CN111808843A - DNA extraction kit and extraction method - Google Patents

Abstract

The invention discloses a DNA extraction kit and an extraction method, wherein the kit comprises the following components: erythrocyte lysate, leukocyte lysate, proteinase K solution, ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and TE solution. The extraction method comprises the steps of removing anucleate components from a whole blood sample by using a red blood cell lysate, adding a white blood cell lysate and proteinase K to lyse cells and release nucleic acid, and finally removing impurities by using an ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and a TE solution to obtain a DNA extraction product. The kit has simple components, no toxic or harmful components, less required samples and good extraction effect; the extraction method is simple and rapid to operate, special equipment is not needed for assistance, the extraction can be completed only by using a centrifugal tube and a centrifugal machine, and the whole extraction process can be completed only in 40 minutes; the concentration and purity of the extracted DNA product meet the requirements.

Description

DNA extraction kit and extraction method

Technical Field

The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a DNA extraction kit and an extraction method.

Background

Nucleic acid is a biological macromolecular compound formed by polymerizing a plurality of nucleotides, and can be divided into ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) according to different chemical compositions, and is widely existed in all animals, plants, microorganisms and organisms. With the development of molecular biology and the wide application of nucleic acid detection, the extraction and purification of nucleic acid are the basis of various molecular biological researches and clinical tests. Human blood samples are one of the most commonly used human samples in molecular biology research and clinical testing because of their simple and readily available clinical sampling. The rapid and effective extraction and purification of human blood DNA is helpful for clinical in vitro detection.

The existing nucleic acid extraction methods mainly comprise phenol chloroform extraction method, centrifugal column method and magnetic bead method. The phenol chloroform extraction method is the most classical method, but the operation is complex and time-consuming, and the method contains harmful organic solvents, is harmful to human bodies and easily pollutes the environment; the centrifugal column method and the magnetic bead method are the methods which are commonly used at present, but the operation is complicated and the cost is high due to the special adsorption column or the magnetic adsorption instrument.

With the wide application of nucleic acid detection, nucleic acid extraction and purification technology is also developing, and various human blood nucleic acid extraction kits are proposed at home and abroad. At present, the nucleic acid extraction and purification kits have the disadvantages of complex operation, need of special instruments or equipment, high cost, even harm and the like, and are not beneficial to biological research and clinical detection application. Therefore, there is a need for a simple, rapid, non-hazardous reagent-free, human blood DNA extraction kit that does not require any special equipment or instruments, and whose product is suitable for clinical in vitro testing.

Disclosure of Invention

In order to solve the various problems in the prior art, the invention provides a DNA extraction kit, which can simply, quickly and effectively extract human blood DNA, has short extraction time, does not contain harmful components, and does not need special equipment or instrument assistance. The invention also provides a DNA extraction method.

The DNA extraction kit provided by the invention comprises the following components: erythrocyte lysate, leukocyte lysate, proteinase K solution, ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and TE solution;

the erythrocyte lysate contains per liter: 2-20g of ammonium chloride, 0.1-5g of potassium bicarbonate, 0.1-1g of disodium ethylene diamine tetraacetate and deionized water as a solvent;

the leukocyte lysate is 0.5-5% of lauryl sodium sulfate aqueous solution by mass percent;

the concentration of the proteinase K in the proteinase K solution is 0.05-2.00 mg/L;

the concentration of ammonium acetate in the ammonium acetate solution is 2.5-10 mol/L;

the 75% ethanol is ethanol water solution with 75% ethanol by volume percentage.

The method adopts the combination of sodium dodecyl sulfate and proteinase K to crack cells and degrade proteins and ribozymes. The addition of ammonium acetate has two effects, on the one hand, it can promote the precipitation of part of protein, and on the other hand, it can provide proper salt ions to promote the precipitation of DNA in ethanol. The method can precipitate DNA by centrifugation in an environment of providing ammonium acetate and ethanol, thereby eliminating the need for an adsorption column. The sodium dodecyl sulfate and the protease K are adopted to degrade the protein, and the ammonium acetate ethanol is adopted to remove impurities such as the protein, so that high-purity DNA can be provided, and harmful reagents such as phenol, chloroform and the like are avoided.

Preferably, in the DNA extraction kit, the pH of the erythrocyte lysate is 7.0-8.0.

Preferably, in the DNA extraction kit, the TE solution is an aqueous solution containing Tris-HCl and EDTA, the pH value is 8.0, the concentration of Tris-HCl is 1mol/L, and the concentration of EDTA is 0.5 mol/L.

The DNA extraction method provided by the invention is completed by using any one of the DNA extraction kits.

Preferably, the DNA extraction method comprises the steps of removing anucleate components from a whole blood sample by using a red blood cell lysate, adding a white blood cell lysate and proteinase K to lyse cells and release nucleic acid, and finally removing impurities by using an ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and a TE solution to obtain a DNA extraction product.

Preferably, the DNA extraction method specifically includes the following steps:

(1) placing a whole blood sample in a centrifugal tube, adding erythrocyte lysate, uniformly mixing, placing, centrifuging again, and removing supernatant to obtain a precipitate;

(2) adding protease K solution and leukocyte lysate into the precipitate, resuspending the precipitate, and performing water bath at 65 ℃ for 10 min;

(3) adding ammonium acetate solution, mixing, centrifuging, collecting supernatant, adding precooled anhydrous ethanol, mixing, standing at room temperature, centrifuging, and removing supernatant to obtain precipitate;

(4) adding 75% ethanol into the precipitate obtained in the step (3), uniformly mixing, centrifuging, and removing the supernatant to obtain a precipitate;

(5) and (4) airing the precipitate in the step (4) at room temperature until the ethanol is completely volatilized, adding a TE solution, carrying out water bath at 65 ℃ for 5min after heavy suspension, and centrifuging to enable the solution to be concentrated to the bottom of the tube, thus obtaining the DNA extraction product.

Preferably, in the DNA extraction method, the whole blood sample is used in an amount of 200. mu.L.

Preferably, in the DNA extraction method, the whole blood sample is a fresh human peripheral blood sample anticoagulated with EDTA K2/K3.

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

the DNA extraction kit has simple reagent components, does not contain harmful organic components such as phenol, chloroform and the like, and all the reagent components are safe and harmless.

The method of combining ammonium acetate and ethanol is utilized to remove impurities and precipitate DNA at the same time, thereby ensuring the purity of the extracted product DNA.

The whole kit does not need to use a special adsorption column to adsorb DNA in the extraction process, the used consumables are simple, and only a centrifugal tube with the volume specification of 1.5ml and without nuclease pollution is needed; and special instruments such as a magnetic frame are not needed, and the extraction process can be completed only by a common centrifuge.

The blood samples required by extraction are very few, the extraction requirement can be met only by 200 mu L, the concentration range of the extracted DNA is 31.7-94.5 ng/mu L, 260/280 is between 1.8-2.0, and the concentration and the purity are high. And the biological properties of the DNA are not destroyed.

The extraction method is simple and quick, is easy to be mastered by operators, can finish the extraction of human blood DNA in 40 minutes in the whole process, and has more advantages in batch operation.

Detailed Description

The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.

The techniques used in the following examples are, unless otherwise specified, conventional techniques known to those skilled in the art; the instruments, reagents, etc. used, unless otherwise specified in this specification, are publicly available to those of skill and research in the art.

EXAMPLE 1 composition of DNA extraction kit and method of use

(1) The main reagents and consumables required by the kit comprise:

the erythrocyte lysate has the following formula: 2-20g of ammonium chloride, 0.1-5g of potassium bicarbonate and 0.1-1g of disodium ethylene diamine tetraacetate, adding deionized water to dissolve, adjusting the pH value to 7.0-8.0, and fixing the volume to 1L.

② leukocyte lysate, the formula is 0.5-5% of sodium dodecyl sulfate.

And the concentration of the proteinase K is 0.05-2 mg/mL.

And fourthly, the concentration of the ammonium acetate in the ammonium acetate solution is 2.5-10 mol/L.

Absolute ethyl alcohol and 75% ethyl alcohol.

Sixthly, the formula of the TE solution is Tris-HCl 1mol/L, EDTA0.5mol/L and pH 8.0.

Seventhly, 1.5mL of centrifugal tube without nuclease pollution.

(2) The main operation process of the kit is as follows:

putting 200 mu L of whole blood sample into a 1.5mL centrifugal tube, adding 800 mu L of erythrocyte lysate, turning upside down for 3-5 times, mixing uniformly, standing at room temperature for 5min, and turning upside down and mixing uniformly for 1-2 times during the standing period. Centrifuge at 12000rpm for 2min, and discard the supernatant.

② adding 10 mu L of LProteinase K solution and 200 mu L of leukocyte lysate, and whirling to ensure that the precipitate is fully resuspended. Water bath at 65 deg.c for 10 min.

③ adding 100 mu L of ammonium acetate solution, mixing evenly, centrifuging, taking supernatant, adding 400 mu L of precooled absolute ethyl alcohol, mixing evenly, and standing for 5min at room temperature. Centrifuge at 12000rpm for 10 min.

Fourthly, the supernatant is discarded, 1mL of 75 percent ethanol is added, the mixture is inverted from top to bottom for 3 to 5 times, and the mixture is centrifuged at 12000rpm for 2 min.

Fifthly, abandoning the supernatant, and airing for 5-10min at room temperature to completely volatilize the residual ethanol.

Sixthly, 100 mu LTE is added, vortex is carried out for a short time, and water bath at 65 ℃ is carried out for 5 min. The solution was collected to the bottom of the tube by centrifugation and stored at 4 ℃ or-20 ℃.

Example 2 DNA extraction and Effect detection of peripheral blood samples from healthy persons

In this example, a healthy human peripheral blood sample was used for DNA extraction, DNA concentration and purity were measured with a ultramicro spectrophotometer (NanoDrop2000), and PCR amplification effect of the DNA extract was further detected with MTHFR (C677T) gene polymorphism detection reagent. The details are as follows:

(1) sample preparation

Collecting 0.5mL of fresh human peripheral blood, adding into an EDTAK2/K3 anticoagulation tube, immediately and gently inverting and mixing for 5 times, and storing at 2-8 deg.C.

(2) DNA extraction

The DNA extraction procedure was the same as in section (2) of example 1.

(3) DNA concentration and purity measurement

DNA concentration and purity were measured using a ultramicro spectrophotometer (NanoDrop2000) on 15 samples of human blood. The DNA concentration range of 15 samples is 31.7-94.5 ng/mu L, 260/280 is between 1.8-2.0, and the specific detection results are shown in Table 1:

TABLE 1DNA extraction concentration and purity measurements

(4) Detection of PCR amplification effect of DNA extraction product

15 human blood samples were subjected to DNA extraction using the kit and nucleic acid extraction kit (YC-B) (GmbH, Wuhanhai Jili Biotech Co., Ltd.), and the PCR amplification effect of the DNA extraction product was further detected using MTHFR (C677T) gene polymorphism detection reagent as the extraction product. After the two methods are used for extracting DNA, the genotype results detected by PCR are consistent, and the detection results are shown in a table 2:

TABLE 2 comparison of PCR test results of samples

Note: the number Y represents the YC-B extraction method, and the number H represents the extraction method of the kit.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (8)

1. A DNA extraction kit is characterized by comprising the following components: erythrocyte lysate, leukocyte lysate, proteinase K solution, ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and TE solution;

the erythrocyte lysate contains per liter: 2-20g of ammonium chloride, 0.1-5g of potassium bicarbonate, 0.1-1g of disodium ethylene diamine tetraacetate and deionized water as a solvent;

the leukocyte lysate is 0.5-5% of lauryl sodium sulfate aqueous solution by mass percent;

the concentration of the proteinase K in the proteinase K solution is 0.05-2.00 mg/L;

the concentration of ammonium acetate in the ammonium acetate solution is 2.5-10 mol/L;

the 75% ethanol is ethanol water solution with 75% ethanol by volume percentage.

2. The DNA extraction kit of claim 1, wherein the pH of the red blood cell lysate is 7.0-8.0.

3. The DNA extraction kit according to claim 1, wherein the TE solution is an aqueous solution containing Tris-HCl and EDTA, and has a pH of 8.0, a Tris-HCl concentration of 1mol/L and an EDTA concentration of 0.5 mol/L.

4. A method for DNA extraction, which comprises using the DNA extraction kit according to any one of claims 1 to 3.

5. The DNA extraction method according to claim 4, wherein the whole blood sample is first freed from non-nuclear components by using a red blood cell lysate, then a white blood cell lysate and proteinase K are added to lyse cells and release nucleic acids, and finally, an ammonium acetate solution, absolute ethyl alcohol, 75% ethyl alcohol and TE solution are used to remove impurities, thereby obtaining a DNA extraction product.

6. The DNA extraction method according to claim 5, comprising the following steps:

(1) placing a whole blood sample in a centrifugal tube, adding erythrocyte lysate, uniformly mixing, placing, centrifuging again, and removing supernatant to obtain a precipitate;

(2) adding protease K solution and leukocyte lysate into the precipitate, resuspending the precipitate, and performing water bath at 65 ℃ for 10 min;

(3) adding ammonium acetate solution, mixing, centrifuging, collecting supernatant, adding precooled anhydrous ethanol, mixing, standing at room temperature, centrifuging, and removing supernatant to obtain precipitate;

(4) adding 75% ethanol into the precipitate obtained in the step (3), uniformly mixing, centrifuging, and removing the supernatant to obtain a precipitate;

(5) and (4) airing the precipitate in the step (4) at room temperature until the ethanol is completely volatilized, adding a TE solution, carrying out water bath at 65 ℃ for 5min after heavy suspension, and centrifuging to enable the solution to be concentrated to the bottom of the tube, thus obtaining the DNA extraction product.

7. The DNA extraction method according to claim 6, wherein the whole blood sample is used in an amount of 200. mu.L.

8. The DNA extraction method according to claim 6, wherein the whole blood sample is a fresh human peripheral blood sample anticoagulated with EDTAK 2/K3.