a nucleic acid in a solution,
For example, a virus having only a trace amount in serum can be indirectly proved by amplifying and detecting a nucleic acid that is a gene of the virus.

[0004] However, there are some problems when this PCR method is used for daily inspection in a clinical setting. In particular, the importance of nucleic acid extraction and purification steps in pretreatment has been pointed out (Oshima et al., JJCLA, 22 (2), 145-15).
0 (1997)). This is due to the effects of inhibitors that could not be removed during nucleic acid purification, including hemoglobin in blood,
Surfactants and the like used in the extraction step are known. In addition, the extraction step requires a complicated operation using a manual technique and a great deal of labor by a skilled person. Therefore, this is an obstacle to introducing a new genetic test into a laboratory in a hospital, and automation of this process is eagerly desired.

[0005] In addition, in institutions that conduct molecular biology research, plasmid DNA is frequently used as a material for performing gene recombination operations. Similarly, automation of the process of extracting and purifying nucleic acids is desired.

[0006] As a known method for recovering a nucleic acid from a biological sample containing nucleic acids in a highly purified state free of an inhibitory factor, a surfactant is allowed to act on a biological sample in the presence of a protease. Nucleic acid is released, mixed with phenol (and chloroform), centrifuged to separate the aqueous and organic phases several times, and then the nucleic acid is recovered from the aqueous phase in the form of a precipitate with alcohol. Are known. However, this preparation method has a problem associated with the use of an organic solvent such as phenol which is a toxic substance in the process.

In addition to the above-described method using aqueous / organic phase separation, a method for recovering DNA from agarose gel utilizing the property of nucleic acid binding to the glass surface in the presence of a chaotropic substance (B . Vogelstein and D. Gilles
pie, Proc. Natl. Acad. Sci. USA, 76 (2), 615-619 (197
9)) or a method for recovering plasmid DNA from E. coli (MA Marko, R. Chipperfield and HC Bi
rnboim, Anal. Biochem., 121, 382-387 (1982)).

[0008] Furthermore, Japanese Patent Application Laid-Open No. H11-127854 discloses a simpler method for recovering nucleic acid from biological material.
Exists, but the optimal pH in the process from binding to elution
Is not described. Further, Japanese Patent Application Laid-Open No.
No. -75784 describes that a solution of a chaotropic agent used as a condition for binding ribonucleic acid is preferably a solution having a pH of 6.0 or less, but does not describe a pH in a washing and elution step.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for recovering a nucleic acid containing a nucleic acid quickly, easily and with a high degree of purification without reducing the yield.
Provision of a method suitable for an automatic recovery device for nucleic acid components present in a biological sample, or plasmid DNA from Escherichia coli
The present invention is to provide a method suitable for an automatic recovery device.

[0010]

To achieve the above object, the present invention is configured as follows.

That is, a first step for promoting release of nucleic acid from a nucleic acid-containing material, a second step of mixing a substance which promotes binding of the released nucleic acid and the nucleic acid to the solid phase, a mixture and a nucleic acid binding solid phase A fourth step of separating the solid phase from the liquid, 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. .

The present invention is applicable to any material containing a nucleic acid. In particular, clinical samples such as whole blood, serum, sputum, and urine, or biological samples such as cultured cells and cultured bacteria can be used. Alternatively, a nucleic acid held in a gel after electrophoresis, or the like is suitable. The method of the present invention is also effective for a reaction product such as a DNA amplification enzyme and a material containing a partially purified nucleic acid. The nucleic acid referred to here is double-stranded, 1
It is DNA or RNA having a single-stranded or partially double-stranded or single-stranded structure.

In the first step of the present invention, a method for promoting the release of nucleic acid from the nucleic acid-containing material is a physical method using a mortar, ultrasonic waves, microwaves, or the like, or a chemical method using a surfactant, a denaturing agent, or the like. Methods, biochemical methods using proteolytic enzymes or the like, and methods combining them can be used.

In the second step of the present invention, the substance which promotes the binding of the nucleic acid to the solid phase includes NaI (sodium iodide), KI (potassium iodide), NaClO4, NaS
The use of chaotropic agents such as CN (sodium thiocyanate), GuSCN (guanidine thiocyanate) and GuHCl (guanidine hydrochloride) is preferred. In the present invention, in order to specifically bind a nucleic acid component to a solid phase, for example, when using GuHCl, the final concentration is 4 mol.
/ L or more is desirable.

In the third step of the present invention, the nucleic acid binding solid phase is a substance containing silicon oxide, such as glass particles, silica powder, quartz filter paper, quartz wool, or a crushed product thereof or diatomaceous earth. Although it can be used, in a mixed solution of nucleic acid and chaotropic agent, by increasing the contact probability of the nucleic acid and the solid phase, it is possible to improve the binding efficiency or shorten the binding time, so that the particle size is small. The use of particles is desirable.

In the fourth step of the present invention, the means for separating the solid phase and the liquid can be separated by centrifugation or filtration. However, in the case of automation and equipment, an immunoassay utilizing an antigen-antibody reaction is used. A known B / F (bond form / freeform) separation technique generally used in an apparatus can be applied (for example, Japanese Patent Application Laid-Open No. Hei 8-2942).
No. 4).

In the fifth step of the present invention, the means for washing the solid phase to which the nucleic acid is bound with an acidic solution containing a salt may be a fourth step.
An acid solution containing a salt for washing the solid phase is mixed with the solid phase after the step while maintaining the binding of the nucleic acid to the solid phase, and the solid phase and the liquid are separated as in the fourth step. The cleaning can be performed by performing the following.

It is important that the liquid used for washing the solid phase does not elute the nucleic acids bound to the solid phase, which affects the yield of nucleic acids finally obtained. As a known method, it is recommended to use ethanol at a concentration of at least 75% in order to efficiently perform the above operation at room temperature (CW
Chen and CA Thomas, Jr., Anal.Biochem., 101,339
−341 (1980)) in 50% ethanol at a final concentration of 25 mM.
Good results are obtained even when potassium acetate adjusted to pH 5.0 is added so that

In the sixth step of the present invention, the means for eluting the nucleic acid from the solid phase is achieved by mixing the washed solid phase with an alkaline aqueous solution having a low salt concentration not less than the volume of the solid phase. Since the nucleic acid is transferred from the solid phase to the aqueous phase by this operation, the purified nucleic acid aqueous solution can be obtained by separating the solid phase and the liquid as in the fourth step. The alkaline aqueous solution having a low salt concentration used in the present invention is preferably in a sterilized state. In addition, the alkaline aqueous solution having a low salt concentration used here requires the addition of a substance having a pH buffering ability on the alkaline side, since a very small amount of mixing with the acidic solution used in the washing step cannot be avoided. It is desirable to use a substance that does not cause any trouble when the obtained aqueous nucleic acid solution is used for the next operation. For example, when performing a PCR reaction in the next operation, the use of a Tris buffer solution is good.
When performing the RT-PCR reaction, use of Bicine was good.

Further, in order to increase the yield of the obtained nucleic acid, it is desirable to use the nucleic acid in a state of being heated to 55 to 65 ° C., but if the container used for elution is heated to the same temperature, However, the same effect can be obtained. In the present invention, in order to increase the yield of the nucleic acid, it is desirable to perform the sixth step at least twice.

The nucleic acid recovery method constituted as described above comprises a first step for promoting the release of nucleic acid from the nucleic acid-containing material, and a second step for mixing the released nucleic acid with a substance which promotes the binding of the nucleic acid to the solid phase. A step of contacting the mixture with the nucleic acid-binding solid phase under acidic conditions, a fourth step of separating the solid phase and the liquid, and a step of washing the nucleic acid-bound solid phase with an acidic solution containing a salt. 5 steps, a 6th step of eluting the nucleic acid from the solid phase under alkaline conditions.

As the nucleic acid binding solid phase in the third step, any substance containing silicon oxide, such as glass particles, silica powder, quartz filter paper, quartz wool, or a crushed product thereof or diatomaceous earth, may be used. Is possible, 1-1
In the case of a particle size of about 00 μm, a practically sufficient binding of the nucleic acid to the solid phase can be obtained in several minutes to about 10 minutes. In addition, when diatomaceous earth is used, the cost required for one treatment can be reduced. In addition, in order to increase the contact probability between the nucleic acid and the solid phase, the efficiency of the binding time is further improved and the reproducibility is improved by using a stirring operation in accordance with the specific gravity of the particles used.

As a means for separating the solid phase and the liquid in the fourth step, a B / F separation technique generally used in an immunoanalyzer utilizing an antigen-antibody reaction can be used. It is possible to make use of the equipment technology.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION (Binding rate of nucleic acid to solid phase) Nucleic acid was recovered from an aqueous solution containing nucleic acid according to the flowchart of FIG.

As the nucleic acid-containing material, commercially available purified product pBR3
22 DNA was diluted with TE buffer (pH 8.0). In this case, the first step was skipped because the nucleic acid was already free in the nucleic acid-containing material. In the second step, 100 μl of the nucleic acid aqueous solution was adjusted to various pHs with a MES buffer / or / Tris buffer in a 1.5 ml reaction vessel.
500 μl of a 6 mol / l GuHCl solution was added and stirred. In the third step, 100 mg of quartz particles are added as a nucleic acid binding solid phase,
The solid phase was contacted with the liquid by stirring the solution for a certain period of time with a rotary mixer at room temperature. In the fourth step, a centrifuge is used to separate the solid phase and the liquid to which the nucleic acid is bound, and the solid phase is precipitated by centrifugation at 12000 rpm for 1 minute, and the separated supernatant is used using a spectrophotometer. Then, the binding amount of the nucleic acid was determined. The results are shown in FIG. PH of solution during binding
Causes a difference in the binding rate within a unit time,
It turns out that 6.5 or less is effective.

(Recovery of Nucleic Acid) 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 product pBR322 DNA was diluted with a TE buffer (pH 8.0). In this case, the first step was skipped because the nucleic acid was already free in the nucleic acid-containing material. In the second step, 500 μl of a 6 mol / l GuHCl solution adjusted to pH 4.0 with a MES buffer was added to 100 μl of the nucleic acid aqueous solution and stirred in a 1.5 ml reaction vessel. In the third step, 10 quartz particles are used as the nucleic acid binding solid phase.
0 mg was added, and the solid phase was contacted with the liquid by stirring the solution for a certain period of time with a rotary mixer at room temperature. In the fourth step, a solid phase was precipitated by centrifugation at 12,000 rpm for 1 minute using a centrifuge to separate the solid phase and the liquid to which the nucleic acid was bound, and the supernatant was discarded. In the fifth step, the operation of suspending the solid phase with a 50% aqueous ethanol solution to which potassium acetate adjusted to pH 5.0 was added, centrifuging at 12,000 rpm for 1 minute to obtain a solid phase precipitate, and discarding the supernatant twice. Washing of the solid phase was repeated. In the sixth step, the pH is adjusted to 6.5 to
Various 10 mM Tris buffers prepared in the range of 9.5 were added to the solid phase, elution was carried out by heating at 65 ° C. for 1 minute, and supernatant was obtained by centrifugation at 12,000 rpm for 1 minute. Electrophoresis was performed using a% agarose gel as a support, and after staining with ethidium bromide, the intensity of each band was quantified using a densitograph (manufactured by ATTO), and the recovery rate was calculated based on the amount of nucleic acid in the first step. The results are shown in FIG. It can be seen that the nucleic acid is effectively recovered by using an acidic solution in the binding step and the washing step and eluting with an alkaline solution.

[0027]

Industrial Applicability According to the present invention, nucleic acids containing nucleic acids can be rapidly and simply recovered from a nucleic acid-containing material with a high degree of purification without reducing the yield. And a method suitable for recovering plasmid DNA from Escherichia coli.

[Brief description of the drawings]

FIG. 1 is a flowchart for recovering a nucleic acid according to the present invention.

FIG. 2 is a diagram illustrating the effect of the present invention.

FIG. 3 is a view showing a nucleic acid recovery rate according to the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kenji Yasuda 882-mo, Oji-shi, Hitachinaka-shi, Ibaraki Prefecture Within the measuring instruments group of Hitachi, Ltd. F-term in Hitachi, Ltd. Instrument Group (Reference) 2G045 AA39 BB02 BB12 BB15 BB52 DA12 DA13 DA14 FB15 HA06 4B063 QA01 QQ42 QQ89 QR32

Claims (5)

[Claims] In a method for recovering a nucleic acid from a nucleic acid-containing material, a first step for promoting release of the nucleic acid from the nucleic acid-containing material is performed by mixing a released nucleic acid with a substance that promotes binding of the nucleic acid to a solid phase. A second step of bringing the mixed solution into contact with the nucleic acid-binding solid phase under acidic conditions, a fourth step of separating the solid phase and the liquid, A fifth step of washing, and a sixth step of eluting the nucleic acid from the solid phase under alkaline conditions, wherein the solid phase is a substance containing silica, and the pH of the solution in the third step is 6 or less. A method for recovering a nucleic acid. 2. The method for recovering nucleic acid according to claim 1, wherein a substance having a pH buffering capacity in an acidic solution is used to maintain the pH of the solution in the third step. 3. The method for recovering nucleic acid according to claim 1, wherein the pH of the solution in the sixth step is 8 or more. 4. The method for recovering nucleic acid according to claim 3, wherein a substance having a pH buffering capacity with an alkaline solution is used to maintain the pH of the solution in the sixth step. 5. A first step for promoting release of a nucleic acid from a nucleic acid-containing material, a second step for mixing a substance which promotes binding of the released nucleic acid and the nucleic acid to a solid phase, a mixture and a nucleic acid binding solid phase A fourth step of separating the solid phase and the liquid, a fifth step of washing the nucleic acid-bound solid phase with an acidic solution containing a salt, and a step of bringing the nucleic acid from the solid phase to an alkaline condition. A reagent kit for performing the sixth step eluting below.