WO2022092850A1

WO2022092850A1 - Nucleic acid extraction method using syringe-column elution system

Info

Publication number: WO2022092850A1
Application number: PCT/KR2021/015304
Authority: WO
Inventors: 박희경; 박재신; 이시석; 조시운
Original assignee: 주식회사 시선바이오머티리얼스
Priority date: 2020-10-28
Filing date: 2021-10-28
Publication date: 2022-05-05
Also published as: KR20220056442A

Abstract

The present invention relates to a method for simply and rapidly extracting nucleic acids from a sample by using a plurality of syringe columns that are each filled, in advance, with a different buffer. Compared with conventional nucleic acid extraction methods using spin columns, a nucleic acid extraction method according to the present invention remarkably reduces nucleic acid extraction time by using syringes filled with a buffer enabling simultaneous sample dissolution and nucleic acid binding and a plurality of syringe columns filled with wash buffer and elution buffer, and enables the elution of a buffer into a column without the need for an expensive centrifuge by using syringe pressure, and thus is useful during nucleic acid extraction in a clinical setting in which a molecular-biological diagnosis is required.

Description

Nucleic acid extraction method using syringe-column dissolution system

The present invention relates to a method for extracting nucleic acids, and more particularly, to a method for easily and quickly extracting nucleic acids from a sample using a plurality of syringe columns pre-filled with different buffers.

With the recent development of molecular biology technology, the extraction and analysis of nucleic acids from biological samples or samples containing cells is required in various fields such as diagnosis of diseases using nucleic acids, new drug development, pre-test for virus or bacterial infection, and forensics. Recently, as the cause of disease is interpreted at the genetic level based on the results of human genome research, the demand for manipulation and biochemical analysis of biological samples is gradually increasing for the purpose of curing or preventing human diseases. .

Conventionally, nucleic acid extraction methods have used organic solvents such as phenol and chloroform to extract nucleic acids from prokaryotic and eukaryotic cells or complex biological samples. Nucleic acid extraction using this was enzymatically digested with proteinase K, cells were disrupted with ionic surfactant (SDS), and then the nucleic acid was extracted with phenol or a phenol/chloroform mixture. According to the extraction, the organic solvent phase and the water phase were separated, and the extracted nucleic acid could be separated by precipitating the nucleic acid partitioned to the water side with alcohol (Lis and Schleif, Nucleic Acids Res., 2:383, 1975). . However, phenol or a mixture of phenol/chloroform has a problem that it has a dangerous effect on life or the environment and is considered a hazardous waste, so it has to be handled with care.

Recently, in order to reduce this problem, a kit using a column has become the basis for nucleic acid extraction. This is a method using silica or glass fiber that specifically binds to nucleic acids. Cells are lysed by treatment with a chaotropic reagent, and nucleic acid molecules are used using the principle of structural interaction between water molecules and nucleic acids. is a method for purifying protein from proteins and other intracellular substances (US 5,707,812, Horn et al., Human Gene Ther., 6:565-573, 1995; Chandra et al., Anal. Biochem., 203:169, 1992). . Since glass fiber or silica membrane has a low binding ratio with proteins and cell metabolites, a relatively structurally stable nucleic acid can be obtained at a high concentration. This method is simple compared to the phenol method, but incubates at a temperature of 56 degrees or higher to chemically and physically destroy the cell wall, such as the sample, lysis buffer, and proteinase K (incubation). The downside is that you have to go through the steps. In addition, since a chaotropic reagent or ethanol that strongly inhibits an enzymatic reaction such as PCR is used, these substances must be completely removed, and the operation is complicated and takes time due to the same method as the above conditions.

The principle of nucleic acid extraction using such a column is to purify nucleic acid molecules from proteins and other intracellular substances using the principle of structural interaction between water molecules and nucleic acids using a glass fiber or silica membrane that specifically binds to nucleic acids. For this, a sample incubation step and centrifugation process were essential. Therefore, the conventional nucleic acid extraction kit currently used includes the steps of (1) adding a cell lysis buffer and proteinase K to the sample, (2) incubating at 56 ° C. or higher for a certain period of time ( incubation), (3) adding the dissolved sample to the binding buffer, (4) the first centrifugation step-binding the centrifuged sample to the column, (5) the first washing step, (6) second centrifugation step, (7) second washing step, (8) third centrifugation step, (9) third washing step, (10) fourth centrifugation step, (11) high temperature drying step, ( 12) eluting the nucleic acid from the sample, and (13) the fifth centrifugation step to extract the final nucleic acid such as DNA, RNA, or virus.

Although the above-described method is in the spotlight as a high-purity DNA extraction method, it has a disadvantage in that it takes a lot of time and money because it needs to go through an incubation step and a centrifugation step.

Accordingly, the present inventors have made diligent efforts to develop a more convenient and rapid nucleic acid extraction method than the conventional method. As a result, using a plurality of syringes pre-filled with a buffer capable of dissolution and binding at the same time, washing and elution buffer, respectively, nucleic acid by syringe pressure In the case of eluting , it was confirmed that high-purity nucleic acid can be extracted in a shorter time than the conventional method using incubation and centrifugation, and the present invention was completed.

발명의 요약Summary of the invention

It is an object of the present invention to provide a method for extracting nucleic acids that is simpler and faster than conventional methods for extracting nucleic acids.

Another object of the present invention is to provide a nucleic acid extraction kit that is simpler and faster than the conventional nucleic acid extraction method.

In order to achieve the above object, the present invention provides a method for extracting a nucleic acid comprising the steps of:

(a) adding the sample to the first syringe filled with dissolution and binding buffer, mixing, mounting the first syringe on a column filled with a resin capable of binding to nucleic acid, and applying pressure to the piston of the syringe, binding a nucleic acid to the column;

(b) separating the first syringe from the column, mounting a second syringe filled with washing buffer 1 to the column, and then performing washing by applying pressure to the piston of the syringe; and

(c) separating the second syringe from the column, attaching the third syringe filled with the nucleic acid elution buffer to the column, and then applying pressure to the piston of the syringe to elute the nucleic acid.

The present invention also provides a kit for extraction of nucleic acids comprising;

(i) a column filled with a resin capable of binding to a nucleic acid;

(ii) a first syringe filled with lysis and binding buffer;

(iii) a second syringe filled with washing buffer 1; and

(iv) a third syringe filled with nucleic acid elution buffer.

1 is a syringe according to the present invention - schematically shows the processing process of the column dissolution system.

Figure 2 shows the configuration of the syringe-column dissolution kit according to the present invention.

발명의 상세한 설명 및 바람직한 구현예DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

The nucleic acid extraction method using the syringe-column dissolution system according to the present invention is similar to the spin-column manual method, which is a conventional nucleic acid extraction method, but as a “syringe-column dissolution system” using syringe pressure, the buffer is preliminarily used. By grafting a filled syringe (syringe) with a silica column, it has the advantage of being able to conveniently extract the desired nucleic acid in a short time. While it takes 30 minutes to extract nucleic acids using the existing spin column products, when extracting nucleic acids using the syringe-type nucleic acid extraction method of the present invention, nucleic acid extraction is possible within 5 minutes, so nucleic acid can be extracted simply and quickly. can be extracted, and when the human RNase P gene is amplified using the nucleic acid extracted from the clinical sample with the syringe-type nucleic acid extraction method of the present invention and the amplification efficiency is confirmed, higher concentration and lower Ct than conventional spin column products By showing the numerical value, it was confirmed that the nucleic acid extraction efficiency of the syringe-type nucleic acid extraction method of the present invention is equal to or higher than that of the existing product, and nucleic acid extraction is possible significantly faster.

Accordingly, in one aspect, the present invention relates to a method for extracting a nucleic acid comprising the steps of:

In the nucleic acid extraction method of the present invention, additionally, after step (b), (b') after mounting a 2' syringe filled with washing buffer 2 on the column, applying pressure to the piston of the syringe to perform a second washing step can be performed.

The lysis buffer used in the nucleic acid extraction method of the present invention may include guanidium thiocyanate (Guanidinium thiocyanate, GuSCN), preferably guanidine thiocyanate (Guanidine Thiocyanate, GuSCN), Tris-HCl, N -Lauroylsarcosine (N-Lauroylsarcosine sodium salt), EDTA, and may contain isopropyl alcohol (Isopropyl alcohol).

The dissolution and binding buffer of the present invention contains 4~7M concentration of GuSCN, 20~100mM Tris-HCl (pH 6.5~7.5), 1~10% N-Lauroylsarcosine sodium salt, 5~20mM EDTA, and 30~70% of isopropyl alcohol, preferably GuSCN at a concentration of 5 to 6M, Tris-HCl (pH 6.5 to 7.5) at 30 to 70 mM, N-Lauroylsarcosine sodium salt at 2 to 5%, EDTA at 7 to 15 mM and 30-60% isopropyl alcohol.

As the dissolution and binding buffer used in the nucleic acid extraction method of the present invention uses guanidine thiocyanate, sufficient sample dissolution is possible without the need to add Proteinase K or Carrier RNA, which are used for sample dissolution in conventional spin columns, etc.

In the case of the conventional nucleic acid extraction method, the dissolution buffer and the sample are physically mixed by vortexing, heat-treated through a heating block, and then ethanol is added to bind the nucleic acid to a silica column. ) and heat treatment (incubation) are essential, but the nucleic acid extraction method of the present invention enables sample dissolution and nucleic acid binding at the same time without physical stirring (vortex) or heat treatment step, enabling simpler and faster nucleic acid extraction.

In addition, in the case of the conventional nucleic acid extraction method using a spin column, 200 μl of a sample is applied to extract nucleic acids, but in the case of the nucleic acid extraction method of the present invention, it is possible to apply a sample of 400 to 600 μl, which is 2-3 times, A larger amount of nucleic acid can be extracted with one nucleic acid extraction.

The washing buffer 1 used in the nucleic acid extraction method of the present invention may include GuHCl, Tris-HCl, EDTA and ethanol.

Washing buffer 1 of the present invention may contain GuHCl at a concentration of 2 to 6M, Tris-HCl (pH 6.8 to 7.8) at 5 to 40 mM, EDTA at 1 to 10 mM, and ethanol at 30 to 70%, preferably 3 GuHCl at a concentration of ~4M, Tris-HCl (pH 6.8~7.8) at 10~30mM, EDTA at 2~5mM, and ethanol at 40~60% may be included.

The washing buffer 2 used in the nucleic acid extraction method of the present invention may include purified water, and the purified water may be nuclease free water.

The elution buffer used in the nucleic acid extraction method of the present invention may include purified water, and the purified water may be nuclease free water.

In the present invention, the resin capable of binding to the nucleic acid may be selected from the group consisting of silica.

According to an embodiment of the present invention, the nucleic acid extraction method using the nucleic acid extraction kit may be performed through the following steps (see FIGS. 1 and 2 ).

i) Using a pipette or a disposable pipette, apply the required amount of the nucleic acid-containing liquid sample to Syringe 1 pre-filled with lysis and binding buffers.

ii) Invert Syringe 1 or stir (shaking) to allow the sample to react with the dissolution and binding buffer filled in Syringe 1 (room temperature).

(iii) After combining the nucleic acid extraction column (luer lock) filled with the nucleic acid binding resin and the column adapter (screw type) (see FIG. 2) in syringe 1, press the piston of syringe 1 to release the nucleic acid dissolved in the sample to the column to be bound.

(iv) After separating the column (including adapter) to which the nucleic acid is bound, and combining it with syringe 2 previously filled with wash buffer 1, press the syringe piston as in (iii) to allow the wash buffer to pass through the column to remove impurities do.

(v) After separating the combined column (including adapter) and combining with syringe 2' pre-filled with wash buffer 2, press the syringe piston as in iv) to allow wash buffer 2 to pass through the column to remove impurities.

(vi) After separating the bound column (including the adapter) and combining it with syringe 3 pre-filled with the elution buffer, press the piston to the extent that the silica membrane in the column is wet, and then the nucleic acid is purified with nuclease Allow to stand for 1 minute at room temperature to dissolve well in free water.

vii) Press the piston of syringe 3 to allow the nucleic acid to elute.

In the case of the nucleic acid extraction kit according to the present invention, it is possible to extract nucleic acids using a clinical sample 2 to 3 times larger than that of a conventional spin column, so that a larger amount of nucleic acids can be extracted with one nuclear phase extraction.

In one aspect of the present invention, when the nucleic acid extraction method according to the present invention was used, a desired nucleic acid could be extracted from a clinical sample within 4 minutes, and using the same clinical sample, a conventional spin column ((QIAamp DSP Virus spin Kit, When using Quiagen, USA), it took 30 minutes to extract the nucleic acid, and it was confirmed that when the nucleic acid extraction method according to the present invention is used, the time for extracting the nucleic acid from the sample can be significantly reduced (Example) 1 and Comparative Example 1. In addition, it was confirmed that nucleic acid can be extracted very easily because there is no step of using a heating block and centrifuge equipment, which are heat treatment equipment. In the case of extraction, it was confirmed that the concentration of the extracted nucleic acid was higher than in the case of using the conventional spin column (Table 4).

In another aspect, the present invention relates to a kit for extraction of nucleic acids comprising;

(i) a column filled with a resin capable of binding to a nucleic acid;

(ii) a first syringe filled with lysis and binding buffer;

(iii) a second syringe filled with washing buffer 1; and

(iv) a third syringe filled with nucleic acid elution buffer.

The nuclear phase extraction kit of the present invention may additionally include a 2' syringe filled with (v) wash buffer 2 .

The lysis buffer used in the nucleic acid extraction method of the present invention may include guanidine thiocyanate (GuSCN), preferably guanidine thiocyanate (GuSCN), Tris-HCl, N-lau may include loylsarcosine (N-Lauroylsarcosine sodium salt), EDTA and isopropyl alcohol.

The dissolution and binding buffer of the present invention contains 4~7M concentration of GuSCN, 20~100mM Tris-HCl (pH 6.5~7.5), 1~10% N-Lauroylsarcosine sodium salt, 5~20mM EDTA, and 30~70% of isopropyl alcohol, preferably GuSCN at a concentration of 5 to 6M, Tris-HCl (pH 6.5 to 7.5) at 30 to 70 mM, N-Lauroylsarcosine sodium salt at 2 to 5%, EDTA at 7 to 15 mM and 40-60% isopropyl alcohol.

The dissolution and binding buffer used in the nucleic acid extraction method of the present invention uses guanidine thiocyanate, so there is no need to add Proteinase K or Carrier RNA to aid in the binding of a small amount of RNA, which is used for sample dissolution in a conventional spin column, etc. Sufficient sample dissolution and binding are possible simultaneously.

In the case of the existing nucleic acid extraction method, after mixing with a dissolution buffer, heat treatment for reaction is performed, and then ethanol is added to bind the nucleic acid to a silica column. and nucleic acid binding are possible at the same time, enabling simpler and faster nucleic acid extraction.

In addition, there is no heat treatment step that requires a heating block. By pressing a syringe piston without centrifugation, very simple nucleic acid extraction is possible compared to the conventional extraction method.

Washing buffer 1 of the present invention may contain GuHCl at a concentration of 2 to 6M, Tris-HCl (pH 6.8 to 7.8) at 5 to 40 mM, EDTA at 1 to 10 mM, and ethanol at 30 to 70%, preferably 3 GuHCl at a concentration of ~4M, Tris-HCl (pH 6.8~7.8) at 10~30mM, EDTA at 2~5mM, and ethanol at 40~65% may be included.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Nucleic acid extraction using a syringe-type nucleic acid extraction kit according to the present invention

For the clinical sample used in this example, a liquid sample was used that scraped the epidermis in the oral cavity by the Buccal swab sampling method and stored the swab in VTM (Virus Transport Medium, Korea, Sunsun Biomaterials).

400 μl of the clinical sample obtained by the above method was added to syringe 1 filled with 1 ml of lysis & binding buffer, mixed by inverting, and left at room temperature for 2 minutes.

The composition of the lysis and binding buffer used in this Example is shown in Table 1.

After combining syringe 1 with a nucleic acid extraction column filled with nucleic acid binding resin (luer lock) and a column adapter (screw type) (see FIG. 2), press the piston of syringe 1 to bind the nucleic acid dissolved in the sample to the column (binding) ) was made. After separating the column (including the adapter) to which the nucleic acid was bound, and combining it with syringe 2 previously filled with washing buffer 1, the piston of syringe 2 was pressed to allow the washing buffer to pass through the column to remove impurities. After separating the combined column (including adapter) and combining with syringe 2' previously filled with washing buffer 2, the piston of syringe 2' was pressed to allow washing buffer 2 to pass through the column to remove impurities.

The compositions of washing buffer 1 and washing buffer 2 used in this example are shown in Tables 2 and 3, respectively.

After separating the combined column (including the adapter) and combining it with syringe 3 pre-filled with the elution buffer, press the piston to the extent that the silica membrane in the column is wetted, and then the nucleic acid is stored in nuclease free water ) and leave at room temperature for 1 minute to dissolve well.

After standing at room temperature for 1 minute, a new tube containing the eluted nucleic acid was connected to the column, and the piston of syringe 3 was pressed to elute the nucleic acid.

The nucleic acid extraction process took a total of 3 minutes and 30 seconds (including 2 minutes standing at room temperature).

Comparative Example 1: Nucleic acid extraction using spin column type kit

In this comparative example, nucleic acids were extracted from clinical samples using a commercially available spin column type nucleic acid extraction kit (QIAamp DSP Virus spin Kit, Quiagen, USA).

200 μl of the clinical sample obtained in the same manner as in Example 1, 200 μl of AL buffer, 20 μl of Proteinase K (20 mg/mL), and 4 μl of carrier RNA were added to the EP tube and mixed by vortexing for 15 seconds , and left at 56 °C for 15 minutes. Then, it was left still at room temperature for 2 minutes. After adding 250 μl of 100% ethanol, vortex for 15 seconds, transfer the mixture to a spin column, centrifuge at 6,000 x g for 1 minute, remove the solution passing through the column, and put the first wash buffer (AW1) on the spin column. ), the first wash was performed by centrifugation at 6,000 x g for 1 minute, and 500 μl of the second wash buffer (AW2) was added to the spin column, and the second wash was performed by centrifugation at 6,000 x g for 1 minute. carried out. A third washing was performed by adding 500 μl of ethanol to the spin column, followed by centrifugation at 6,000 x g for 1 minute, and further centrifugation at 20,000 x g for 3 minutes to completely remove ethanol, followed by heating at 56 ° C for 2 minutes. . After adding 50 μl of AVE (elution buffer) and reacting at room temperature for 1 minute, centrifugation was performed at 20,000xg for 1 minute to elute the nucleic acids.

The nucleic acid extraction process using a spin column took a total of 30 minutes.

Example 2: Confirmation of Efficiency of Nucleic Acid Extraction Kit Using RT-PCR

Using the nucleic acid derived from the clinical sample extracted in Example 1 and Comparative Example 1, the liver RNase P gene was amplified to confirm the extraction efficiency.

The kit used for RNase P amplification was the U-TOP™ COVID-19 Detection Kit (Korea, Sunsun Biomaterials), and the amplification method and primer sequence are shown in the table below.

As a result, as shown in Table 6, when using the syringe-type nucleic acid extraction kit (SEASEUN SC™ V Extraction System) of the present invention than when using a spin column (QIAamp DSP Virus spin Kit, Quiagen, USA), It was confirmed that the concentration of the extracted nucleic acid was higher and the Ct level of the RNase P product was lower, so that many PCR products were amplified.

The nucleic acid extraction method using the syringe-column elution system according to the present invention significantly reduces the nucleic acid extraction time (about 85% reduction) compared to the conventional nucleic acid extraction method using a spin column, and without expensive heating blocks and centrifuges. , it is possible to elute the buffer to the column using syringe pressure, which is useful for nucleic acid extraction in clinical sites requiring molecular biology diagnosis.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

An electronic file is attached.

Claims

It relates to a method for extracting nucleic acids comprising the steps of:

(a) adding the sample to the first syringe filled with dissolution and binding buffer, mixing, mounting the first syringe on a column filled with a resin capable of binding nucleic acid, and applying pressure to the piston of the syringe, binding a nucleic acid to the column;

(b) separating the first syringe from the column, mounting a second syringe filled with washing buffer 1 to the column, and then performing washing by applying pressure to the piston of the syringe; and

(c) separating the second syringe from the column, attaching the third syringe filled with the nucleic acid elution buffer to the column, and then applying pressure to the piston of the syringe to elute the nucleic acid.
According to claim 1, additionally, after step (b), (b') after mounting a 2' syringe filled with washing buffer 2 to the column, applying pressure to the piston of the syringe to perform a second washing is performed A method characterized by being.
The method of claim 1 , wherein the dissolution and binding buffer comprises Guanidine Thiocyanate (GuSCN).
4. The method of claim 3, wherein the dissolution and binding buffer comprises guanidine thiocyanate (GuSCN), Tris-HCl, N-Lauroylsarcosine sodium salt, EDTA and isopropyl alcohol. A method characterized in that.
The method of claim 1, wherein the wash buffer 1 comprises GuHCl, Tris-HCl, EDTA and ethanol.
The method of claim 2, wherein the wash buffer 2 comprises nuclease free water.
The method of claim 1, wherein the elution buffer comprises nuclease free water.
The method of claim 1, wherein the resin capable of binding to the nucleic acid is selected from the group consisting of silica.
A kit for extracting nucleic acids comprising;

(i) a column filled with a resin capable of binding to a nucleic acid;

(ii) a first syringe filled with lysis and binding buffer;

(iii) a second syringe filled with washing buffer 1; and

(iv) a third syringe filled with nucleic acid elution buffer.
The kit for extracting nucleic acids according to claim 9, further comprising (v) a 2' syringe filled with wash buffer 2.
10. The kit of claim 9, wherein the lysis and binding buffer comprises guanidine thiocyanate (GuSCN).
12. The method of claim 11, wherein the dissolution buffer comprises guanidine thiocyanate (GuSCN), Tris-HCl, N-Lauroylsarcosine sodium salt, EDTA and isopropyl alcohol. A kit for nucleic acid extraction.
10. The kit of claim 9, wherein the wash buffer 1 comprises GuHCl, Tris-HCl, EDTA, and ethanol.
The kit for extracting nucleic acids according to claim 10, wherein the washing buffer 2 comprises nuclease free water.
10. The kit of claim 9, wherein the elution buffer comprises nuclease free water.
The kit for extracting nucleic acids according to claim 9, wherein the resin capable of binding to the nucleic acid is selected from the group consisting of silica.