阅读说明：本技术 一种同时提取dna和rna的试剂盒及其使用方法 (Kit for simultaneously extracting DNA and RNA and use method thereof ) 是由 卢秀劲 王小芳 于 2020-07-29 设计创作，主要内容包括：本发明涉及一种同时提取DNA和RNA的试剂盒及其使用方法。本发明的试剂盒包括以下试剂：细胞裂解液、核酸结合液、核酸吸附材料、洗脱液A、去蛋白漂洗液、去盐分漂洗液和洗脱液B。本发明通过选择性的洗脱DNA,将RNA继续保留在吸附材料上,达到分级分离DNA与RNA的效果。本发明区别于用于同时提取DNA和RNA的TRIzol法,所用的试剂种类少,不使用苯酚、氯仿等有毒溶剂,且能够用于核酸纯化仪上,具有快速、高效、高通量、环保、可自动化同时提取DNA和RNA的优点。(The invention relates to a kit for simultaneously extracting DNA and RNA and a using method thereof. The kit of the invention comprises the following reagents: cell lysate, nucleic acid binding solution, nucleic acid adsorption material, eluent A, deproteinized rinsing solution, desalted rinsing solution and eluent B. The invention can keep RNA on the adsorbing material by selectively eluting DNA, thereby achieving the effect of separating DNA and RNA by grades. The method is different from a TRIzol method for simultaneously extracting DNA and RNA, uses few reagents, does not use toxic solvents such as phenol, chloroform and the like, can be used on a nucleic acid purification instrument, and has the advantages of rapidness, high efficiency, high flux, environmental protection and capability of automatically and simultaneously extracting DNA and RNA.)

1. A kit for simultaneously extracting DNA and RNA is characterized by comprising the following reagents: cell lysate, nucleic acid binding solution, nucleic acid adsorption material, eluent A, deproteinized rinsing solution, desalted rinsing solution and eluent B.

2. The kit of claim 1, wherein the cell lysate comprises the following components: 1-20% (v/v) of surfactant, 1-5 mol/L of protein denaturant and 0.1-1 mol/L of nuclease inhibitor/L, pH stabilizer.

3. The kit according to claim 2, wherein the surfactant is at least one of sodium dodecyl sarcosinate, sodium dodecyl benzene sulfonate, cetyl trimethyl ammonium bromide, Triton X-100, Tween 20;

the protein denaturant is at least one of guanidine hydrochloride, guanidine isothiocyanate or urea;

the nuclease inhibitor is at least one of ethylene diamine tetraacetic acid, ethylene glycol-bis- (2-aminoethylether) tetraacetic acid, mercaptoethanol, TCEP or dithiothreitol;

the pH stabilizer is at least one of trihydroxymethyl aminomethane, 4-hydroxyethyl piperazine ethanesulfonic acid, 3-morpholine propanesulfonic acid and N-tri (hydroxymethyl) methylglycine.

4. The kit according to claim 1, wherein the nucleic acid binding solution is at least one of a solution containing a guanidine salt and an alcohol solution; the concentration of the guanidine salt in the solution containing the guanidine salt is 0.1-8 mol/L, and the volume percentage of alcohol in the alcohol solution is 40-80%.

5. The kit according to claim 4, wherein the guanidine salt is at least one of guanidine hydrochloride and guanidine isothiocyanate, and the concentration of the guanidine salt is 1-4 mol/L;

the alcohol in the alcohol solution is ethanol, polyethylene glycol or isopropanol, and the volume percentage of the alcohol in the alcohol solution is 40-60%.

6. The kit according to claim 1, wherein the nucleic acid adsorbing material is a silica purification column or silica ferroferric oxide nano magnetic beads.

7. The kit according to claim 1, wherein the eluent A is at least one of a chloride-containing solution and an alcohol solution; the concentration of chloride in the solution containing chloride is 0.05-6 mol/L, and the volume percentage of alcohol in the alcohol solution is 5-40%.

8. The kit according to claim 7, wherein the chloride is at least one of sodium chloride, ammonium chloride and magnesium chloride;

the alcohol in the alcohol solution is ethanol or isopropanol, and the volume percentage of the alcohol in the alcohol solution is 4-30%.

9. The kit according to claim 1, wherein the concentration of guanidine salt in the deproteinizing rinse liquid is 1-4 mol/L, and the volume percentage of ethanol in the deproteinizing rinse liquid is 10-50%; the desalting rinse solution is 75% ethanol solution; the eluent B is DEPC treated water.

10. The use method of the kit according to claims 1 to 9, characterized by comprising the following steps:

(1) adding lysis solution into a sample, uniformly mixing and centrifuging to obtain a supernatant;

(2) adding the supernatant obtained in the step (1) into a nucleic acid binding solution, uniformly mixing, and transferring to a nucleic acid adsorption material;

(3) when the nucleic acid adsorbing material is a siliceous purification column, centrifuging the nucleic acid adsorbing material in the step (2) to remove centrifugate, adding deproteinized rinsing liquid into the nucleic acid adsorbing material, and centrifuging to retain the adsorbing material;

when the nucleic acid adsorbing material is silicon dioxide ferroferric oxide nano magnetic beads, placing the nucleic acid adsorbing material obtained in the step (2) in a magnetic field, removing liquid and retaining the adsorbing material;

(4) DNA elution: adding the eluent A into the nucleic acid adsorbing material treated in the step (3), and centrifuging to collect filtrate;

(5) adding an eluent A into the nucleic acid adsorbing material treated in the step (4), centrifuging and retaining the nucleic acid adsorbing material, and drying;

(6) RNA elution: adding the eluent B into the nucleic acid adsorbing material treated in the step (5), and centrifuging to obtain RNA;

(7) DNA purification: and (4) adding the filtrate obtained in the step (4) into the nucleic acid binding solution with the same volume, uniformly mixing, transferring to an adsorption material, centrifugally adsorbing DNA, removing the filtrate, adding desalted eluent, centrifuging at room temperature, repeating the process once, drying at room temperature, and adding eluent B to obtain the DNA.

Technical Field

The invention relates to the field of genetic engineering, in particular to a kit for simultaneously extracting DNA and RNA and a using method thereof.

Background

DNA and RNA are collectively called as nucleic acid, the nucleic acid is the most basic research object in molecular biology research and application, and how to obtain high-quality nucleic acid becomes an important and basic experimental technology. The current technical route aims at extracting DNA or RNA independently, and if the DNA and the RNA are extracted simultaneously, the experimental materials are divided into two parts. The techniques currently used for nucleic acid extraction mainly include: phenol chloroform extraction, silica gel column separation, salting out, and magnetic bead method. These techniques are basically only capable of extracting DNA or RNA, and cannot extract DNA and RNA simultaneously from the same sample.

The classical method for extracting RNA, TRIzol, is currently the most commonly used method for simultaneous extraction of DNA and RNA. The TRIzol method, namely a guanidine-phenol-chloroform method, is to prepare guanidine salt and phenol into a single solution, dissociate nucleic acid after cell lysis, add chloroform for extraction, separate an aqueous phase from an organic phase after centrifugation, allow RNA to exist in the aqueous phase and DNA to exist in the organic phase, and further achieve the effect of simultaneously extracting DNA and RNA. The method is complex to operate, and phenol, chloroform and the like are toxic organic solvents, so that the method is not environment-friendly and cannot realize high-throughput automatic extraction.

The invention with the patent number ZL201510982621.9 (application person is Beijing Edley Biotechnology Co., Ltd.) discloses a reagent for simultaneously extracting DNA and RNA, an extraction method and a communication method. The invention has complex operation and uses phenol chloroform and other toxic organic solvents, and the method is to elute RNA from total nucleic acid, because RNA and siliceous material are more closely adsorbed, it is difficult to ensure RNA to be specifically eluted without eluting DNA.

At present, with the maturity of the second-generation sequencing technology, more and more samples are clinically used for disease prediction and diagnosis by using the second-generation sequencing technology, and how to rapidly, efficiently and simultaneously extract DNA and RNA at high flux has important significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a kit and an extraction method for simultaneously extracting DNA and RNA, which are rapid, efficient, environment-friendly and high in flux.

In order to achieve the purpose, the invention adopts the following technical scheme:

a kit for simultaneously extracting DNA and RNA comprises the following reagents: cell lysate, nucleic acid binding solution, nucleic acid adsorption material, eluent A, deproteinized rinsing solution, desalted rinsing solution and eluent B.

Cell lysis solution is used for lysing cells and dissociating nucleic acid in the cells; the nucleic acid binding solution can create an environment for binding nucleic acid and the adsorption material, so that the nucleic acid is specifically adsorbed on the adsorption material, and impurities such as protein and the like are removed through specific selection of the adsorption material; the deproteinized rinsing liquid and the desalted rinsing liquid can further remove trace impurities, so that the purity of the nucleic acid sample is ensured; the nucleic acid adsorbing material can adsorb nucleic acid, and DNA and RNA can be eluted separately by using different eluents according to the difference of adsorption force.

Preferably, the cell lysate comprises the following components: 1-20% (v/v) of surfactant, 1-5 mol/L of protein denaturant and 0.1-1 mol/L of nuclease inhibitor/L, pH stabilizer.

Preferably, the surfactant is at least one of sodium dodecyl sarcosinate, sodium dodecyl benzene sulfonate, hexadecyl trimethyl ammonium bromide, triton X-100 and Tween 20.

Preferably, the protein denaturant is at least one of guanidine hydrochloride, guanidine isothiocyanate or urea.

The protein denaturing agent can efficiently denature and inactivate proteins such as nuclease, thereby protecting nucleic acids from hydrolysis.

Preferably, the nuclease inhibitor is at least one of ethylenediaminetetraacetic acid, ethylene glycol-bis- (2-aminoethylether) tetraacetic acid, mercaptoethanol, TCEP, and dithiothreitol.

Preferably, the pH stabilizer is at least one of tris (hydroxymethyl) aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, 3-morpholine propanesulfonic acid and N-tris (hydroxymethyl) methylglycine.

Nuclease inhibitors can inhibit the activity of intracellular nucleases, protecting nucleic acids from hydrolysis.

Preferably, the nucleic acid binding solution is at least one of a solution containing a guanidine salt and an alcohol solution; the concentration of the guanidine salt in the solution containing the guanidine salt is 0.1-8 mol/L, and the volume percentage of alcohol in the alcohol solution is 40-80%.

Preferably, the guanidine salt is at least one of guanidine hydrochloride and guanidine isothiocyanate, and the concentration of the guanidine salt is 1-4 mol/L.

Preferably, the alcohol in the alcohol solution is ethanol, polyethylene glycol or isopropanol, and the volume percentage of the alcohol in the alcohol solution is 40-60%.

Preferably, the nucleic acid adsorbing material is a silica purification column or silica ferroferric oxide nano magnetic beads.

Preferably, the eluent A is at least one of a chloride-containing solution and an alcohol solution; the concentration of chloride in the solution containing chloride is 0.05-6 mol/L, and the volume percentage of alcohol in the alcohol solution is 5-40%.

The DNA eluent is based on the different bonding strength between DNA and RNA and siliceous material, and can specifically release DNA from the adsorbing material, and ensure the RNA and the adsorbing material to be continuously and stably bonded, thereby achieving the purpose of separating RNA from DNA.

Preferably, the chloride is at least one of sodium chloride, ammonium chloride or magnesium chloride; the alcohol in the alcohol solution is ethanol or isopropanol, and the volume percentage of the alcohol in the alcohol solution is 4-30%.

Preferably, the deproteinizing rinsing liquid is at least one of guanidine salt or ethanol solution; the desalting rinse solution is an ethanol solution; the eluent B is DEPC treated water.

The use method of the kit for simultaneously extracting DNA and RNA comprises the following steps:

(1) adding lysis solution into a sample, uniformly mixing and centrifuging to obtain a supernatant;

(2) adding the supernatant obtained in the step (1) into a nucleic acid binding solution, uniformly mixing, and transferring to a nucleic acid adsorption material;

(3) when the nucleic acid adsorbing material is a siliceous purification column, centrifuging the nucleic acid adsorbing material in the step (2) to remove centrifugate, adding deproteinized rinsing liquid into the nucleic acid adsorbing material, and centrifuging to retain the adsorbing material;

when the nucleic acid adsorbing material is silicon dioxide ferroferric oxide nano magnetic beads, placing the nucleic acid adsorbing material obtained in the step (2) in a magnetic field, removing liquid and retaining the nucleic acid adsorbing material;

(4) DNA elution: adding the eluent A into the nucleic acid adsorbing material treated in the step (3), and centrifuging to collect filtrate;

(5) adding an eluent A into the nucleic acid adsorbing material treated in the step (4), centrifuging and retaining the adsorbing material, and drying;

(6) RNA elution: adding the eluent B into the nucleic acid adsorbing material treated in the step (5), and centrifuging to obtain RNA;

(7) DNA purification: and (4) adding the filtrate obtained in the step (4) into the nucleic acid binding solution with the same volume, uniformly mixing, transferring to an adsorption material, centrifugally adsorbing DNA, removing the filtrate, adding desalted eluent, centrifuging at room temperature, repeating the process once, drying at room temperature, and adding eluent B to obtain the DNA. Preferably, the rotation speed of the centrifugation is 12000rpm, and the time of the centrifugation is 1 min.

Preferably, the drying temperature is 20-25 ℃, and the drying time is 1 min.

Compared with the prior art, the invention has the beneficial effects that:

the kit does not contain toxic organic solvents such as phenol, chloroform and the like, and has environment-friendly components; compared with the traditional TRIzol method, the method is simple to operate, the purity of the extracted DNA and RNA is high, and the method has the advantages of rapidness, high efficiency and capability of simultaneously extracting the DNA and the RNA. In addition, the kit can be used on a nucleic acid purification instrument, is automatically operated, can realize high-throughput extraction of DNA and RNA, and meets the requirement of second-generation sequencing.

Drawings

FIG. 1 is a 1% agarose gel electrophoresis of DNA and RNA extracted according to the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described below by referring to specific examples.