CN111549023A - Magnetic nano material and application thereof in extraction of human whole blood genome DNA - Google Patents
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Abstract
The invention discloses a magnetic nano material and application thereof in extraction of human whole blood genome DNA, the magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide, the concentration of genome DNA extracted from 100 mu l of human whole blood genome by the magnetic nano material is about 1 mu g, the concentration of genome DNA extracted from 100 mu l of human whole blood genome by a conventional kit method is about 0.5 mu g, and the concentration of human whole blood genome extracted by the magnetic nano material is higher. The genome DNA extracted from the human whole blood genome provided by the magnetic nano material prepared by the invention is subjected to PCR amplification experiment, and the DNA fragment of about 680bp of the mitochondrial cytochrome oxidase COI gene is amplified by PCR. The human whole blood genome DNA extracted by the novel magnetic nano material does not influence the subsequent PCR experiment.
Description
Technical Field
The invention belongs to the field of nano materials and application thereof, and particularly relates to a magnetic nano material and application thereof in extraction of human whole blood genome DNA.
Background
DNA is the most important biological information molecule as a carrier of genetic information, and is a major subject of molecular biology research. The extraction of genomic DNA is the basis of many molecular biological experiments, and the acquisition of high molecular weight and high purity genomic DNA is an important prerequisite for research in routine experiments such as genome sequencing, Southern hybridization, PCR amplification, BCA library construction, molecular cloning, etc. With the deep development of molecular biology technology, the detection and diagnosis of diseases at the gene level has become a basic technical means for clinical diagnosis; especially, individual identification, paternity test and the like in forensic medicine have higher requirements on the integrity and purity of the extracted genome DNA. Blood is readily available and reliable, and therefore, many studies have used blood as a material to extract complete genomic DNA. In consideration of various factors such as experiment cost and work efficiency, researchers want to obtain human whole blood genome DNA as soon as possible for long-term storage, and meet the needs of long-term, repeated and various researches.
Genomic DNA extraction generally follows several principles: 1. ensuring the integrity of the extracted DNA fragments; 2. substances having an inhibitory effect on DNA should not be present after purification; 3. the pollution of organic solvent and metal ions is reduced as much as possible; 4. reducing substances such as protein, polysaccharide, lipid and the like; 5. reduce contamination by other nucleic acid molecules. For the extraction of human whole blood genome DNA, the classical method is to digest by SDS and proteinase k and repeatedly extract by phenol chloroform, and the process not only uses poisonous phenol and chloroform, but also has the disadvantages of complex operation, time and labor consumption, and is very easy to cause the loss and pollution of samples.
Disclosure of Invention
In order to solve the problems, the invention provides a magnetic nano material and application thereof in extraction of human whole blood genome DNA, the magnetic nano material has the advantages of simple and convenient operation, higher concentration of extracted genome DNA, no influence on subsequent PCR experiments and the like, and the specific content of the magnetic nano material is as follows:
the invention aims to provide a magnetic nano material, which has the technical points that: the magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide.
In some embodiments of the present invention, the method for preparing the magnetic nanomaterial comprises the following steps:
the method comprises the following steps: weighing zero-valent nano iron and graphene oxide, respectively placing the weighed zero-valent nano iron and graphene oxide in purified water, and performing ultrasonic dispersion treatment to obtain a suspension containing zero-valent nano iron and a suspension containing graphene oxide;
step two: and (3) pouring the turbid liquid containing the zero-valent nano iron and the turbid liquid containing the graphene oxide obtained in the step one into the same beaker, uniformly mixing to obtain a mixed turbid liquid, then placing the mixed turbid liquid on a shaking table for coating treatment, and finally placing the mixed turbid liquid treated by the shaking table in an oven for drying to obtain the magnetic nano material.
In some embodiments of the present invention, the zero-valent nano-iron is a composite of zero-valent nano-iron and polymethyl methacrylate.
In some embodiments of the present invention, the ultrasonic treatment conditions in the first step of the preparation method of a magnetic nanomaterial are as follows: 1400 ℃ and 2000HZ, 100 ℃ and 200W, the ultrasonic time is 2-4h, and the ultrasonic temperature is 40-60 ℃.
In some embodiments of the invention, the concentration of the suspension containing zero-valent nano-iron in the first step of the preparation method of the magnetic nano-material is 600-800mg/L, and the concentration of the suspension containing graphene oxide is 200-800 mg/L.
In some embodiments of the present invention, in the second step of the method for preparing a magnetic nanomaterial, the weight ratio of the suspension containing zero-valent nano-iron to the suspension containing graphene oxide is (2-5): 5.
in some embodiments of the present invention, in the second step of the preparation method of a magnetic nanomaterial, the temperature of the shaking table is set to be 40-60 ℃, the rotation speed of the shaking table is 150-170rpm, and the coating time is 5-10 min.
In some embodiments of the invention, the drying temperature of the oven in the second step of the preparation method of the magnetic nano material is 40-60 ℃, and the drying time of the oven is 1-6 h.
Another object of the present invention is to provide an application of a magnetic nanomaterial, which is characterized in that: the magnetic nano material can be used for quickly extracting genomic DNA from human whole blood genome.
In some embodiments of the present invention, the extracting human whole blood genomic DNA using the magnetic nanomaterial comprises the following steps:
the method comprises the following steps: adding 80-120 mu L of human whole blood sample into 160-200 mu L of lysis solution, and then placing the lysis solution in a water bath at 50-60 ℃ for lysis for 8-12 min;
step two: adding 5-15mg of magnetic nano material into the lysis solution in the step one, adding 200-300 mu L of binding buffer solution, reversing and uniformly mixing for 0.5-2min, and standing for 2-4 min;
step three: performing magnetic separation on the liquid treated in the second step by using a magnet to obtain a magnetic nano material-DNA complex and a supernatant, removing the supernatant, washing the magnetic nano material-DNA complex twice by using 60-80% ethanol, drying, adding 100-300 mu LTE buffer solution, eluting in water bath at 60-70 ℃ for 8-12min, then centrifuging at 10000-14000rpm for 0.6-1.4min, and transferring the supernatant into a new EP tube, wherein the EP tube is the extracted human whole blood genome DNA;
the lysis solution in the first step of the method for extracting the human whole blood genome DNA by adopting the magnetic nano material contains 2 wt% of SDS, 5 muL of RNase and 5 muL of protease;
the binding buffer solution in the second step of the method for extracting human whole blood genome DNA by using the magnetic nano material contains 15 wt% of PEG8000 and 1.25mol/L of NaCl.
Compared with the prior art, the invention has the beneficial effects that:
1. the concentration of the genomic DNA extracted from 100 mu l of human whole blood genome by the magnetic nanomaterial is about 1 mu g, while the concentration of the genomic DNA extracted from 100 mu l of human whole blood genome by the conventional kit method is about 0.5 mu g, and the concentration of the human whole blood genome extracted by the magnetic nanomaterial is higher.
2. The genome DNA extracted from the human whole blood genome provided by the magnetic nano material prepared by the invention is subjected to PCR amplification experiment, and the DNA fragment of about 680bp of the mitochondrial cytochrome oxidase COI gene is amplified by PCR. The human whole blood genome DNA extracted by the novel magnetic nano material does not influence the subsequent PCR experiment.
Drawings
FIG. 1 shows genomic DNA extracted from magnetic nanomaterials in human whole blood and by a kit method according to the present invention;
FIG. 2 shows PCR amplification of genomic DNA extracted from human whole blood according to the present invention.
In fig. 1, M: 1 kbmarker;
1,2: human Na2EDTA anticoagulant whole blood genome DNA extracted from the magnetic nano material;
3,4: human heparin sodium anticoagulant whole blood genome DNA extracted from the magnetic nano material;
5,6: human Na2EDTA anticoagulant whole blood genome DNA extracted by a kit method;
7,8: human heparin sodium anticoagulation whole blood genome DNA extracted by a kit method.
In fig. 2, M: marker;
1,2: human EDTA-2Na anticoagulated whole blood genome DNACR result extracted from the magnetic nano material;
3,4: DNACR (deoxyribonucleic acid-polymerase chain reaction) results of the anticoagulant whole blood genome of the human heparin sodium extracted from the magnetic nano material;
5,6: a DNACR result of the human EDTA-2Na anticoagulated whole blood genome extracted by the kit method;
7,8: a DNAPCR result of the anticoagulant whole blood genome of the human heparin sodium extracted by a kit method; 9 negative control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.
Example 1
A magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide, and the preparation method of the magnetic nano material comprises the following steps:
the method comprises the following steps: weighing zero-valent nano iron and graphene oxide, respectively placing the zero-valent nano iron and the graphene oxide in purified water, and performing ultrasonic dispersion treatment (the ultrasonic treatment conditions are 1800HZ and 150W, the ultrasonic time is 3h, and the ultrasonic temperature is 50 ℃), so as to obtain suspension containing 500mg/L zero-valent nano iron and suspension containing graphene oxide, wherein the concentration of the suspension is 700 mg/L;
step two: and (2) pouring the suspension containing the zero-valent nano-iron and the suspension containing the graphene oxide (the suspension containing the zero-valent nano-iron: the suspension containing the graphene oxide is (3.5: 5)) into the same beaker, uniformly mixing to obtain a mixed suspension, then placing the mixed suspension on a shaking table (the shaking table is set to be at a temperature of 50 ℃, a rotating speed of 160rpm and a coating time of 7.5min) for coating, and finally placing the mixed suspension treated by the shaking table in an oven for drying (the oven drying condition is that the temperature is 50 ℃ and the drying time is 3.5h) to obtain the magnetic nano-material.
The magnetic nano material can be used for quickly extracting genome DNA from human whole blood genome, and the method for extracting the human whole blood genome DNA by adopting the magnetic nano material comprises the following steps:
the method comprises the following steps: adding 100 μ L of human whole blood sample into 180 μ L of lysate (containing 2 wt% SDS, 5 μ L RNase and 5 μ L protease), and subjecting to 55 deg.C water bath for 10 min;
step two: adding 10mg of magnetic nano material into the lysis solution in the step one, adding 250 mu L of binding buffer solution (the binding buffer solution contains 15 wt% of PEG8000 and 1.25mol/L of NaCl), reversing, uniformly mixing for 1min, and standing for 3 min;
step three: and (3) carrying out magnetic separation on the liquid treated in the second step by using a magnet to obtain a magnetic nano material-DNA complex and a supernatant, removing the supernatant, washing the magnetic nano material-DNA complex twice by using 70% ethanol, drying, adding 200 mu LTE buffer solution, eluting in a water bath at 65 ℃ for 10min, centrifuging at 12000rpm for 1min, and transferring the supernatant into a new EP (European medical science) tube, wherein the EP tube is the extracted human whole blood genome DNA.
Example 2
A magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide, and the preparation method of the magnetic nano material comprises the following steps:
the method comprises the following steps: weighing zero-valent nano-iron and graphene oxide, respectively placing the zero-valent nano-iron and graphene oxide in purified water, and performing ultrasonic dispersion treatment (the ultrasonic treatment conditions are 1400HZ and 100W, the ultrasonic time is 4h, and the ultrasonic temperature is 60 ℃), so as to obtain a suspension liquid containing the zero-valent nano-iron and the graphene oxide, wherein the concentration of the suspension liquid is 600mg/L, and the concentration of the suspension liquid is 200 mg/L;
step two: and (3) pouring the suspension containing the zero-valent nano-iron and the suspension containing the graphene oxide (the suspension containing the zero-valent nano-iron: the suspension containing the graphene oxide is poured into the same beaker (5: 5)) to be uniformly mixed to obtain a mixed suspension, then placing the mixed suspension on a shaking table (the shaking table is set to be at 40 ℃, the rotating speed is 170rpm, and the coating time is 10min) to be coated, and finally placing the mixed suspension treated by the shaking table in an oven to be dried (the oven drying condition is 40 ℃, and the drying time is 6h) to obtain the magnetic nano-material.
The magnetic nano material can be used for quickly extracting genome DNA from human whole blood genome, and the method for extracting the human whole blood genome DNA by adopting the magnetic nano material comprises the following steps:
the method comprises the following steps: adding 80 μ L of human whole blood sample into 160 μ L of lysate (containing 2 wt% SDS, 5 μ L RNase and 5 μ L protease), and subjecting to 50 deg.C water bath for lysis for 8 min;
step two: adding 5mg of magnetic nano material into the lysis solution in the step one, adding 200 mu L of binding buffer solution (the binding buffer solution contains 15 wt% of PEG8000 and 1.25mol/L of NaCl), reversing, uniformly mixing for 0.5min, and standing for 4 min;
step three: and (3) carrying out magnetic separation on the liquid treated in the second step by using a magnet to obtain a magnetic nano material-DNA complex and a supernatant, removing the supernatant, washing the magnetic nano material-DNA complex twice by using 60% ethanol, drying, adding 100 mu LTE buffer solution, eluting in a water bath at 60 ℃ for 8min, centrifuging at 10000rpm for 0.6min, and transferring the supernatant into a new EP (European patent application) tube, wherein the EP tube is the extracted human whole blood genome DNA.
Example 3
A magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide, and the preparation method of the magnetic nano material comprises the following steps:
the method comprises the following steps: weighing zero-valent nano-iron and graphene oxide, respectively placing the weighed zero-valent nano-iron and graphene oxide in purified water, and performing ultrasonic dispersion treatment (the ultrasonic treatment conditions are 2000HZ and 200W, the ultrasonic time is 2h, and the ultrasonic temperature is 40 ℃) to obtain 800mg/L suspension containing zero-valent nano-iron and 800mg/L suspension containing graphene oxide;
step two: and (2) pouring the suspension containing the zero-valent nano-iron and the suspension containing the graphene oxide (the suspension containing the zero-valent nano-iron: the suspension containing the graphene oxide is (1: 1)) into the same beaker, uniformly mixing to obtain a mixed suspension, then placing the mixed suspension on a shaking table (the shaking table is set to be at 60 ℃, the rotating speed is 150rpm, and the coating time is 10min) for coating, and finally placing the mixed suspension treated by the shaking table in an oven for drying (the oven drying condition is 60 ℃, and the drying time is 1h) to obtain the magnetic nano-material.
The magnetic nano material can be used for quickly extracting genome DNA from human whole blood genome, and the method for extracting the human whole blood genome DNA by adopting the magnetic nano material comprises the following steps:
the method comprises the following steps: mu.L of human whole blood sample is added into 200. mu.L of lysate (the lysate contains 2 wt% SDS, 5. mu.L of RNase and 5. mu.L of protease), and then the lysate is placed into a water bath at 60 ℃ for lysis for 12 min;
step two: adding 15mg of magnetic nano material into the lysis solution in the step one, adding 300 mu L of binding buffer solution (the binding buffer solution contains 15 wt% of PEG8000 and 1.25mol/L of NaCl), reversing, uniformly mixing for 2min, and standing for 2 min;
step three: and (3) carrying out magnetic separation on the liquid treated in the second step by using a magnet to obtain a magnetic nano material-DNA complex and a supernatant, removing the supernatant, washing the magnetic nano material-DNA complex twice by using 80% ethanol, drying, adding 300 mu LTE buffer solution, eluting in a 70 ℃ water bath for 12min, centrifuging at 14000rpm for 0.6min, and transferring the supernatant into a new EP (European patent application) tube, wherein the EP tube is the extracted human whole blood genome DNA.
Examples of the experiments
Using example 1 as an example, the human whole blood genomic DNA extracted in example 1 was quantified by electrophoresis and UV, and the results are shown in FIGS. 1 and 2, and FIG. 1 shows that the magnetic nanomaterial can be extracted from human whole blood into human whole blood genomic DNA. The ultraviolet quantitative result shows that the concentration of the genome DNA extracted from 100 mu l of human whole blood genome by the magnetic nano material is about 1 mu g, the concentration of the genome DNA extracted from 100 mu l of human whole blood genome by the kit method is about 0.5 mu g, and the concentration of the genome extracted by the kit method is close to that described in the specification. FIG. 2 shows PCR amplification experiments of extracted genomic DNA from human whole blood, which amplified a DNA fragment of about 680bp of the mitochondrial cytochrome oxidase COI gene. FIG. 2 shows clear bands, which illustrate that the human whole blood genome DNA extracted by the magnetic nanomaterial of the present invention does not affect the subsequent PCR experiment.
The present invention provides only one magnetic nanomaterial and its application in extraction of human whole blood genome DNA, and the method and way for implementing the technical scheme are many, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should be regarded as the protection scope of the present invention, and all the components not specified in the embodiments can be implemented by the existing technology.
Claims (10)
1. A magnetic nanomaterial characterized by: the magnetic nano material is prepared by dispersing, coating and drying zero-valent nano iron and graphene oxide.
2. A magnetic nanomaterial according to claim 1, wherein: the preparation method of the magnetic nano material comprises the following steps:
the method comprises the following steps: weighing zero-valent nano iron and graphene oxide, respectively placing the weighed zero-valent nano iron and graphene oxide in purified water, and performing ultrasonic dispersion treatment to obtain a suspension containing zero-valent nano iron and a suspension containing graphene oxide;
step two: and (3) pouring the turbid liquid containing the zero-valent nano iron and the turbid liquid containing the graphene oxide obtained in the step one into the same beaker, uniformly mixing to obtain a mixed turbid liquid, then placing the mixed turbid liquid on a shaking table for coating treatment, and finally placing the mixed turbid liquid treated by the shaking table in an oven for drying to obtain the magnetic nano material.
3. A magnetic nanomaterial according to claim 2, wherein: the zero-valent nano-iron is a compound of zero-valent nano-iron and polymethyl methacrylate.
4. A magnetic nanomaterial according to claim 2, wherein: the ultrasonic treatment conditions in the first step are as follows: 1400 ℃ and 2000HZ, 100 ℃ and 200W, the ultrasonic time is 2-4h, and the ultrasonic temperature is 40-60 ℃.
5. A magnetic nanomaterial according to claim 2, wherein: the concentration of the suspension containing the zero-valent nano-iron in the first step is 600-800mg/L, and the concentration of the suspension containing the graphene oxide is 200-800 mg/L.
6. A magnetic nanomaterial according to claim 2, wherein: the weight ratio of the suspension containing the zero-valent nano iron to the suspension containing the graphene oxide in the second step is (2-5): 5.
7. a magnetic nanomaterial according to claim 2, wherein: the temperature of the shaking table in the second step is 40-60 ℃, the rotation speed of the shaking table is 150-170rpm, and the coating time is 5-10 min.
8. A magnetic nanomaterial according to claim 2, wherein: and in the second step, the drying temperature of the drying oven is 40-60 ℃, and the drying time of the drying oven is 1-6 h.
9. A magnetic nanomaterial according to any of claims 1 to 8, wherein: the magnetic nano material can be used for quickly extracting genomic DNA from human whole blood genome.
10. An application of a magnetic nano material in extraction of human whole blood genome DNA is characterized in that: the method for extracting the human whole blood genome DNA by adopting the magnetic nano material comprises the following steps:
the method comprises the following steps: adding 80-120 mu L of human whole blood sample into 160-200 mu L of lysis solution, and then placing the lysis solution in a water bath at 50-60 ℃ for lysis for 8-12 min;
step two: adding 5-15mg of magnetic nano material into the lysis solution in the step one, adding 200-300 mu L of binding buffer solution, reversing and uniformly mixing for 0.5-2min, and standing for 2-4 min;
step three: performing magnetic separation on the liquid treated in the second step by using a magnet to obtain a magnetic nano material-DNA complex and a supernatant, removing the supernatant, washing the magnetic nano material-DNA complex twice by using 60-80% ethanol, drying, adding 100-300 mu LTE buffer solution, eluting in water bath at 60-70 ℃ for 8-12min, then centrifuging at 10000-14000rpm for 0.6-1.4min, and transferring the supernatant into a new EP tube, wherein the EP tube is the extracted human whole blood genome DNA;
the lysis solution in the step one contains 2 wt% of SDS, 5 mu L of RNase and 5 mu L of protease;
the binding buffer in step two contains 15 wt% PEG8000 and 1.25mol/L NaCl.
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