KR-20260062955-A - Nucleic acid purification method and kit for nucleic acid purification

KR20260062955AKR 20260062955 AKR20260062955 AKR 20260062955AKR-20260062955-A

Abstract

The present invention aims to provide a method for conveniently purifying nucleic acid with high purity from a sample containing nucleic acid in a short time, and a kit for purification. The method for purifying nucleic acid according to the present invention comprises a process of contacting a sample containing nucleic acid with a zeolite and a proton receptor.

Inventors

나카가와 시미즈 유키
사토 가즈히코

Assignees

에이껜 가가꾸 가부시끼가이샤

Dates

Publication Date: 20260507
Application Date: 20240813
Priority Date: 20230901

Claims (15)

A method for purifying nucleic acids comprising a process of contacting a sample containing nucleic acids with a zeolite and a proton receptor.
In paragraph 1, The above proton receptor is a general formula (1) (In the formula, R1 , R2 , and R3 are each independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms that may have a substituent, and a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms that may have a substituent, wherein a nitrogen atom, a sulfur atom, or an oxygen atom is interposed between the carbon atoms, and the substituent is a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an imino group, a guanidyl group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, and a heterocyclic group having 3 to 6 carbon atoms, wherein the substituent comprises at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as constituent atoms of the ring, and is selected from the group consisting of a heterocyclic group having 3 to 6 carbon atoms that may have a substituent which is an aliphatic hydrocarbon having 1 to 6 carbon atoms, and In the case where an aliphatic hydrocarbon group having 1 to 6 carbon atoms, which may have a substituent, has a substituent that is an aromatic hydrocarbon group having 6 to 12 carbon atoms, the aliphatic hydrocarbon group having 1 to 6 carbon atoms is an aliphatic hydrocarbon group having 2 to 6 carbon atoms, and the aromatic hydrocarbon group is not bonded to the carbon atom closest to the N atom in general formula (1) of the aliphatic hydrocarbon group having 2 to 6 carbon atoms, R1 and R2 , R2 and R3 , or R1 and R3 may be combined to form a ring structure, wherein the ring structure is a 5 to 7-membered ring and may additionally include a nitrogen atom, a sulfur atom, or an oxygen atom, and if the ring structure has a double bond, it includes a nitrogen atom other than the N atom in general formula (1), In general formula (1), the total number of first, second, or third amino groups is greater than the total number of -COOH and -SO₃H . A method, which is a compound represented by
In paragraph 1, Method in which the above proton receptor is trishydroxymethylaminomethane, and/or 2-morpholinothenesulfonic acid.
In any one of paragraphs 1 through 3, A method in which the above sample is derived from at least one specimen selected from the group consisting of blood, bodily fluid, urine, feces, sputum, saliva, nasal discharge, smear fluid, amniotic fluid, and mouthwash.
In any one of paragraphs 1 through 3, A method in which, in the above contact process, at least a portion of the nucleic acid is not adsorbed onto the zeolite.
In any one of paragraphs 1 through 3, A method in which the zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, beta-type zeolite, and mordenite-type.
In any one of paragraphs 1 through 3, A method in which the zeolite is at least one selected from the group consisting of ferrierite- NH4 type zeolite, ferrierite-K type zeolite, ZSM- 5 - NH4 type zeolite, Y-Na type zeolite, beta-NH4 type zeolite, mordenite-H type zeolite, mordenite-Na type, and ferrierite-H type zeolite.
In any one of paragraphs 1 through 3, A method in which the nucleic acid is RNA and/or DNA.
A kit for purifying nucleic acids, comprising a zeolite and a proton receptor.
In Paragraph 9, The above proton receptor is a general formula (1) (In the formula, R1 , R2 , and R3 are each independently a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms that may have a substituent, and a chain aliphatic hydrocarbon group having 2 to 10 carbon atoms that may have a substituent, wherein a nitrogen atom, a sulfur atom, or an oxygen atom is interposed between the carbon atoms, and the substituent is a hydroxyl group, a carboxyl group, an amino group, a sulfonic acid group, an imino group, a guanidyl group, an aromatic hydrocarbon group having 6 to 12 carbon atoms, and a heterocyclic group having 3 to 6 carbon atoms, wherein the substituent comprises at least one atom selected from the group consisting of a nitrogen atom, a sulfur atom, and an oxygen atom as constituent atoms of the ring, and is selected from the group consisting of a heterocyclic group having 3 to 6 carbon atoms that may have a substituent which is an aliphatic hydrocarbon having 1 to 6 carbon atoms, and In the case where an aliphatic hydrocarbon group having 1 to 6 carbon atoms, which may have a substituent, has a substituent that is an aromatic hydrocarbon group having 6 to 12 carbon atoms, the aliphatic hydrocarbon group having 1 to 6 carbon atoms is an aliphatic hydrocarbon group having 2 to 6 carbon atoms, and the aromatic hydrocarbon group is not bonded to the carbon atom closest to the N atom in general formula (1) of the aliphatic hydrocarbon group having 2 to 6 carbon atoms, R1 and R2 , R2 and R3 , or R1 and R3 may be combined to form a ring structure, wherein the ring structure is a 5 to 7-membered ring and may additionally include a nitrogen atom, a sulfur atom, or an oxygen atom, and if the ring structure has a double bond, it includes a nitrogen atom other than the N atom in general formula (1), In general formula (1), the total number of first, second, or third amino groups is greater than the total number of -COOH and -SO₃H . Kit, a compound represented by
In Paragraph 9, The above proton receptor is trishydroxymethylaminomethane, and/or 2-morpholinothanesulfonic acid, kit.
In any one of paragraphs 9 through 11, A kit in which the above zeolite is at least one selected from the group consisting of a ferrierite-type zeolite, a ZSM-5-type zeolite, a Y-type zeolite, a beta-type zeolite, and a mordenite-type.
In any one of paragraphs 9 through 11, A kit in which the zeolite is at least one selected from the group consisting of ferrierite- NH4 type zeolite, ferrierite-K type zeolite, ZSM- 5 - NH4 type zeolite, Y-Na type zeolite, beta-NH4 type zeolite, mordenite-H type zeolite, mordenite-Na type, and ferrierite-H type zeolite.
In any one of paragraphs 9 through 11, The above kit is a kit for purifying nucleic acids derived from at least one specimen selected from the group consisting of blood, fluid, urine, feces, sputum, saliva, nasal discharge, smear fluid, amniotic fluid, and mouthwash, and further comprises a nucleic acid extraction reagent comprising a surfactant.
In any one of paragraphs 9 through 11, A kit in which the above nucleic acid is RNA and/or DNA.

Description

Nucleic acid purification method and kit for nucleic acid purification The present invention relates to a nucleic acid purification method and a kit for nucleic acid purification. Currently, nucleic acid amplification technologies, represented by PCR and LAMP methods, have permeated all fields of biology, including molecular biology and medicine, and are widely used in applications such as genetic diagnosis, DNA analysis, food inspection, environmental hygiene inspection, and animal and plant inspection. When amplifying nucleic acids in a sample, it is typically necessary to extract and purify the nucleic acids from the sample. Methods for extracting and purifying nucleic acids from a sample include, for example, using commercially available nucleic acid extraction kits. On the other hand, while using a general commercially available nucleic acid extraction kit (e.g., QIAamp Viral RNA Mini Kit (manufactured by Qiagen)) yields nucleic acids of high purity, it requires multiple devices such as high-speed centrifuges and heating heaters, making it difficult to use the kit unless the operating environment is equipped with such facilities. Furthermore, the process is cumbersome as it involves dozens of steps, requiring approximately one hour for the extraction and purification of nucleic acids (e.g., Non-patent Literatures 1-2). In nucleic acid amplification technology, when the number of test samples is large, it is desirable to prepare samples suitable for nucleic acid amplification in a short time; therefore, extraction and purification methods that involve cumbersome operations are difficult to apply. Furthermore, while commercially available nucleic acid extraction kits (e.g., Kaneka Simple DNA Extraction Kit, Template Prepper for DNA, Nippon Jinsa, ISOSPIN Viral RNA) allow for rapid extraction and purification of nucleic acids, the purity of the extracted nucleic acids may be low. Moreover, even when using such kits, heating operations may be required or the operation process may involve dozens of steps, making it difficult to claim that nucleic acids can be extracted and purified simply and quickly with high purity. In light of these issues, the market demands a method that extracts and purifies nucleic acids with high purity simply and quickly without requiring large equipment. In addition, methods for isolating and purifying nucleic acids from cell lysates, etc., using zeolites are also known. For example, Patent Document 1 discloses a method for preparing a sample for nucleic acid amplification used for amplifying nucleic acids contained in a biological sample, comprising an extraction step of adding a nucleic acid extraction reagent containing an anionic surfactant and/or alkali to a biological sample to obtain a nucleic acid extract, and a step of contacting the nucleic acid extract with a zeolite, wherein the zeolite is a proton-type zeolite and the substance adsorbed to the zeolite is removed. Patent Document 2 discloses a method for pre-treating a clarified lysate using a zeolite in a method for isolating nucleic acids from a biological solution (e.g., cell lysate), wherein the zeolite is added to a cell lysate treated with an alkaline reagent such as sodium dodecyl sulfate (SDS), thereby adsorbing SDS, etc., and the cell lysate can be clarified by centrifugation. The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments. [Nucleic Acid Purification Method] The method for purifying nucleic acid according to the present embodiment comprises a process of contacting a sample containing nucleic acid with a zeolite and a proton receptor. The contact process involves contacting a sample containing nucleic acid with a zeolite and a proton receptor, wherein the zeolite adsorbs substances other than nucleic acid. As shown in the examples described below, by contacting a sample containing nucleic acid with a zeolite and a proton receptor, the adsorption effect of substances other than nucleic acid on the zeolite is enhanced, so nucleic acid can be purified from a sample containing nucleic acid with high purity in a short time. Samples containing nucleic acid A sample containing nucleic acid is an aqueous solution or aqueous suspension in which nucleic acid and a substance other than nucleic acid are dissolved or suspended. The type of nucleic acid included in the sample is not particularly limited and may be DNA or RNA, and may be single-stranded or double-stranded. The origin of the nucleic acid is also not limited and may be, for example, nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of the present invention can particularly purify RNA easily and with high purity. The pH of the sample containing nucleic acid is not particularly limited. Substances other than nucleic acids include proteins (e.g., enzymes, glycoproteins, polypeptides, etc.), lipids, sugars (e.g., monosac