阅读说明：本技术 一种利用角蛋白酶提取dna的方法 (method for extracting DNA by using keratinase ) 是由 张丹 侯庆唐 夏冬景 何凤琴 于 2019-09-22 设计创作，主要内容包括：本发明公开了一种利用角蛋白酶提取DNA的方法,该发明通过分解角蛋白获得DNA,指纹中含有皮肤角质层细胞脱落的物质,角质层细胞是一些角化死亡的细胞,含有少量尚未完全降解的基因组DNA和大量角蛋白,与角质层细胞类似,指甲、毛发也含有少量尚未完全降解的基因组DNA和大量角蛋白,而角蛋白分子内部含有丰富的二硫键,使得肽链内部和肽链之间发生交联,形成立体网状结构,使角蛋白难以溶解以及分解,加入角蛋白酶利用酶的专一性使得样品中的角蛋白被角蛋白酶分解为可溶性的多肽,从而释放样品中的DNA,并经过一系列裂解、结合、漂洗、洗脱等DNA提取步骤,实现DNA提取的目的,提取得到的DNA纯度高,方法便捷,具有很好的推广前景。(the invention discloses a method for extracting DNA by keratinase, which obtains DNA by decomposing keratin, a fingerprint contains substances shed by skin stratum corneum cells, the stratum corneum cells are cells with keratinized death, contain a small amount of genomic DNA which is not completely degraded and a large amount of keratin, are similar to the stratum corneum cells, nails and hairs also contain a small amount of genomic DNA which is not completely degraded and a large amount of keratin, keratin molecules contain rich disulfide bonds, the inside of peptide chains and peptide chains are crosslinked to form a three-dimensional network structure, the keratin is difficult to dissolve and decompose, the keratin in a sample is decomposed into soluble polypeptide by the keratinase by the specificity of enzyme by adding the keratinase, thereby releasing the DNA in the sample, and the purpose of DNA extraction is realized by a series of DNA extraction steps of cracking, combining, rinsing, eluting and the like, the extracted DNA has high purity, and the method is convenient and has good popularization prospect.)

1. a method for extracting DNA by using keratinase, which is characterized in that: the method comprises the following steps:

A. sampling, treating the sample, adding keratinase and lysis solution into the sample, uniformly mixing, heating, digesting and lysing;

B. transferring the supernatant after high-speed centrifugation to a new centrifugal tube, adding a binding solution into the centrifugal tube, and binding DNA through a DNA binding carrier;

C. Rinsing the DNA-binding carrier with an ethanol solution;

D. The DNA was eluted with a eluent.

2. the method for extracting DNA using keratinase according to claim 1, wherein: the sample in the step A is one of hair, nails and fingerprints, and is specifically processed by the following method:

1) hair: taking one hair, removing the hair follicle part, cutting the part which is 3-4 cm long and close to the hair follicle end, and putting the cut part into a centrifugal tube;

2) Nail: taking a nail A, cutting into small pieces, and putting into a centrifuge tube;

3) Fingerprint: wiping the fingerprint with a swab stained with purified water, breaking the swab head and putting the swab head into a centrifuge tube.

3. the method for extracting DNA using keratinase according to claim 1, wherein: the concentration of the keratinase in the step A is 5Units/ul, and the dosage of the keratinase is 10 ul; the lysis solution in the step A comprises the following components: 10mmol/l Tris-HCl, sodium dodecyl sulfate with m/v concentration of 0.1%, 1mmol/l EDTA, 10.0 pH value of lysis solution and 300ul dosage of lysis solution.

4. the method for extracting DNA using keratinase according to claim 1, wherein: the components of the binding solution in the step B are as follows: 4.5mol/l of guanidinium isothiocyanate, 100mmol/l of sodium citrate, the pH value of the binding solution is 5.5, and the dosage of the binding solution is 450 ul.

5. The method for extracting DNA using keratinase according to claim 1, wherein: and the DNA binding carrier in the step B is one of magnetic beads, silica beads and silica gel molds.

6. The method for extracting DNA using keratinase according to claim 5, wherein: the specific steps for extracting DNA by magnetic beads are as follows:

1.1, sampling, processing a sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in a water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

1.2: placing the centrifuge tube filled with the first mixed solution in the step 1.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding a binding solution and magnetic beads, placing the centrifuge tube on an oscillator, shaking for 5-15min at the room temperature at the rotation speed of 1300rpm, placing the centrifuge tube on a magnetic frame, standing for 5-10min, and removing the supernatant to obtain the magnetic beads of a first intermediate;

1.3: adding a rinsing liquid into the centrifugal tube with the magnetic beads in the step 1.2, placing the centrifugal tube on an oscillator, oscillating for 5-10min at room temperature with the rotation speed of 1300rpm, placing the centrifugal tube on a magnetic frame, standing for 5-10min, removing supernatant, adding the rinsing liquid again, oscillating for 5-10min at room temperature with the rotation speed of 1300rpm, placing the centrifugal tube on the magnetic frame, standing for 5-10min, and removing supernatant to obtain the magnetic beads containing the second intermediate;

1.4: and (3) putting the magnetic beads containing the second intermediate prepared in the step (1.3) into a new centrifugal tube, adding eluent, shaking for 10-15min under the conditions that the temperature is 65 ℃ and the rotating speed is 1300rpm, and taking out the magnetic beads to prepare the DNA solution.

7. The method for extracting DNA using keratinase according to claim 5, wherein: the specific steps for extracting DNA by silica beads are as follows:

2.1, sampling, processing the sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

2.2: placing the centrifuge tube filled with the first mixed solution in the step 2.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding the binding solution and the silica beads, placing the centrifuge tube on an oscillator, shaking for 5-15min at the room temperature at the rotation speed of 1300rpm, placing the centrifuge tube into the centrifuge, centrifuging for 1-2min at the room temperature at the rotation speed of 8000rpm, and removing the supernatant to obtain silica beads containing a third intermediate;

2.3: adding the rinsing liquid into the centrifugal tube filled with the silica beads in the step 2.2, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the centrifugal tube, adding the rinsing liquid again, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the centrifugal tube, and centrifuging for 3min at the rotating speed of 12000rpm to obtain the silica beads containing the fourth intermediate;

2.4: and (3) putting the silica beads containing the fourth intermediate in the step 2.3 into a new centrifugal tube, adding an eluent, standing for 3min at room temperature, centrifuging for 1min at the rotation speed of 12000rpm, and taking out the silica beads to obtain the DNA solution.

8. the method for extracting DNA using keratinase according to claim 5, wherein: the specific steps of extracting DNA by a silica gel mold are as follows:

3.1, sampling, processing the sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in a water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

3.2: putting the centrifuge tube filled with the first mixed solution in the step 3.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding the binding solution, stirring for 5-15min to prepare a second mixed solution, adding the second mixed solution into a silica gel membrane centrifugal column placed into a collecting tube, centrifuging for 2min at the rotation speed of 12000rpm, and removing the liquid in the collecting tube to obtain a silica gel membrane containing a fifth intermediate;

3.3: adding the rinsing liquid into the collecting pipe with the silica gel membrane in the step 3.2, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the collecting pipe, adding the rinsing liquid again, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the collecting pipe, and centrifuging for 3min at the rotating speed of 12000rpm to obtain a silica gel membrane centrifugal column containing a sixth intermediate;

3.4: and (3) putting the silica gel membrane centrifugal column containing the sixth intermediate in the step 3.3 into a new centrifugal tube, dripping eluent on the silica gel membrane in the center of the centrifugal column, standing for 3min at room temperature, centrifuging for 1min at the rotation speed of 12000rpm, and taking out the centrifugal column to obtain the DNA solution.

9. The method for extracting DNA using keratinase according to claim 1, wherein: the rinsing liquid in the step C is ethanol with the concentration of 75% v/v, and the using amount of the rinsing liquid is 500 ul.

10. the method for extracting DNA using keratinase according to claim 1, wherein: the eluent in the step D is one of water or TE buffer solution with the pH value of 8.0, and the dosage of the eluent is 30 ul.

Technical Field

The invention belongs to the technical field of DNA extraction, and particularly relates to a method for extracting DNA by using keratinase.

Background

In the biological evidence of suspect remaining in the crime scene, the fingerprint, hair, nail and the like occupy a very large proportion, and the case inspector needs to solve the problem of extracting DNA of the fingerprint, hair, nail and the like when trying to acquire the genetic information of the suspect from the evidence, wherein the fingerprint contains substances shed by skin cuticle cells, the cuticle cells are cells with keratinization death, contain a small amount of genomic DNA which is not completely degraded and a large amount of keratin, and similar to the cuticle cells, the nail and hair also contain a small amount of genomic DNA which is not completely degraded and a large amount of keratin, and how to digest the keratin and release trace DNA in the keratin is the key for successfully extracting DNA from the fingerprint, hair and nail;

a DNA extraction method commonly used at the present stage is an organic method, the organic method utilizes the characteristic that DNA is easily soluble in water and insoluble in an organic solution, an organic solvent is used for extracting and mixing a sample with pure water, protein molecules in the sample and water are hydrated to form a hydration layer on the surface, so that the protein molecules enter an aqueous solution to form a stable colloidal solution, the organic solvent is added for centrifugally extracting the DNA, the extracted DNA is characterized by double chains and high purity, but a certain amount of toxic organic reagent is added in the process of extracting the DNA, the toxic organic reagent is volatilized during DNA extraction, the toxic organic reagent causes damage to human bodies and environmental pollution, and a DNA centrifugal tube needs to be replaced ordinarily, so that the DNA is easily polluted in the process of extracting the DNA, the detection effect is influenced, and meanwhile, the DNA centrifugal tube is replaced for multiple times, the coding disorder of a container is easily caused, and the DNA sequence cannot be accurately;

Chinese invention patent CN102382821A discloses a method for extracting DNA, the invention is through after the sample is washed and dried by absolute ethyl alcohol and distilled water, after digestion in PCR buffer solution with SDS and protease K, add RNase to remove RNA, then add CTAB binding protein under the high salt environment, finally extract through chloroform isoamyl alcohol, isopropanol is precipitated, wash with ethanol solution, get DNA sample, the invention is DNA extracted to make DNA in the sample release fully, it is effectual to extract, but the invention in extracting DNA process, use a certain amount of poisonous organic solvent, apt to cause the health damage of operating personnel and cause the pollution to the environment, add too much organic reagent, will cause certain influence while amplifying and amplifying the DNA chain, influence the subsequent determination to DNA sequence.

Disclosure of Invention

The invention aims to provide a method for extracting DNA by using keratinase, which comprises the steps of firstly sampling, then processing a sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate, wherein the keratinase is an enzyme capable of specifically degrading natural keratin, can decompose keratin which is not dissolved in water into soluble polypeptide, fingerprints comprise substances shed by skin stratum corneum cells, the stratum corneum cells are cells with keratinization death, contain a small amount of genomic DNA and a large amount of keratin which are not completely degraded, hair and nails contain a small amount of genomic DNA and a large amount of keratin which are not completely degraded, the keratinase is used for decomposing the keratin, the lysate is used for extracting soluble protein from the sample, the protein is separated by centrifugation, binding solution is added, the DNA is obtained by centrifugation through a silica gel membrane centrifuge column, and rinsing solution is added for precipitating the DNA, centrifuging for many times to make the DNA attached to the silica gel membrane, and separating the DNA from the silica gel membrane by using a eluent to obtain a DNA solution; the method does not use a large amount of toxic solvent, does not harm the life safety of operators and does not pollute the environment in the extraction process, does not use a large amount of toxic solvent and does not influence the subsequent DNA sequence determination, does not pollute the DNA when the DNA loading and taking container is replaced for a few times, and has simple and convenient operation and high purity of extracted DNA.

The technical problems to be solved by the invention are as follows:

1. common methods for extracting DNA are an organic method and a Chelex100 method, the organic method is a classical DNA extraction method, the extracted DNA is characterized by double chains and high purity, but a certain amount of toxic organic reagent is added in the process of extracting the DNA, the toxic organic reagent volatilizes when the DNA is extracted, the harm is caused to human bodies, the environmental pollution is caused, DNA loading and taking containers need to be replaced for many times in the process of extracting the DNA, the pollution is easily caused to the DNA, and the subsequent DNA sequence determination result is influenced;

2. the common method for extracting DNA needs to consume a large amount of reagents in the extraction process, the extraction method is complex, the extraction time is long, the purity of the extracted DNA is not high, the extracted DNA is polluted by RNA, protein and phenol, and the DNA sequence cannot be accurately measured;

3. The Chelex100 method is used for DNA extraction, and a sample for extracting DNA needs to be washed for many times, so that partial DNA in the sample is lost, the DNA extraction work is completed, a DNA sequence cannot be accurately determined when the DNA sequence is determined, and the work efficiency of the DNA determination is seriously influenced.

The purpose of the invention can be realized by the following technical scheme:

a method for extracting DNA by using keratinase, comprising the following steps:

A. sampling, treating the sample, adding keratinase and lysis solution into the sample, uniformly mixing, heating, digesting and lysing;

B. transferring the supernatant after high-speed centrifugation to a new centrifugal tube, adding a binding solution into the centrifugal tube, and binding DNA through a DNA binding carrier;

C. Rinsing the DNA-binding carrier with an ethanol solution;

D. the DNA was eluted with a eluent.

Further, the sample in the step A is one of hair, nail and fingerprint, and is processed by the following method:

1) hair: taking one hair, removing the hair follicle part, cutting the part which is 3-4 cm long and close to the hair follicle end, and putting the cut part into a centrifugal tube;

2) nail: taking a nail A, cutting into small pieces, and putting into a centrifuge tube;

3) fingerprint: wiping the fingerprint with a swab stained with purified water, breaking the swab head and putting the swab head into a centrifuge tube.

Further, the DNA binding carrier in the step B is one of magnetic beads, silica beads and silica gel molds.

Further, the specific steps of extracting DNA by magnetic beads are as follows:

1.1, sampling, processing a sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in a water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

1.2: placing the centrifuge tube filled with the first mixed solution in the step 1.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding a binding solution and magnetic beads, placing the centrifuge tube on an oscillator, shaking for 5-15min at the room temperature at the rotation speed of 1300rpm, placing the centrifuge tube on a magnetic frame, standing for 5-10min, and removing the supernatant to obtain a first intermediate;

1.3: adding a rinsing liquid into the centrifugal tube with the magnetic beads in the step 1.2, placing the centrifugal tube on an oscillator, oscillating for 5-10min at room temperature with the rotation speed of 1300rpm, placing the centrifugal tube on a magnetic frame, standing for 5-10min, removing supernatant, adding the rinsing liquid again, oscillating for 5-10min at room temperature with the rotation speed of 1300rpm, placing the centrifugal tube on the magnetic frame, standing for 5-10min, and removing supernatant to obtain the magnetic beads containing the second intermediate;

1.4: and (3) placing the magnetic beads containing the second intermediate prepared in the step (1.3) into a new centrifugal tube, shaking for 10-15min at the temperature of 65 ℃ and the rotation speed of 1300rpm, and removing the magnetic beads to obtain the DNA solution.

further, the specific steps of extracting DNA by silica beads are as follows:

2.1, sampling, processing the sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

2.2: placing the centrifuge tube filled with the first mixed solution in the step 2.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding the binding solution and silica beads, placing the centrifuge tube on an oscillator, shaking for 5-15min at the room temperature at the rotation speed of 1300rpm, placing the centrifuge tube into the centrifuge, centrifuging for 1-2min at the room temperature at the rotation speed of 8000rpm, and removing the supernatant to obtain a third intermediate;

2.3: adding the rinsing liquid into the centrifugal tube filled with the silica beads in the step 2.2, centrifuging for 1min at the rotating speed of 12000rpm, removing supernatant liquid in the centrifugal tube, adding the rinsing liquid again, centrifuging for 1min at the rotating speed of 12000rpm, removing the supernatant liquid in the centrifugal tube, and centrifuging for 3min at the rotating speed of 12000rpm to obtain silica beads containing a fourth intermediate;

2.4: and (3) putting the silica beads containing the fourth intermediate in the step 2.3 into a new centrifugal tube, adding eluent, standing for 3min at room temperature, centrifuging for 1min at the rotation speed of 12000rpm, and taking out a centrifugal column to prepare the DNA solution.

Further, the specific steps of extracting DNA through a silica gel membrane are as follows:

3.1, sampling, processing the sample, adding the processed sample into a centrifuge tube, adding keratinase and lysate into the centrifuge tube, stirring for 5-15min, and heating in a water bath for 3-6h at the temperature of 50 ℃ to prepare a first mixed solution;

3.2: putting the centrifuge tube filled with the first mixed solution in the step 3.1 into a centrifuge, centrifuging for 1min at the rotation speed of 12000rpm, transferring the supernatant into a new centrifuge tube, adding the binding solution, stirring for 5-15min to prepare a second mixed solution, adding the second mixed solution into a silica gel membrane centrifugal column put into a collecting tube, centrifuging for 2min at the rotation speed of 12000rpm, and removing the liquid in the collecting tube to obtain a fifth intermediate;

3.3: adding the rinsing liquid into the collecting pipe filled with the fifth intermediate in the step 3.2, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the collecting pipe, adding the rinsing liquid again, centrifuging for 1min at the rotating speed of 12000rpm, removing the liquid in the collecting pipe, and centrifuging for 3min at the rotating speed of 12000rpm to obtain a silica gel membrane centrifugal column containing the sixth intermediate;

3.4: and (3) putting the silica gel membrane centrifugal column containing the sixth intermediate in the step 3.3 into a new centrifugal tube, dripping eluent on the silica gel membrane in the center of the centrifugal column, standing for 3min at room temperature, centrifuging for 1min at the rotation speed of 12000rpm, and taking out the centrifugal column to obtain the DNA solution.

Further, the concentration of keratinase in step A is 5Units/ul, and the dosage of keratinase is 10 ul.

further, the lysate in step a comprises the following components: 10mmol/l Tris-HCl, 0.1% (m/v) sodium dodecyl sulfate, 1mmol/l EDTA, 10.0 pH value of lysis solution and 300ul dosage of lysis solution.

Further, the components of the binding solution in the step B are as follows: 4.5mol/l of guanidinium isothiocyanate, 100mmol/l of sodium citrate, the pH value of the binding solution is 5.5, and the dosage of the binding solution is 450 ul.

Further, the rinsing liquid in step C is 75% (v/v) ethanol, and the amount of the rinsing liquid is 500 ul.

Further, the eluent in the step D is one of water or TE buffer solution with the pH value of 8.0, and the dosage of the eluent is 30 ul.

the invention has the beneficial effects that:

1. adding keratinase and a lysis solution into a sample to dissolve protein contained in the sample, separating DNA from the protein, adding a binding solution to distribute the DNA and the protein in different media, centrifuging to remove the protein, adding a rinsing solution to precipitate the DNA from the binding solution after centrifuging or shaking for many times to be attached to an adsorption carrier, and adding an eluent to separate the DNA from the DNA binding carrier to obtain a DNA solution, wherein a large amount of toxic organic reagent is not added in the process of extracting the DNA, so that the method does not influence the health of operators and pollute the environment, does not influence the amplification of subsequent DNA, further ensures the effect of DNA sequencing, does not need to replace DNA centrifuge tubes for many times, and reduces the risk of DNA pollution;

2. The sample comprises fingerprints, nails and hair, wherein the fingerprints contain substances shed by skin stratum corneum cells, the stratum corneum cells are keratinized dead cells and contain a small amount of incompletely degraded micro-genomic DNA and a large amount of keratin, the nails and the hair also contain a small amount of incompletely degraded micro-genomic DNA and a large amount of keratin, the keratin can be divided into two types of soft keratin and hard keratin, the soft keratin exists in skin and other cell tissues, the hard keratin is a main component forming hair, nails and the like, keratin molecules contain rich disulfide bonds inside keratin molecules, cross-linking occurs between peptide chains and peptide chains to form a three-dimensional network structure, the natural keratin is difficult to dissolve and decompose, the keratin in the sample is decomposed into soluble polypeptide by adding keratinase by utilizing the specificity of the enzyme, so that the micro-DNA in the sample is released, and then, the DNA combination carrier is used for centrifugation or oscillation, functional groups with DNA adsorption are fixed on the DNA combination carrier, and the combination liquid, rinsing liquid and eluent are added for repeated centrifugation or oscillation so as to separate the DNA from impurities.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.