阅读说明：本技术 一种利用磁珠法提取血液基因组dna的试剂盒及其应用 (Kit for extracting blood genome DNA by using paramagnetic particle method and application thereof ) 是由 张立岩 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种利用磁珠法提取血液基因组DNA的试剂盒及其应用,属于分子生物学领域。本发明的目的是为了提供一种安全快速从血液中提取基因组DNA的试剂盒,该试剂盒包括结合液、红细胞裂解液、磁珠、磁珠漂洗液和DNA洗脱液。本发明试剂盒不包含有机溶剂,对全血细胞进行裂解后,通过磁珠吸附DNA分子,再通过漂洗去除杂质,最后对磁珠上的DNA分子进行洗脱,获得高质量的基因组DNA。该试剂盒具有操作简单快速、提取效率高、成本低廉的优点,提取的血液基因组DNA纯度高,能够满足后续如PCR扩增、基因测序等科研或临床诊断分析。(The invention discloses a kit for extracting blood genome DNA by a paramagnetic particle method and application thereof, belonging to the field of molecular biology. The invention aims to provide a kit for safely and quickly extracting genomic DNA from blood, which comprises a binding solution, a red blood cell lysate, magnetic beads, a magnetic bead rinsing solution and DNA eluent. The kit does not contain an organic solvent, after cracking the whole blood cells, the DNA molecules are adsorbed by the magnetic beads, impurities are removed by rinsing, and finally the DNA molecules on the magnetic beads are eluted to obtain high-quality genome DNA. The kit has the advantages of simple and rapid operation, high extraction efficiency and low cost, and the extracted blood genome DNA has high purity, and can meet the requirements of follow-up scientific research or clinical diagnosis and analysis such as PCR amplification, gene sequencing and the like.)

1. A kit for extracting blood DNA by using a magnetic bead technology is characterized in that the kit comprises components of a red blood cell lysate, a magnetic bead rinsing solution, a DNA eluent, a binding solution and magnetic beads, wherein the binding solution comprises SDS, Tris-HCl and EDTA, the magnetic bead rinsing solution is an ethanol aqueous solution, the DNA eluent comprises Tris-HCl and EDTA, and the red blood cell lysate comprises Tris-HCl, NaCl and MgCl₂The magnetic beads are hydroxyl magnetic beads.

2. The kit according to claim 1, wherein the erythrocyte lysate has a concentration of Tris-HCl 0.01mol/L, a pH of 7.6, a concentration of NaCl 0.01mol/L, and MgCl₂The concentration of (B) is 0.005 mol/L.

3. The kit of claim 1, wherein the volume fraction of ethanol solution in the bead rinse is 70%.

4. The kit of claim 1, wherein the DNA eluate has a Tris-HCl concentration of 10mM, a PH of 7.5, and an EDTA concentration of 0.1 mM.

5. The kit of claim 1, wherein the concentration of the magnetic beads is 300 nM.

6. The kit according to claim 1, wherein the binding solution has a concentration of Tris-HCl of 10mM, pH8, EDTA of 14mM, and SDS of 0.4% by mass.

7. Use of the kit of any one of claims 1 to 6 for the extraction of blood DNA.

8. The application of claim 7, wherein the specific steps of the application are as follows:

(1) adding 100-400 mul of whole blood into a 1.5mL centrifuge tube, adding 20 mul of proteinase K, then adding 300 mul of binding solution, uniformly mixing, standing at 65 ℃ for 15 minutes, uniformly mixing every 1-3 minutes, and totally mixing for 5 times;

(2) adding 350 mu L of isopropanol into the solution obtained in the step (1), uniformly mixing, adding 50 mu L of magnetic bead solution, uniformly mixing, standing at room temperature for 7 minutes, uniformly mixing every 1 minute for 5 times in total;

(3) performing instantaneous centrifugation on the solution obtained in the step (2), collecting liquid at the bottom of the tube, placing the collected liquid on a magnetic frame to stand for 2 minutes, sucking and discarding all the solution, adding 700 mu L of magnetic bead rinsing liquid, resuspending the magnetic beads, washing the magnetic beads for 2 times, and sucking and discarding all the solution;

(4) and (2) placing the magnetic beads in air for drying for 10-15 minutes, adding 50-100 mu LDNA eluent after the surfaces of the magnetic beads have no obvious water bead luster and no residual liquid, after the magnetic beads are resuspended, placing the magnetic beads at 58 ℃ for incubation for 5 minutes, then placing the magnetic beads on a magnetic frame for standing for 1-2 minutes, and placing supernate into a new centrifugal tube to finish the extraction of DNA in whole blood.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a kit for extracting blood genome DNA by a paramagnetic particle method and application thereof.

Background

The traditional method for extracting the DNA of the blood at present is derived from 'cracking of freshly extracted or cryopreserved blood cells' of 'molecular cloning experimental guidelines', the method mainly utilizes phenol/chloroform to extract the DNA, but the method has low purity of the extracted DNA, is easy to be polluted by plasma protein, has long experimental operation time, and in addition, the used reagent has toxic action on human bodies and the environment; although the centrifugal column method can improve the purity of DNA, the centrifugal method can adsorb only large DNA fragments due to the affinity problem of the affinity column.

Disclosure of Invention

The invention aims to provide a method for safely and quickly extracting genomic DNA from blood, which overcomes the problems that harmful solvents in the conventional common method have the harm to human bodies, time and labor are saved, the cost is saved, and the purity of the extracted genomic DNA is high. The invention provides a kit for extracting blood DNA by using a magnetic bead technology, which comprises components of a red blood cell lysate, a magnetic bead rinsing solution, a DNA eluent, a binding solution and magnetic beads, wherein the binding solution comprises SDS, Tris-HCl and EDTA, the magnetic bead rinsing solution is an ethanol aqueous solution, the DNA eluent comprises Tris-HCl and EDTA, the red blood cell lysate comprises Tris-HCl, NaCl and MgCl₂The magnetic beads are hydroxyl magnetic beads.

Further, the erythrocyte lysate has a Tris-HCl concentration of 0.01mol/L, a pH value of 7.6, a NaCl concentration of 0.01mol/L, and MgCl₂The concentration of (B) is 0.005 mol/L.

Further defined, the volume fraction of the ethanol solution in the bead rinse solution is 70%.

Further defined, the DNA eluate has a Tris-HCl concentration of 10mM, a pH of 7.5, and an EDTA concentration of 0.1 mM.

Further defined, the concentration of the magnetic beads is 300 nM.

Further, the binding solution has a concentration of Tris-HCl of 10mM, a PH of 8, an EDTA concentration of 14mM, and an SDS mass fraction of 0.4%.

The invention also provides application of the kit for extracting the blood DNA by using the magnetic bead technology in extracting the blood DNA.

Further defined, the specific steps of the application are as follows:

(1) adding 100-400 mul of whole blood into a 1.5mL centrifuge tube, adding 20 mul of proteinase K, then adding 300 mul of binding solution, uniformly mixing, standing at 65 ℃ for 15 minutes, uniformly mixing every 1-3 minutes, and totally mixing for 5 times;

(2) adding 350 mu L of isopropanol into the solution obtained in the step (1), uniformly mixing, adding 50 mu L of magnetic bead solution, uniformly mixing, standing at room temperature for 7 minutes, uniformly mixing every 1 minute for 5 times in total;

(3) performing instantaneous centrifugation on the solution obtained in the step (2), collecting liquid at the bottom of the tube, placing the collected liquid on a magnetic frame to stand for 2 minutes, sucking and discarding all the solution, adding 700 mu L of magnetic bead rinsing liquid, resuspending the magnetic beads, washing the magnetic beads for 2 times, and sucking and discarding all the solution;

(4) and (2) placing the magnetic beads in air for drying for 10-15 minutes, adding 50-100 mu LDNA eluent after the surfaces of the magnetic beads have no obvious water bead luster and no residual liquid, after the magnetic beads are resuspended, placing the magnetic beads at 58 ℃ for incubation for 5 minutes, then placing the magnetic beads on a magnetic frame for standing for 1-2 minutes, and placing supernate into a new centrifugal tube to finish the extraction of DNA in whole blood.

Has the advantages that: 1. the method is simple and quick to operate, the whole blood does not need to be pretreated, the extraction operation can be completed quickly within 1h, and the extraction time of the whole blood genome DNA is greatly shortened.

2. The kit does not use organic solvents such as chloroform, phenol and the like, and can greatly reduce the physical damage to experimenters.

3. The extraction method of the kit can effectively reduce the loss of nucleic acid in the extraction process, greatly increase the extraction efficiency, and ensure that the genome DNA product has high purity and more complete fragments.

4. The sample has wide applicability: genomic DNA can be directly extracted from samples such as fresh or frozen anticoagulated blood (EDTA, heparin and the like), a leucocyte layer, blood clots, saliva, an oral swab and the like.

Drawings

FIG. 1 is an agarose gel electrophoresis strip of genomic DNA of human whole blood extracted according to the present invention, wherein M is Marker, 100uL is a sample from which 100uL of human whole blood DNA is extracted, 200uL is a sample from which 200uL of human whole blood DNA is extracted, 300uL is a sample from which 300uL of human whole blood DNA is extracted, and 400uL is a sample from which 400uL of human whole blood DNA is extracted;

FIG. 2 is a graph showing the results of extracting genomic DNA from a leukocyte layer, a blood clot, saliva and a buccal swab in accordance with the present invention, wherein the abscissa is the cycle number and the ordinate is the fluorescence intensity.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1.

The kit for extracting the DNA of the blood by utilizing the magnetic bead technology comprises the components of red blood cell lysate, magnetic bead rinsing liquid, DNA eluent, binding solution and magnetic beads.

(1) Binding liquid: 0.4% SDS, 10mM Tris-HCl (pH8.0), 14mM EDTA (pH 8.0).

(2) Erythrocyte lysate: 0.01 mol/LTris-HCLpH7.6; 0.01 mol/LNaCl; 0.005mol/LMgCl 2.

(3) Magnetic bead rinsing liquid: 70 percent ethanol solution by volume fraction.

(4) DNA eluent: 10mM Tris HCl and 0.1mM EDTA, final pH 7.5.

(5) Specification of magnetic beads: 300nM hydroyxomagnetic beads.

Example 2 method for extracting genomic DNA from blood Using the magnetic bead method

3 parts of human frozen anticoagulated blood genomic DNA was extracted using the kit in example 1.

1. Preparing a clean 1.5mL centrifuge tube, sequentially adding 20 μ L of proteinase K (20mg/mL) and 100 μ L of human whole blood sample, finally adding 300 μ L of lysis/binding solution GAL, and vortexing for 30s to mix uniformly; standing at 65 ℃ for 15min, and turning upside down every 1-3 min for 5 times to mix uniformly.

2. Taking the solution in the last step out of the water bath, standing at room temperature for about 3min, adding 350 μ L isopropanol, and mixing uniformly for about 10s by vortex; adding 50 μ LDNA combined magnetic beads, mixing by vortexing or turning upside down for 20s, standing at room temperature for 7min, and turning upside down for 5 times.

3. And (5) performing instantaneous centrifugation for 2s, and collecting liquid at the bottom of the tube.

4. The centrifuge tube was transferred to a magnetic rack, placed for 2min to adsorb magnetic beads, and carefully pipetted off all solutions.

5. Adding 700 mu L of washing solution (added with absolute ethyl alcohol), uniformly mixing by vortex, completely suspending the magnetic beads on the tube wall in the solution, instantly centrifuging for 2s, collecting the liquid on the tube wall, transferring the liquid to a magnetic frame, standing for 1min, or carefully sucking and discarding all the solution when the solution is completely clarified.

6. Repeat step 5 once.

7. Adding 700 μ L of rinsing solution (added with anhydrous ethanol), vortex mixing, suspending the magnetic beads in the solution completely, centrifuging for 2s instantly to collect the liquid on the tube wall, transferring to a magnetic frame, standing for 1min, or carefully removing all the solution when the solution is completely clarified.

8. Repeat step 7 once.

9. And taking the centrifugal tube off the magnetic frame, performing instantaneous centrifugation for 3s to collect residual liquid on the tube wall, placing the tube on the magnetic frame for tens of seconds to adsorb magnetic beads, removing all residual liquid by using a 200 mu L pipette tip, keeping the cover of the centrifugal tube in an open state, and drying the tube in the air for 10-15 min. Note that: the drying time is related to the addition amount of magnetic beads, air humidity and environment temperature, the actual drying time is flexibly increased and decreased, no obvious water bead gloss exists on the surfaces of the magnetic beads, no residual liquid is left in a centrifugal tube, and excessive drying of the magnetic beads can cause that DNA is difficult to elute and the yield is influenced.

10. Adding 50-100 mu L of eluent, swirling for 1min to resuspend magnetic beads, placing the centrifuge tube in a water bath at 58 ℃ to incubate for 5min, and shaking the centrifuge tube every 2-3 min to accelerate DNA dissolution.

11. And (3) performing instantaneous centrifugation for 2s to collect magnetic beads and liquid on the tube wall, transferring the centrifuge tube to a magnetic frame, standing for 1-2 min, and carefully transferring the DNA solution to a new centrifuge tube with the volume of 1.5 mL. The DNA solution can be stored temporarily at 2-8 ℃ for 1-2 days, and the DNA solution is required to be placed at-20 ℃ for long-time storage.

Example 3 method for extracting genomic DNA from blood Using the magnetic bead method

3 parts of human cryoanticoagulated blood genomic DNA were extracted using the kit in example 1: methods referring to example 2, 200. mu.L of human whole blood sample.

Example 4 method for extracting genomic DNA from blood Using the magnetic bead method

3 parts of human cryoanticoagulated blood genomic DNA were extracted using the kit in example 1: methods referring to example 2, 300. mu.L of human whole blood sample.

Example 5 extraction of genomic DNA from blood Using the magnetic bead method

3 parts of human cryoanticoagulated blood genomic DNA were extracted using the kit in example 1: methods referring to example 2, 400 μ L of human whole blood samples.

The experimental effect was verified using the following experiment:

genomic experimental results were extracted for different human whole blood volumes (100. mu.L, 200. mu.L, 300. mu.L, 400. mu.L). The agarose gel concentration is 1%, the sample is loaded with 3.0 μ L, M is 15KbDNAmarker, the sample is loaded with 5 μ L, the voltage is 130V, and the electrophoresis time is 20 min. The result is shown in figure 1, the obtained whole genome DNA product has good parallelism, clear band and no tailing and impurity band, and shows that the extracted genome DNA has good integrity, no other DNA pollution, high DNA product content and good purity.

Respectively extracting total DNA of human cells, a leucocyte layer, a blood clot, saliva and an oral swab, diluting and properly diluting the total DNA, amplifying an internal reference gene ACTB, and obtaining a cell sample: 10^6293T cells, blood clots: 1g blood clot, anticoagulation: 1mL, saliva dilution: 5mL, throat swab extract: 1mL gave a band that was amplified efficiently, and the results are shown in FIG. 2.