Nucleic acid eluent and method for eluting nucleic acid using same

Abstract

A nucleic acid bound to a nucleic acid capture carrier is recovered at a high concentration while increasing the recovery rate. The nucleic acid bound to the nucleic acid capture carrier is recovered using a nucleic acid eluent containing a polar solvent that dissolves the nucleic acid and a hydrophobic solution that performs liquid-liquid layer separation from the polar solvent.

Description

Technical Field

[0001] The present invention relates to a nucleic acid elution solution for eluting nucleic acids from a carrier adsorbed with nucleic acids, and an elution method for nucleic acids using the nucleic acid elution solution. Background Art

[0002] In recent years, information obtained by nucleic acid analysis such as cancer genome examination using a next-generation sequencing (NGS) system has been effectively used in various fields such as medical treatment, clinical examination, pharmaceutical industry, and food industry. In this nucleic acid analysis, extraction of nucleic acids from various biological samples such as blood, tissues, and cultured cells has become an essential pretreatment.

[0003] As a nucleic acid extraction method, a method based on the property that nucleic acids bind to silica in the presence of a chaotropic agent or a method based on the property that nucleic acids bind to silica in the presence of an organic solvent is generally used instead of a method using harmful organic solvents such as phenol and chloroform. Using these methods, a nucleic acid extraction method using a nucleic acid capture tip having a solid phase containing silica as a nucleic acid capture carrier and a method using magnetic beads (nucleic acid capture carrier) whose surface is covered with silica have been reported. These methods include a step of binding nucleic acids to a nucleic acid capture carrier and an elution step of eluting nucleic acids from the nucleic acid capture carrier using an elution solution.

[0004] In the method using magnetic beads, after the elution step, the magnetic beads are recovered from the elution solution using a magnet. As an example, a method can be cited in which an elution solution containing magnetic beads is aspirated into a pipette tip, the magnetic beads are left in the pipette tip using a magnet, and only the elution solution is discharged from the pipette tip. In addition, as another example, a method can be cited in which a rod-shaped magnet (which may be covered with a cover) is inserted into an elution solution containing magnetic beads to recover the magnetic beads from the elution solution.

[0005] In addition, Non-Patent Document 1 has disclosed a method in which nucleic acids are recovered from a silica gel membrane by passing an elution solution through the silica gel membrane adsorbed with nucleic acids. In the method disclosed in Non-Patent Document 1, the elution solution is passed through the silica gel membrane by a method such as centrifugation or aspiration. At this time, in order to recover the elution solution remaining on the silica gel membrane, mineral oil is passed through the silica gel membrane by the same method such as centrifugation or aspiration. By passing the mineral oil through the silica gel membrane, the elution solution remaining on the silica gel membrane is reliably recovered.

[0006] In addition, Patent Documents 1 to 3 disclose systems for eluting nucleic acids captured by magnetic beads using magnetic beads such as disclosed in Patent Document 4. In the systems disclosed in Patent Documents 1 to 3, aqueous solutions such as washing liquid and elution liquid are arranged in a column via an oil layer, and the magnetic beads are passed through the column, thereby washing the magnetic beads and eluting nucleic acids from the magnetic beads. That is, by providing an oil layer in the column, aqueous solutions such as washing liquid and elution liquid are prevented from mixing.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2017-184626

[0010] Patent Document 2: Japanese Patent Application Publication No. 2016-067274

[0011] Patent Document 3: Japanese Patent Application Publication No. 2014-093988

[0012] Patent Document 4: Japanese Patent Application Publication No. 2017-176023

[0013] Non-patent literature

[0014] Non-patent literature 1: npj Precision Oncology volume 4, Article number: 3 (2020)

[0015] Problems to be solved by the invention

[0016] However, when nucleic acids are recovered using a nucleic acid capture carrier such as magnetic beads as described above, it is preferred to use a system with excellent nucleic acid recovery efficiency. In particular, when analyzing trace amounts of nucleic acids contained in biological samples, if the nucleic acid recovery efficiency is low, it is considered that there is a disadvantage that the analysis using the recovered nucleic acids cannot be accurately performed. On the other hand, when the amount of the eluent is increased in order to efficiently recover the nucleic acids captured by the nucleic acid capture carrier, the nucleic acid concentration in the eluent becomes low, and there is also a disadvantage that complicated operations such as the nucleic acid must be concentrated before the subsequent analysis.

[0017] However, in the above-mentioned existing system, the only method to improve the nucleic acid recovery efficiency from the nucleic acid capture carrier is to increase the liquid volume of the elution solution, and there is a problem that high-concentration nucleic acids cannot be recovered in the elution solution. Therefore, in view of the above-mentioned actual situation, the present invention aims to provide a nucleic acid elution solution that can achieve excellent nucleic acid recovery efficiency when using a nucleic acid capture carrier to recover nucleic acids and can recover high-concentration nucleic acids in the elution solution, and provide a method for eluting nucleic acids using the nucleic acid elution solution.

[0018] Means for solving problems

[0019] In order to achieve the above-mentioned purpose, the present inventors conducted in-depth research and found that by setting the nucleic acid elution solution used to elute nucleic acid from the nucleic acid capture carrier to a mixed solvent comprising a polar solvent capable of dissolving nucleic acid and a hydrophobic solution that undergoes liquid-liquid layer separation with the polar solvent, the nucleic acid captured by the nucleic acid capture carrier can be recovered in the polar solvent at a high concentration, thereby completing the present invention.

[0020] The present invention includes the following contents.

[0021] [1] A nucleic acid eluting solution, characterized in that the nucleic acid bound to the nucleic acid capturing carrier is eluted;

[0022] The nucleic acid eluting solution comprises a polar solvent that dissolves nucleic acid and a hydrophobic solution that performs liquid-liquid layer separation with the polar solvent.

[0023] [2] The nucleic acid elution solution according to [1], characterized in that the kinematic viscosity of the hydrophobic solution at 40°C is 17 mm 2 / s or less.

[0024] [3] The nucleic acid elution solution according to [1], characterized in that the kinematic viscosity of the hydrophobic solution at 25°C is 10 mm 2 / s or less.

[0025] [4] The nucleic acid elution solution according to [1], characterized in that the hydrophobic solution contains at least one component selected from silicone oil, fluorine-based oil and liquid paraffin.

[0026] [5] A method for eluting nucleic acid, characterized in that a nucleic acid-capturing carrier having captured nucleic acid is brought into contact with a nucleic acid-eluting solution, and nucleic acid is eluted from the nucleic acid-capturing carrier.

[0027] The nucleic acid eluting solution comprises a polar solvent that dissolves nucleic acid and a hydrophobic solution that performs liquid-liquid layer separation on the polar solvent.

[0028] [6] The method for eluting nucleic acid according to [5], characterized in that the hydrophobic solution contained in the nucleic acid eluting solution has a kinematic viscosity of 17 mm at 40°C. 2 / s or less.

[0029] [7] The method for eluting nucleic acid according to [5], characterized in that the hydrophobic solution contained in the nucleic acid elution solution has a kinematic viscosity of 10 mm at 25°C. 2 / s or less.

[0030] [8] The method for eluting nucleic acid according to [5], characterized in that the hydrophobic solution contained in the nucleic acid elution solution contains at least one component selected from silicone oil, fluorine-based oil and liquid paraffin.

[0031] [9] The method for eluting nucleic acid according to [5], characterized in that after the step of contacting the nucleic acid capture carrier with the nucleic acid elution solution, the nucleic acid capture carrier is separated from the nucleic acid elution solution and the nucleic acid is recovered in the polar solvent of the nucleic acid elution solution.

[0032]

[10] The method for eluting nucleic acid according to [5] is characterized in that it includes: bringing a biological sample prepared from an organism to be examined into contact with a nucleic acid capture carrier, and capturing nucleic acid from the biological sample onto the nucleic acid capture carrier.

[0033]

[11] A nucleic acid processing device, comprising a reaction container for contacting a nucleic acid capturing carrier having captured nucleic acid with a nucleic acid elution solution, and a separation device for separating either the nucleic acid capturing carrier or the nucleic acid elution solution from the reaction container,

[0034] The nucleic acid eluting solution comprises a polar solvent that dissolves nucleic acid and a hydrophobic solution that performs liquid-liquid layer separation on the polar solvent.

[0035]

[12] The nucleic acid processing device according to

[11] is characterized in that the above-mentioned nucleic acid capture carrier is a magnetic bead, the above-mentioned separation device has a magnetic body part containing a magnetic material, and the above-mentioned separation device pulls the above-mentioned nucleic acid capture carrier toward the above-mentioned magnetic body part to separate the above-mentioned nucleic acid capture carrier from the above-mentioned nucleic acid elution solution in the above-mentioned container.

[0036] Effects of the Invention

[0037] According to the nucleic acid eluting solution of the present invention, the amount of liquid can be increased by using a hydrophobic solution when eluting nucleic acid from a nucleic acid capturing carrier, and nucleic acid captured by the nucleic acid capturing carrier can be recovered in a polar solvent at a high concentration.

[0038] In addition, since the nucleic acid elution method of the present invention uses a nucleic acid elution solution containing a polar solvent that dissolves nucleic acids and a hydrophobic solution, the hydrophobic solution can be used to increase the liquid volume when eluting nucleic acids from the nucleic acid capture carrier, and the nucleic acid captured by the nucleic acid capture carrier can be recovered in the polar solvent at a high concentration.

[0039] In addition, since the nucleic acid processing device of the present invention uses a nucleic acid elution solution containing a polar solvent for dissolving nucleic acids and a hydrophobic solution, it is possible to increase the liquid volume of the nucleic acid elution solution in the reaction container when eluting nucleic acids from the nucleic acid capture carrier, and to recover the nucleic acid captured by the nucleic acid capture carrier into the polar solvent at a high concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a flow chart showing an example of a nucleic acid extraction method to which the present invention is applied.

[0041] Figure 2 This is a schematic diagram showing an example of a system for extracting nucleic acid using magnetic particles.

[0042] Figure 3 This is a schematic diagram showing another example of a system for extracting nucleic acid using magnetic particles.

[0043] Figure 4 This is a characteristic diagram showing the relationship between the amount of nucleic acid elution solution and the DNA recovery rate.

[0044] Figure 5 This is a characteristic diagram showing the relationship between the amount of nucleic acid elution solution and the recovery rate of the nucleic acid elution solution.

[0045] Figure 6 This is a characteristic diagram showing the relationship between the composition conditions of the nucleic acid elution solution and the DNA recovery rate.

[0046] Figure 7 This is a characteristic diagram showing the relationship between the amount of hydrophobic solution added to the nucleic acid elution solution and the DNA recovery rate.

[0047] Figure 8 This is a characteristic diagram showing the relationship between the amount of the hydrophobic solution added to the nucleic acid elution solution and the recovery rate of the nucleic acid elution solution. DETAILED DESCRIPTION

[0048] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0049] The nucleic acid elution solution of the present invention comprises a polar solvent for dissolving nucleic acid and a hydrophobic solution for liquid-liquid layer separation with the polar solvent. The nucleic acid elution solution can elute nucleic acid from the nucleic acid capture carrier by contacting with the nucleic acid capture carrier that captures nucleic acid. By using the nucleic acid elution solution of the present invention, the recovery rate of nucleic acid from the nucleic acid capture carrier (nucleic acid recovery rate) can be greatly improved.

[0050] The so-called polar solvent is a polar solvent that can dissolve hydrophilic nucleic acids, and is also called a high-polarity solvent. As a polar solvent, an aqueous solution with a low salt concentration or pure water can be used in typical cases. As an example, a solvent containing 10 mmol / L Tris (pH 8.5) and 0.1 mmol / L EDTA can be used as a polar solvent.

[0051] The so-called hydrophobic solution is a solution with non-polar molecules as the main component and the property of liquid-liquid layer separation with the above-mentioned polar solvent. Therefore, the nucleic acid eluent containing a hydrophobic solution and a polar solvent becomes a state in which the layer containing the hydrophobic solution is separated from the layer containing the polar solvent. The composition of the hydrophobic solution is not particularly limited, but it is preferably composed of oil, that is, a hydrocarbon compound as the main component. As such a hydrophobic solution, for example, a liquid containing an oil selected from silicone oil, fluorine oil and liquid paraffin can be cited. It should be noted that the hydrophobic solution is not limited to a solution consisting of a single composition, but can also be a mixed solution consisting of multiple compositions. As silicone oil, dimethyl silicone oil, methyl hydrogen silicone oil, methyl phenyl silicone oil, cyclic dimethyl silicone oil, etc. can be used.

[0052] In addition, as a hydrophobic solution, the kinematic viscosity at 40°C is preferably 17 mm 2 / s or less, more preferably 14mm 2 / s or less, and the most preferred kinematic viscosity at 25°C is 10 mm 2 The kinematic viscosity of the hydrophobic solution at 40°C is 17 mm 2 / s or less, 14mm 2 / s or less, or the kinematic viscosity at 25°C is 10mm 2 / s or less, the nucleic acid recovery rate can be further improved. It should be noted that the kinematic viscosity of the hydrophobic solution is specified as a value at a given temperature, and has the property that the kinematic viscosity decreases as the temperature increases. Therefore, by setting the temperature when using the nucleic acid elution solution to above 40°C, even if the kinematic viscosity at 40°C is greater than 17 mm / s, the recovery rate of the nucleic acid can be further improved. 2 / s hydrophobic solution can also achieve excellent nucleic acid recovery rate.

[0053] The nucleic acid elution solution constructed as described above can be used to elute nucleic acid from a nucleic acid capture carrier to which nucleic acid is bound. The nucleic acid elution solution of the present invention can be used, for example, in Figure 1 It should be noted that the nucleic acid elution solution of the present invention is Figure 1 In the flowchart shown in FIG. 4 , the nucleic acid elution solution is used for step 4: elution step from the nucleic acid capture carrier. Figure 1 The application is shown in the flowchart.

[0054] In the present nucleic acid extraction method, first, in step 1, a dissolving reagent for preparing a sample containing nucleic acid and a binding reagent for binding nucleic acid to a nucleic acid binding carrier are prepared, and the dissolving reagent and the binding reagent are added to a biological sample such as an animal cell, respectively. It should be noted that the dissolving reagent and the binding reagent can also be prepared as a liquid and then added to the biological sample. According to step 1, the nucleic acid contained in the biological sample becomes dissolved in the solution, and a sample containing nucleic acid can be prepared. The so-called nucleic acid here means deoxyribonucleic acid and ribonucleic acid having a double-stranded or single-stranded structure or partially having a double-stranded or single-stranded structure. In the present nucleic acid extraction method, the biological sample is not limited to animal cells, and microbial cells such as bacteria and fungi, plant cells or viruses can also be used. In particular, the biological sample is expected to be a biological sample such as whole blood, plasma, serum, sputum, urine, cultured cells, cultured bacteria, etc.

[0055] It should be noted that, in step 1, heating, stirring, and the like may be appropriately applied to promote the dissolution of the biological sample. Alternatively, after the biological sample is dissolved with the dissolving agent, the solid component may be removed by centrifugation.

[0056] As the substance that promotes the binding of nucleic acid to the nucleic acid binding solid phase contained in the above-mentioned binding reagent, there can be mentioned NaI (sodium iodide), KI (potassium iodide), NaClO4 (sodium perchlorate), NaSCN (sodium thiocyanate), GuSCN (guanidine thiocyanate), GuHCl (guanidine hydrochloride) and other chaotropic agents. The binding reagent is not limited to these, as long as it is a substance that promotes the binding of nucleic acid to the nucleic acid binding solid phase.

[0057] Then, in the present nucleic acid extraction method, as step 2, a nucleic acid capture carrier is brought into contact with a sample containing nucleic acid prepared in step 1, so that the nucleic acid is bound to the nucleic acid binding carrier. As nucleic acid capture carriers, glass particles, silica particles, magnetic particles coated with silica, quartz filter paper, quartz wool or their broken products, or diatomaceous earth, etc. can be cited. In other words, as a nucleic acid capture carrier, any substance containing silicon oxide or an organic polymer having a hydroxyl group on the surface can be used. Nucleic acid capture carriers are not limited to them, as long as they are substances that capture nucleic acids.

[0058] In particular, the present invention can be applied to a method for extracting nucleic acid using magnetic particles coated with silica (also referred to as magnetic beads or magnetic beads). Specifically, Figure 2As shown, first, a plurality of magnetic particles 2 are mixed with a sample 1 containing nucleic acid prepared in step 1 in a reaction container 3. In this way, the nucleic acid contained in the sample 1 can be bound to the magnetic particles 2. At this time, the magnetic particles 2 are sucked and discharged together with the sample 1 using a pipette tip 4, so that the sample 1 can be effectively brought into contact with the magnetic particles 2. At this time, the pipette tip 4 is directly or indirectly equipped with a pipette, a syringe, a pump, etc. in an airtight state, and these are used to perform vacuum decompression, pressurization, or centrifugation, so that the sample 1 can be sucked and discharged.

[0059] Afterwards, if Figure 2 As shown, while the magnetic particles 2 are attracted to the pipette tip 4 together with the sample 1, the magnetic body 5 is brought close to the side of the pipette tip 4. As a result, the magnetic particles 2 in the pipette tip 4 are captured by the inner wall of the pipette tip 4. In this state, the sample 1 in the pipette tip 4 is discharged to the reaction container 3, thereby separating the magnetic particles 2 bound with nucleic acid from the sample 1.

[0060] In addition, as another example, Figure 3 As shown, first, a plurality of magnetic particles 2 are mixed with a sample 1 containing nucleic acid prepared in step 1 in a reaction container 3. In this way, the nucleic acid contained in the sample 1 can be bound to the magnetic particles 2. At this time, by stirring the sample 1 containing the magnetic particles 2, the sample 1 and the magnetic particles 2 can be effectively brought into contact.

[0061] Afterwards, if Figure 3 As shown, the magnetic rod 7 is inserted into the cylindrical cover 6 and immersed in the reaction container 3, so that the magnetic particles 2 in the sample 1 can be captured on the bottom and side surfaces of the cover 6. Thereafter, the magnetic rod 7 is removed from the reaction container 3 while being inserted into the cylindrical cover 6, so that the magnetic particles 2 bound with nucleic acid can be separated from the sample 1.

[0062] In addition, the magnetic rod 7 may be pulled out from the outer cover 6 while the magnetic particles 2 in the sample 1 are captured on the bottom and side surfaces of the outer cover 6, thereby returning the magnetic particles 2 captured on the bottom and side surfaces of the outer cover 6 to the sample 1. The operation of capturing the magnetic particles 2 in the sample 1 on the bottom and side surfaces of the outer cover 6 and then returning the magnetic particles 2 captured on the bottom and side surfaces of the outer cover 6 to the sample 1 may be repeated, thereby effectively bringing the sample 1 and the magnetic particles 2 into contact with each other.

[0063] It should be noted that, although not shown, a nucleic acid capture carrier can also be arranged inside a pipette tip, and a solution containing nucleic acid is attracted to the pipette tip, thereby combining the nucleic acid in the solution. In this case, the solution is repeatedly attracted and discharged, so that the solution can be effectively contacted with the nucleic acid capture carrier arranged in the pipette tip. In addition, although not shown, a nucleic acid capture column filled with a nucleic acid capture carrier can also be used. In this case, a sample containing nucleic acid can be added to the upper part of the column, and the sample liquid is passed through by pressurization, centrifugation, vacuum decompression, etc., so that the nucleic acid capture carrier is in contact with the sample.

[0064] Then, in the present nucleic acid extraction method, as step 3, the nucleic acid capture carrier bound with the nucleic acid is washed to remove non-specific binding substances from the nucleic acid capture carrier. The reagent for washing is not particularly limited, and for example, any reagent can remove the reagent added in step 1 and impurities from the nucleic acid capture carrier while maintaining the binding of the nucleic acid to the nucleic acid capture carrier. As the reagent for washing, organic compounds such as lower alcohols and low molecular weight ketones can be used. As the reagent for washing, for example, ethanol and isopropanol can be used, and ethanol with a concentration of 70% or more is particularly preferred.

[0065] It should be noted that the method for contacting the cleaning reagent with the nucleic acid capturing carrier is not particularly limited, but the method used when contacting the nucleic acid-containing sample with the nucleic acid capturing carrier described above (for example, Figure 2 , 3 method shown).

[0066] Then, in the present nucleic acid extraction method, as step 4, the nucleic acid bound to the nucleic acid capture carrier is eluted from the nucleic acid capture carrier. Specifically, the nucleic acid elution solution is brought into contact with the nucleic acid capture carrier to which the nucleic acid is bound, thereby recovering the nucleic acid in the nucleic acid elution solution. There is no particular limitation on the method for bringing the nucleic acid elution solution into contact with the nucleic acid capture carrier, but the method used when bringing the nucleic acid-containing sample into contact with the nucleic acid capture carrier can be appropriately applied (for example, Figure 2 , 3 method shown).

[0067] Here, the nucleic acid eluting solution of the present invention comprises a polar solvent and a hydrophobic solution for dissolving nucleic acid, wherein the nucleic acid is dissolved in the polar solvent. Thus, by using the nucleic acid eluting solution of the present invention, nucleic acid at a high concentration can be eluted in a polar solvent.

[0068] For example, in accordance with Figure 2 , 3When the method shown in the figure makes the nucleic acid capture carrier contact with the nucleic acid elution solution, it is difficult to elute all or most of the nucleic acids from the nucleic acid capture carrier unless the entire nucleic acid capture carrier such as the magnetic particles 2 is contacted with the nucleic acid elution solution. Figure 2 , 3 In order to elute all or most of the nucleic acid from the nucleic acid capturing carrier by a method other than the method of , it is important to ensure that the nucleic acid elution solution is in contact with the nucleic acid capturing carrier.

[0069] In addition, Figure 2 In the method shown, when the nucleic acid elution liquid is small, the inner diameter of the pipette tip is preferably reduced corresponding to the amount of liquid that can be sucked. However, if the inner diameter is too small, the nucleic acid capture carrier will be blocked in the pipette tip, so it is preferred to ensure a certain size. Thus, while maintaining a certain inner diameter, it is preferred to limit the amount of nucleic acid elution liquid that can be sucked to the pipette tip.

[0070] exist Figure 3 In the method shown above, the nucleic acid elution solution is preferably filled to a height where the magnetic rod covers the nucleic acid capturing carrier, and the liquid volume is preferably ensured to a certain height. At the same time, the amount of the nucleic acid elution solution is preferably limited.

[0071] Since the nucleic acid eluting solution of the present invention contains a hydrophobic solution and is stirred, the polar solvent can be suppressed to a small amount even if the amount of liquid that can reliably contact the nucleic acid capture carrier is used. In other words, by using the nucleic acid eluting solution of the present invention, the nucleic acid eluting solution can be reliably contacted with the nucleic acid capture carrier to elute all or most of the nucleic acid from the nucleic acid capture carrier, and a high concentration of nucleic acid can be dissolved in the polar solvent.

[0072] By using the nucleic acid elution solution of the present invention in this way, the recovery rate of nucleic acid from the nucleic acid capture carrier bound with nucleic acid can be improved, and a high-concentration nucleic acid solution can be obtained. It should be noted that in the obtained nucleic acid solution, the layer of the hydrophobic solution and the layer of the polar solvent containing nucleic acid can be separated and the layer of the polar solvent can be used for subsequent analysis, but the two layers of the hydrophobic solution layer and the polar solvent layer containing nucleic acid can also be directly used for subsequent analysis.

[0073] The analysis using the obtained nucleic acid solution is not particularly limited, but examples thereof include sequence analysis using a next generation sequencer, electrophoresis, high performance liquid chromatography mass spectrometry, etc. When applied to these specific analyses, both the hydrophobic solution layer and the polar solvent layer containing nucleic acids can be used directly.

[0074] Example

[0075] Hereinafter, the present invention will be described in further detail using examples; however, the technical scope of the present invention is not limited to the following examples.

[0076] [Example 1]

[0077] 1. Purpose

[0078] In this example, the relationship between nucleic acid concentration, the amount of nucleic acid elution solution, and extraction efficiency was investigated, and then a concentration method for extracting nucleic acid with a small amount of liquid and high concentration was studied. Specifically, a liquid in which a hydrophobic solution (oil) was added to a polar solvent (main component: water) was used as a nucleic acid elution solution. The property that DNA can be dissolved in a polar solvent but not in a hydrophobic solution (oil) was utilized.

[0079] In this embodiment, the purpose is to elute high-concentration DNA with a smaller liquid volume (less than 50 μL) than before by using a nucleic acid elution solution containing a hydrophobic solution. It should be noted that if the recovery rate of DNA is the same as before due to the addition of the hydrophobic solution, it can be considered that the DNA is concentrated in the polar solvent compared with the past.

[0080] 2. Target

[0081] In this embodiment, as an example, a nucleic acid preparation system capable of handling liquid biopsy is used, in which free DNA in blood (hereinafter referred to as cfDNA) is used as a target.

[0082] 3. Materials

[0083] 3-1. Reagents

[0084] Table 1 shows the kinematic viscosity and flash point (product catalog-SDS included value) of the hydrophobic solution (oil) used in the nucleic acid elution solution. Hydrophobic solutions (oils) A, B, and D are silicone oils with a dimethylpolysiloxane structure, which are extremely inactive chemically and have excellent heat resistance, cold resistance, and viscosity stability. There are various viscosity lists. Hydrophobic solutions (oils) C and E (mineral oils) are hydrophobic, low-viscosity oils that are added to the PCR reaction solution to prevent evaporation of the reaction solution during PCR, and do not hinder the PCR reaction after nucleic acid extraction.

[0085]

[0086] 3-2. Simulated plasma samples

[0087] A PCR product of 140 bp in length was added to human plasma containing no cfDNA or DNA as simulated cfDNA (hereinafter referred to as simulated cfDNA). The concentration of simulated cfDNA added was prepared according to each experiment.

[0088] 4. Methods

[0089] 4-1. cfDNA extraction method

[0090] 4-1-1. cfDNA extraction method 1

[0091] The extraction of simulated cfDNA was performed. An example of the extraction method used in this embodiment is shown in Figure 3 That is, first, a reagent that makes the protein in the blood solubilized is added to bind the DNA in the sample to the magnetic beads. A plastic rod-shaped comb is used to stir the sample by moving it up and down. If the sample is immersed in the solution with a magnet placed in the comb, the magnetic beads bound to the DNA are adsorbed at the front end of the comb. Then, with a magnet placed in the comb, the adsorbed magnetic beads are moved to the cleaning solution while remaining unchanged. If the comb is left in the cleaning solution and the magnet is lifted, the magnetic beads are released into the cleaning solution. Similarly, stirring, adsorption of magnetic beads, and movement of magnetic beads are repeated, and cleaning is performed using multiple cleaning solutions. Then, the magnetic beads are moved to a nucleic acid elution solution, and the DNA bound to the magnetic beads is eluted into the nucleic acid elution solution. After the DNA is released, the magnetic beads are adsorbed to the magnet via the comb and separated from the nucleic acid elution solution.

[0092] The specific steps of cfDNA extraction are shown below: First, two 24-deep-well plates are prepared, and each reagent is dispensed into the designated locations of the plates according to Table 2.

[0093]

[0094] Then, 5 mL of the simulated plasma sample was added to columns A and B of plate 1, and the mixture was gently mixed after pipetting. After that, nucleic acid extraction was performed. After the nucleic acid extraction was completed, the DNA extracted to column A of plate 2 was immediately recovered into a 500 μL tube for storage.

[0095] 4-1-2. cfDNA extraction method 2

[0096] In addition, cfDNA extraction was performed using different methods. The specific steps are shown below.

[0097] In 1 mL of the simulated plasma sample, the reagents were mixed as shown in Table 3. In the case of multiple samples, batch processing was performed.

[0098]

[0099] Then, incubate at 60°C for 20 minutes. Take out from the incubator every 5 minutes and stir for 1 minute. Then, let stand on ice for 5 minutes. Finally, mix the samples in a buffer containing magnetic beads as shown in Table 4. In the case of multiple samples, batch processing is performed.

[0100]

[0101] After the solution in the tube is fully mixed, let it stand on the magnet for 5 minutes. In this state, completely remove the supernatant. Remove the tube from the magnet, add 1 mL of the dissolving binding reagent per 1 mL of the sample in the tube containing the magnetic beads and stir, then transfer the sample to a new 1.5 mL tube. Fix the tube to the magnet again, return the recovered supernatant to the original tube, further recover the remaining magnetic beads, and add them to the previous 1.5 mL tube. While the tube is fixed to the magnet, completely remove the supernatant. Remove the tube from the magnet, add 1 mL of the dissolving binding reagent, and stir for 30 seconds. Let the stirred sample stand on the magnet for 2 minutes, or let it stand on the magnet until the color of the supernatant becomes transparent. Completely remove the supernatant while the tube is fixed to the magnet. Remove the tube from the magnet, add 1 mL of 80% ethanol, and stir for 30 seconds. Let the stirred sample stand on the magnet for 2 minutes, or let it stand on the magnet until the color of the supernatant becomes transparent. The supernatant was completely removed while the tube was fixed to the magnet.

[0102] The tube is fixed to the magnet and left to stand for 3 to 5 minutes. After drying, the residual liquid is completely removed. The tube is removed from the magnet and a polar solvent is added to dissolve the binding reagent. In addition, a hydrophobic solution (oil) is added according to the experimental conditions. The amount of liquid corresponds to each experimental condition.

[0103] Thereafter, the mixture was stirred for 5 minutes, and the stirred sample was allowed to stand on a magnet for 2 minutes, or until the color of the supernatant became transparent, and the supernatant was recovered.

[0104] 4-1-3. Evaluation of Nucleic Acid Elution Buffer

[0105] The amount of nucleic acid elution buffer and DNA concentration were determined, and the DNA recovery rate, which is synonymous with nucleic acid extraction efficiency, was calculated.

[0106] Regarding the amount of nucleic acid eluate, the weight of the empty tube and the tube after the nucleic acid eluate was recovered were measured, and the amount of nucleic acid eluate was calculated based on the difference. The amount of simulated cfDNA added to the simulated plasma sample was set to the amount of liquid taken with a pipette. The recovery rate of the nucleic acid eluate was calculated according to the following calculation formula.

[0107]

[0108] Regarding DNA concentration, the DNA concentration used in the experiment and the DNA concentration in the nucleic acid eluate were measured using a fluorophotometer. The same experiment was performed on the sample without adding simulated cfDNA to the simulated plasma sample, and the measured value was subtracted as the background.

[0109] The DNA recovery rate was calculated by multiplying the liquid volume by the DNA concentration. Then, the DNA recovery rate was calculated according to the following calculation formula.

[0110] [Mathematical formula 2]

[0111]

[0112] 5. Effect of the amount of nucleic acid elution buffer

[0113] The simulated plasma sample to which simulated cfDNA was added to achieve 15 ng / 5 mL of plasma was processed using a nucleic acid elution device, and DNA was eluted under three conditions of nucleic acid elution solution volumes of 50, 100, and 200 μL. Figure 4 The DNA recovery rate for each volume of the nucleic acid elution solution is shown in the figure. The DNA recovery rates of 50 μL and 100 μL were 63.8% and 58.9% respectively, and no significant difference was observed. On the other hand, the DNA recovery rate of 200 μL was increased to 77.7%. Figure 5 The recovery rate of the nucleic acid elution solution is shown in Figure 3 In the manner shown, when the nucleic acid eluent is set to 50, 100, and 200 μL, the volume is reduced compared to the assumed volume, becoming 80.4-91.5%. In addition, the more the amount of nucleic acid eluent increases, the higher the recovery rate of the nucleic acid eluent. This shows that if the amount of nucleic acid eluent decreases, the proportion of nucleic acid eluent remaining in the magnetic beads increases.

[0114] 6. Effect of hydrophobic solution in nucleic acid elution buffer

[0115] 6-1. Comparison of kinematic viscosity of hydrophobic solutions (oils)

[0116] Nucleic acids were eluted manually from 1 mL of simulated plasma sample to which simulated cfDNA was added in a manner to achieve 50 to 120 ng / 1 mL of plasma. The nucleic acid elution solution used was a solution of 50 μL or 100 μL of polar solvent and 50 μL of hydrophobic solution (oil) added to 50 μL of polar solvent. Four types of hydrophobic solutions (oils) with different kinematic viscosities were compared, except for C in Table 1. Figure 6 The DNA recovery rate is shown in the table. Figure 6 As shown in the figure, the DNA recovery rate of 50 μL of nucleic acid elution solution without adding hydrophobic solution (oil) was 62.7% on average. 2 / s (25°C) of hydrophobic solution (oil: A, B, D) with a DNA recovery rate of 72.6-77.6%, which is the same as or higher than that of nucleic acid elution solution without adding hydrophobic solution (oil). On the other hand, when adding hydrophobic solution with a kinematic viscosity of 14-17 mm2 / s (38℃), the average DNA recovery rate was 32.8%, and the DNA recovery rate was significantly reduced.

[0117] The experimental conditions in which the DNA recovery rate did not increase or decreased were considered to be due to insufficient mixing conditions of the hydrophobic solution (oil) and the polar solvent. It was found that the DNA recovery rate could be increased by optimizing these conditions.

[0118] 6-2. Comparison of the amount of hydrophobic solution (oil) added

[0119] Nucleic acid was extracted from 1 mL of human plasma sample to which simulated cfDNA was added in an amount of about 0.75 ng / 1 mL of plasma using 4-1-2. cfDNA extraction method 2. The nucleic acid eluent used was a nucleic acid eluent containing only 50 μL of polar solvent, a solution containing 50 or 150 μL of hydrophobic solution (oil) added to the nucleic acid eluent, a nucleic acid eluent containing only 15 μL of polar solvent, and a solution containing 35, 85, or 185 μL of hydrophobic solution (oil) added to the nucleic acid eluent. The hydrophobic solution (oil) used was a solution with a kinematic viscosity of 5 mm 2 / s (C has no measurement temperature in the SDS, B is 25°C) C and B. Based on the previous study, B was selected due to its low viscosity and high flash point (102°C).

[0120] Figure 7 The DNA recovery rate under each condition is shown in the figure. When the nucleic acid eluent is only 50 μL of polar solvent, the average is 77.7%. When a hydrophobic solution (oil) is further added, the DNA recovery rate increases to 93.7-97.4%. No difference due to the type of hydrophobic solution (oil) is observed. The DNA recovery rate when the nucleic acid eluent is only 15 μL of polar solvent is 70.1% on average. When the amount of hydrophobic solution (oil) added is further set to 85 and 185 μL, the DNA recovery rates are 82.8-87.7% and 77.8-94.2%, respectively, which is higher than the nucleic acid eluent without the addition of hydrophobic solution (oil). Regarding the type of oil, the recovery rate is higher in case of C than in B.

[0121] Figure 8 The recovery rate of the nucleic acid eluate recovered under various conditions is shown in the table. The recovery rate of the amount of nucleic acid eluate is 69.3-93.5%, which is smaller than the set amount of nucleic acid eluate of 15 μL or 50 μL. In the case of 15 μL of nucleic acid eluate, the recovery rate of the nucleic acid eluate is increased by adding the hydrophobic solution C. Correspondingly, the DNA recovery rate also shows an increasing trend. This shows that the amount of residual nucleic acid eluate in the magnetic beads, etc. is reduced.

[0122] 7. Study of results

[0123] As described above, the results of this example clearly show that by using a nucleic acid eluting solution containing a polar solvent that dissolves nucleic acids and a hydrophobic solution, nucleic acids can be eluted at a high concentration even if the amount of polar solvent is small (e.g., less than 50 μL). The kinematic viscosity of the hydrophobic solution suggests that although 10 mm is obviously preferred, 2 / s or less, but even outside this range, nucleic acid can be eluted at a high concentration by studying the mixing conditions with a polar solvent.

[0124] Regarding the amount of hydrophobic solution added, the DNA recovery rate tends to increase when the amount of hydrophobic solution added is large. However, it is suggested that even if the amount of hydrophobic solution is small, evaporation during operation and residues when separating the magnetic beads from the nucleic acid elution solution can be reduced, and nucleic acids can be eluted at a high concentration.

Claims

1. A nucleic acid elution solution, characterized in that: The nucleic acid bound to the nucleic acid capture carrier is eluted. The nucleic acid eluting solution comprises a polar solvent for dissolving nucleic acid and a hydrophobic solution for performing liquid-liquid layer separation with the polar solvent.

2. The nucleic acid elution solution according to claim 1, characterized in that The kinematic viscosity of the hydrophobic solution at 40°C is 17 mm 2 / s or less.

3. The nucleic acid elution solution according to claim 1, characterized in that The kinematic viscosity of the hydrophobic solution at 25°C is 10 mm 2 / s or less.

4. The nucleic acid elution solution according to claim 1, characterized in that The hydrophobic solution contains at least one component selected from silicone oil, fluorine oil and liquid paraffin.

5. A method for eluting nucleic acid, characterized in that: The nucleic acid capturing carrier having captured the nucleic acid is brought into contact with a nucleic acid eluting solution, and the nucleic acid is eluted from the nucleic acid capturing carrier. The nucleic acid eluting solution comprises a polar solvent for dissolving nucleic acid and a hydrophobic solution for performing liquid-liquid layer separation on the polar solvent.

6. The method for eluting nucleic acid according to claim 5, characterized in that: The hydrophobic solution contained in the nucleic acid eluent has a kinematic viscosity of 17 mm at 40°C. 2 / s or less.

7. The method for eluting nucleic acid according to claim 5, characterized in that: The hydrophobic solution contained in the nucleic acid eluent has a kinematic viscosity of 10 mm at 25°C. 2 / s or less.

8. The method for eluting nucleic acid according to claim 5, characterized in that: The hydrophobic solution contained in the nucleic acid elution solution comprises at least one component selected from silicone oil, fluorine oil and liquid paraffin.

9. The method for eluting nucleic acid according to claim 5, characterized in that: After the step of contacting the nucleic acid capturing carrier with the nucleic acid eluting solution, the nucleic acid capturing carrier is separated from the nucleic acid eluting solution, and the nucleic acid is recovered in the polar solvent of the nucleic acid eluting solution.

10. The method for eluting nucleic acid according to claim 5, characterized in that: include: A step of bringing a biological sample prepared from an organism to be examined into contact with a nucleic acid capturing carrier, and capturing nucleic acid from the biological sample on the nucleic acid capturing carrier.

11. A nucleic acid processing device, characterized in that: A reaction vessel for contacting a nucleic acid capturing carrier having captured nucleic acid with a nucleic acid elution solution, and a separation device for separating either the nucleic acid capturing carrier or the nucleic acid elution solution from the reaction vessel, The nucleic acid eluting solution comprises a polar solvent for dissolving nucleic acid and a hydrophobic solution for performing liquid-liquid layer separation on the polar solvent.

12. The nucleic acid processing device according to claim 11, characterized in that: The nucleic acid capture carrier is a magnetic bead. The separation device includes a magnetic body portion containing a magnetic material, and the separation device pulls the nucleic acid capturing carrier toward the magnetic body portion to separate the nucleic acid capturing carrier from the nucleic acid elution solution in the container.

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