阅读说明：本技术 一种dna提取试剂盒及提取方法 (DNA extraction kit and extraction method ) 是由 余小玲 陆青 朱峰 彭杰 黄翔 卢彦羽 肖红涛 赵平锋 于 2020-07-21 设计创作，主要内容包括：本发明公开了一种DNA提取试剂盒及提取方法,试剂盒由以下成分组成：红细胞裂解液、白细胞裂解液、蛋白酶K溶液、醋酸铵溶液、无水乙醇、75％乙醇和TE溶液。提取方法是全血样本先用红细胞裂解液去除无核成分,再加入白细胞裂解液和蛋白酶K裂解细胞并释放核酸,最后采用醋酸铵溶液、无水乙醇、75％乙醇和TE溶液去除杂质,获得DNA提取产物。该试剂盒成分简单,无有毒有害成分,所需样品少,提取效果好；提取方法操作简单、快速,不需要特殊器材辅助,仅使用离心管和离心机即可完成,整个提取过程只需要40分钟即可完成；所提取的DNA产物浓度和纯度均符合要求。(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.)

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.

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.