CN111139234B - Free DNA extraction kit - Google Patents
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- CN111139234B CN111139234B CN201811311248.4A CN201811311248A CN111139234B CN 111139234 B CN111139234 B CN 111139234B CN 201811311248 A CN201811311248 A CN 201811311248A CN 111139234 B CN111139234 B CN 111139234B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention provides a free DNA extraction kit, which is used for extracting free DNA in blood plasma and comprises a liposome magnetic bead solution, a washing solution I, a washing solution II, an eluent and a magnetic separation frame. The liposome magnetic bead solution contains liposome magnetic beads which are used for adsorbing free DNA in plasma and separating the DNA. The liposome magnetic beads in the liposome magnetic bead solution provided by the invention have the characteristics of small particle size, positive charge, high purity and the like, are favorable for being fully combined with free DNA in plasma efficiently, and can separate and extract the free DNA in the plasma to the maximum extent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a free DNA extraction kit.
Background
Liquid biopsy (liquid biopsy), as a branch of in vitro diagnosis, refers to the analysis and diagnosis of diseases such as cancer by testing blood or other body fluids such as urine, saliva, pleural effusion, cerebrospinal fluid, etc. Including Circulating Tumor Cells (CTCs), circulating tumor dna (ctdna), microRNA, and microvesicles. Among them, ctDNA is the most rapidly developed. At present, the main application field of liquid biopsy is blood detection of tumors, namely, the blood is used for prompting the development process of the tumors, and information such as drug resistance and the like is used for guiding individualized and accurate treatment.
In 1948, free DNA (cell free DNA, cfDNA) was first found in human blood. After 30 years, higher levels of cfDNA were found in cancer patients. ctDNA was subsequently found also in cfDNA. Some cfDNA is derived from DNA of normal apoptosis and some ctDNA, and the ratio of ctDNA in cfDNA varies greatly, as low as 0.01% and as high as over 60%, depending on the size and progression of the tumor.
It is generally believed that cfDNA present in the blood circulation system is released into its surrounding microenvironment by apoptosis and necrosis of cancer cells, while its own secretory cell metabolism may also be a latent cfDNA. Necrotic and apoptotic cells are generally cleared by macrophages and other scavenger cells, and phagocytosed necrotic cancer cells can release digested DNA into their tissue environment. In vitro cell culture experiments have shown that macrophages can be activated or die during cfDNA release. The overall content of cfDNA can also fluctuate by clearance, degradation, and other physiological purification processes of the blood and lymphatic circulation systems. Nucleotides present in blood are metabolized and cleared by the liver and kidney of the body, so cfDNA has a certain half-life in blood, generally ranging from 15 minutes to several hours.
Since the content of free DNA (cfDNA) in plasma is small and unstable, it makes the extraction and preservation of cfDNA difficult to perform. Factors influencing the amount of cfDNA extracted are excessive, such as the time of blood sample storage, the temperature of blood sample storage, the centrifugal speed and time of blood sample, and the anticoagulant used in blood sample extraction. It has been found that Ethylenediaminetetraacetic acid (EDTA) has better anticoagulation than citric acid and heparin, and that the secondary centrifugation method facilitates better collection of cfDNA. Studies have shown that if a patient carries about 100g of tumor tissue, this corresponds to 3X 1010About 3.3% of the tumor cells per day enter the blood circulation. On average, the minimum size of this DNA is about 200bp, and the maximum size is 21 kb. Circulating tumor cells in the blood and the micro-deposits produced by their liver and bone marrow all contribute to the release of cfDNA. Compared with normal human, there are large amounts of ctDNA in tumor patients, which is derived from apoptotic or/and necrotic tumor cells, and these DNA fragments derived from tumor tissues or circulating tumor cells carry tumor-specific DNA fragmentsThe sexual DNA sequence enters the blood circulation, forming ctDNA that can be detected in peripheral blood. Related studies have shown that a minimum of 0ng up to more than 1000ng ctDNA per ml of blood is available for cancer patients, with an average of 180ng ctDNA per ml of blood. While in normal healthy people up to 100ng cfDNA per ml of blood can be extracted, on average 30ng per ml.
Studies have shown that different cfDNA extraction methods result in different cfDNA concentrations and purities in the collected blood samples. The traditional methods for extracting cfDNA include phenol-chloroform method, QIAgene kit method, Omega method and traditional magnetic bead method. However, the effect of phenol-chloroform extraction is not ideal, and although the QIAgene kit method and the Omega method can effectively remove various blood components in plasma, such as plasma proteins, cell debris and the like, the loss rate of cfDNA in the process of extracting cfDNA by the two methods reaches 70% -80%. Although the traditional magnetic bead method is superior to the above three methods, the efficiency of extracting cfDNA is highest and the quality is good, but the amount of extracted cfDNA is very limited, and the obtained cfDNA can never meet the requirements of the following experiments.
Research and comparison of the four methods for extracting the cfDNA show that the extraction rate of the magnetic bead method is only superior to that of the phenol-chloroform method, and the loss rate is lower than that of the QIAgene method and the phenol-chloroform method. For extracting small fragment cfDNA, such as mitochondrial cfDNA, the extraction rate of the magnetic bead method is obviously higher than that of the other three methods. The QIAgene kit method and the Omega method more effectively remove various blood components such as plasma proteins, cell debris and the like in plasma, so that the inhibition rate of the blood components is lower than that of other methods, but the loss rate of the two methods reaches 70-80%, so that the sensitivity of extracting cfDNA is greatly reduced. Among the four extraction methods, the paramagnetic particle method has the highest extraction efficiency on free DNA in blood plasma, the lowest loss rate and better repeatability. The kit further improves the method for separating cfDNA by magnetic beads, simplifies the experimental operation steps, is simple and quick to operate, obtains high-purity DNA, and is suitable for extracting small-fragment DNA.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a free DNA extraction kit that can extract cfDNA efficiently and quickly, has a small loss rate of cfDNA during extraction, and has high extraction purity.
The invention provides a free DNA extraction kit, which is characterized by comprising: the preparation method of the liposome magnetic bead solution comprises the following steps: the preparation method of the liposome magnetic bead solution comprises the following steps: step 1, mixing a naked magnetic ball solution containing X mg of naked magnetic balls with a dichloromethane solution to obtain a first liquid, mixing a cholesterol/dichloromethane solution containing 0.02X mg-0.15X mg of cholesterol, a dioleoyl lecithin/dichloromethane solution containing 0.05X mg-0.2X mg of dioleoyl lecithin, a phospholipid polyethylene glycol maleimide/dichloromethane solution containing 0.05X mg-0.2X mg of phospholipid polyethylene glycol maleimide, and a (2, 3-dioleoyl-propyl) -trimethylamine/dichloromethane solution containing 0.05X mg-0.2X mg of (2, 3-dioleoyl-propyl) -trimethylamine to obtain a second liquid, and mixing the first liquid with the dichloromethane solution to obtain a second liquid: adding the first liquid into the second liquid according to the volume ratio of 1: 1-1: 5, and ultrasonically mixing uniformly to obtain a third liquid; step 2, respectively adding 0.05-0.1X mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and 0.05-0.1X mg of N-hydroxysuccinimide solution into the third liquid, and ultrasonically mixing uniformly to obtain primary emulsion; step 3, adding 0.05-0.1X mg of polyvinylpyrrolidone solution into the colostrum and uniformly mixing to form multiple emulsion; step 4, removing dichloromethane in the double emulsion to obtain a fourth liquid; and 5, dialyzing the fourth liquid in a dialysis bag for a first time to obtain a suspension containing liposome magnetic beads.
The free DNA extraction kit provided by the invention can also have the following characteristics: wherein, in the liposome magnetic bead solution, the concentration of the liposome magnetic beads is 10-50 mg/ml.
The free DNA extraction kit provided by the invention can also have the following characteristics: wherein the pH of the liposome magnetic bead solution is 6.5-6.8, and the liposome magnetic bead solution further contains 3.8M guanidine hydrochloride, 1M NaCl, 1% Triton x-100, 0.5% SDS, 10mM Tris and 1mM EDTA.
The free DNA extraction kit provided by the invention can also have the following characteristics: wherein the cut-off molecular weight of the dialysis bag is 8000 kD-12000 kD, and the first time is 24 h-72 h.
The free DNA extraction kit provided by the invention can also have the following characteristics: wherein, in the step 4, the removal of dichloromethane in the double emulsion is carried out by rotary evaporation of a rotary evaporator.
The free DNA extraction kit provided by the invention further comprises: washing liquid I, washing liquid II, eluent and a magnetic separation frame, wherein the washing liquid I is an aqueous solution containing 1M NaCl, 0.5% SDS and 60% ethanol and is used for washing a free DNA sample and washing off redundant substances; the washing solution II is 70% ethanol water solution and is used for washing the free DNA sample for the second time and washing off redundant substances again; the eluent is 10mM Tris-HCl solution with the pH value of 7.0-7.25, and is used for eluting the free DNA sample from the surface of the liposome magnetic sphere and entering the eluent; the magnetic separation frame comprises a separation frame with multiple apertures and an extractable neodymium iron boron permanent magnet with embedded high energy, and is used for carrying out high-efficiency separation on liposome magnetic beads, so that the free DNA can be rapidly separated and purified.
The present invention also provides a method for extracting free DNA in a scientific experiment using the above free DNA extraction kit, characterized in that it comprises the steps of: step A, adding 5ml of sample plasma into a 5ml centrifuge tube, and centrifuging for 10min in a centrifuge at 1500 rpm; step B, taking 1 ml-2 ml of the middle-upper layer liquid of the centrifuge tube to be placed in a 5ml EP tube, adding 15ul of washing liquid I, carrying out vortex mixing for 30sec, placing the tube in a water bath kettle, incubating for 30min at 60 ℃, and carrying out oscillation mixing once every 10min in the incubation process; step C, after the incubation is finished, standing for 5min on ice; step D, adding 30ul of liposome magnetic bead solution, uniformly mixing for 30sec by oscillation, and uniformly mixing for 30min on a 3D uniformly mixing instrument so as to fully combine free DNA with liposome magnetic beads; e, placing the EP tube solution subjected to the step D on a magnetic separation frame, standing until the solution becomes clear, and removing a supernatant; step F, adding 1ml of washing solution II, uniformly mixing for 1min in a vortex mode, resuspending liposome magnetic beads, incubating for 10min at room temperature, placing the solution of the EP tube on a magnetic separation frame after the incubation is finished, standing until the solution becomes clear, and removing supernatant; and G, adding 1ml of eluent into the EP tube, uniformly blowing the magnetic ball by using a liquid transfer gun, uniformly mixing for 5min by vortex, incubating at room temperature for 30min, placing the EP tube on a magnetic separation frame, standing until the solution becomes clear, and recovering the supernatant after magnetic separation, thereby obtaining the free DNA.
Action and Effect of the invention
According to the free DNA extraction kit, naked magnetic beads, cholesterol, phospholipid polyethylene glycol maleimide, (2, 3-dioleoyl-propyl) -trimethylamine, dioleoyl lecithin and the scientific and reasonable proportion of the naked magnetic beads, the cholesterol, the phospholipid polyethylene glycol maleimide, the (2, 3-dioleoyl-propyl) -trimethylamine and the dioleoyl lecithin are adopted in the preparation process of liposome magnetic beads, polyvinylpyrrolidone with a proper proportion is added, then a rotary evaporator is adopted to evaporate dichloromethane in a rotary mode, and dialysis is conducted for a reasonable time through a dialysis bag with a proper molecular weight cutoff, so that the liposome magnetic beads prepared by the method have the characteristics of small particle size, positive charge, high purity and the like, can be effectively and fully combined with free DNA in blood plasma, and can separate and extract the free DNA in the blood plasma to the maximum extent.
Drawings
FIG. 1 is a schematic diagram of liposome magnetic beads in an embodiment of the present invention;
FIG. 2 is a potential diagram of liposome magnetic beads in an embodiment of the present invention;
FIG. 3 is a graph showing the particle size of liposome magnetic beads in an example of the present invention; and
FIG. 4 is a graph showing the peak of ultraviolet absorption in the examples of the present invention.
Detailed Description
In order to make the technical means, creation features, achievement purposes and effects of the invention easy to understand, the following embodiments are specifically described with reference to the accompanying drawings.
The invention provides a free DNA extraction kit, which is used for extracting free DNA in blood plasma and comprises a liposome magnetic bead solution, a washing solution I, a washing solution II, an eluent and a magnetic separation frame.
The pH of the liposome magnetic bead solution in the free DNA extraction kit in this embodiment is 6.5-6.8, and the solution contains 25mg/ml liposome magnetic beads, 3.8M guanidine hydrochloride, 1M NaCl, 1% Triton x-100, 0.5% SDS, 10mM Tris, and 1mM EDTA.
The preparation method of the liposome magnetic bead in the liposome magnetic bead solution is used for adsorbing free DNA in plasma so as to separate the DNA, and comprises the following steps:
step 1, mixing 2ml of bare magnetic ball solution containing 50mg of bare magnetic balls with 1ml of dichloromethane solution to obtain a first liquid, mixing 1ml of cholesterol/dichloromethane solution containing 3mg of cholesterol, 1ml of dioleoyl lecithin/dichloromethane solution containing 6mg of dioleoyl lecithin (DOPC), mixing 1ml of phospholipid polyethylene glycol maleimide/dichloromethane solution containing 6mg of phospholipid polyethylene glycol maleimide (DSPE-PEG-MAL) and 1ml of (2, 3-dioleoyl-propyl) -trimethylamine/dichloromethane solution containing 6mg of (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP) to obtain a second liquid, adding the first liquid into the second liquid, and performing ultrasonic mixing to obtain a third liquid.
And 2, respectively adding 2ml of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) solution with the concentration of 2mg/ml, 2ml of N-hydroxysuccinimide (NHS) solution with the concentration of 2mg/ml and 4ml of deionized water into the third liquid, and ultrasonically mixing uniformly to obtain primary emulsion.
And 3, adding 2ml of polyvinylpyrrolidone (PVP) solution with the concentration of 2mg/ml into the colostrum and uniformly mixing to form multiple emulsion.
And 4, removing dichloromethane in the double emulsion by rotary evaporation through a rotary evaporator to obtain a fourth liquid.
And 5, dialyzing the fourth liquid in a dialysis bag with the molecular weight cutoff of 10000kD for 48 hours to obtain a suspension containing liposome magnetic beads.
FIG. 1 is a schematic diagram of liposome magnetic beads in an embodiment of the present invention.
The liposome magnetic bead prepared in this example is a sphere with positive charges, and the cross section of the liposome magnetic bead passing through the sphere center is shown in fig. 1, and the cross section includes an inner core, a middle ring and an outer ring. The inner layer core comprises a variable number of bare magnetic balls, the middle layer ring is formed by orderly arranging DSPE-PEG-MAL, the outer layer is also formed by orderly arranging DSPE-PEG-MAL, DOPC is orderly inserted into the middle layer ring and the outer layer ring, DOTAP with positive charge is orderly inserted into the outer layer ring, one end of cholesterol is connected with the middle layer ring, and the other end is connected with the outer layer ring.
The liposome magnetic beads prepared in this example were tested for particle size and potential by a malvern laser sizer 3000E (uk).
FIG. 2 is a potential diagram of liposome magnetic beads in an example of the present invention.
As shown in FIG. 2, the abscissa represents the zeta potential of liposome magnetic beads, the ordinate represents the number of liposome magnetic beads, and the zeta potential of liposome magnetic beads is between 16.4 + -0.7 mv, so that the liposome magnetic beads prepared in this example can be better combined with free DNA.
FIG. 3 is a particle size diagram of liposome magnetic beads in an example of the present invention.
As shown in fig. 3, the abscissa represents the size of the liposome magnetic bead, the ordinate represents the distribution density of the liposome magnetic bead in different particle size intervals, the particle size of the liposome magnetic bead, i.e., the diameter, is between 137.1 ± 1.24nm, and the Protein Dispersion Index (PDI) is 0.096 ± 0.021, which indicates that the liposome magnetic bead prepared in this example has small particle size, good dispersibility, and is favorable for adsorbing free DNA in plasma.
FIG. 4 is a graph showing the peak of ultraviolet absorption in the examples of the present invention.
As shown in fig. 4, the abscissa represents the wavelength in nm, and the ordinate represents the absorbance of the liposome magnetic bead, the liposome magnetic bead prepared in this example has three absorption peaks distributed at 195nm, 200nm and 204nm, respectively, which indicates that the prepared liposome magnetic bead is good, and the liposome wraps the magnetic bead inside the microsphere.
The free DNA extraction kit in this embodiment further includes a washing solution I, which is an aqueous solution containing 1M NaCl, 0.5% SDS, and 60% ethanol, and is used to wash the free DNA to wash off excess substances.
The free DNA extraction kit in this embodiment further includes a washing solution II, which is a 70% ethanol aqueous solution, for washing the free DNA for a second time, and washing off the surplus substances again.
The free DNA extraction kit in the embodiment further comprises an eluent, wherein the eluent is a 10mM Tris-HCl solution with the pH value of 7.0-7.25, and is used for eluting the free DNA from the surface of the liposome magnetic sphere and entering the eluent.
The free DNA extraction kit in this embodiment still includes the magnetic separation frame, and this magnetic separation frame contains the separation frame of multiple aperture and the embedded high-energy neodymium iron boron permanent magnet of removable formula, and this magnetic separation frame is used for carrying out high-efficient separation to the liposome magnetic bead to reach the free DNA of quick separation purification.
The following method for extracting free DNA by using the kit in scientific experiments is described by combining the free DNA extraction kit provided in the embodiment, and comprises the following steps:
step A, 5ml of sample plasma was added to a 5ml centrifuge tube and centrifuged at 1500rpm for 10min in a centrifuge.
And step B, taking 1-2 ml of the middle-upper layer liquid of the centrifuge tube to be placed in a 5ml EP tube, adding 15ul of the washing liquid I, uniformly mixing for 30sec in a vortex mode, placing the tube in a water bath kettle for incubation for 30min at the temperature of 60 ℃, and uniformly mixing in a shaking mode every 10min in the incubation process.
And step C, standing for 5min on ice after the incubation is finished.
And D, adding 30ul of liposome magnetic bead solution, uniformly mixing for 30sec by oscillation, and uniformly mixing for 30min on a 3D uniformly mixing instrument so as to fully combine the free DNA with the liposome magnetic beads.
And E, placing the solution of the EP pipe which is subjected to the step D on a magnetic separation frame, standing until the solution becomes clear, and removing a supernatant.
And step F, adding 1ml of washing solution II, uniformly mixing for 1min in a vortex mode, resuspending liposome magnetic beads, incubating for 10min at room temperature, placing the solution of the EP tube on a magnetic separation frame after the incubation is finished, standing until the solution becomes clear, and removing a supernatant.
And G, adding 1ml of eluent into the EP tube, uniformly blowing the magnetic ball by using a liquid transfer gun, uniformly mixing for 5min by vortex, incubating at room temperature for 30min, placing the EP tube on a magnetic separation frame, standing until the solution becomes clear, and recovering the supernatant after magnetic separation, thereby obtaining the free DNA.
Table 1 shows concentration and purity of the isolated DNA extracted by the isolated DNA extraction kit of this example.
TABLE 1
As shown in table 1, Sample name 1 is a blood Sample of ovarian cancer, Sample name 2 is a blood Sample of breast cancer, Sample name 3 is a blood Sample of lung cancer, and Sample name 4 is a blood Sample of brain glioma in children, which are shown in the above table; a260 is the ultraviolet absorption wavelength of DNA, A280 is the ultraviolet absorption wavelength of protein, A230 is the ultraviolet absorption wavelength of the reagents used in the extraction process; the A260/280 ratio is more than 2.0, which indicates that the extracted DNA is purer and less protein impurities; the A260/230 ratio is above 2.0, which indicates that the reagents used in the extraction process have no effect on the DNA. Extracting free DNA in a blood sample by using the free DNA extraction kit of the embodiment; the results show that: the kit has high concentration and good purity of extracted DNA. The reagent used in the kit has little influence on the concentration and purity of the DNA.
Mag-
cfDNA kit Protocl extracts cfDNA from Sample name 2 breast cancer blood samples, using reagents provided with the kit, following the procedures described for the kit.
Table 2 shows the use of Mag-
Table of cfDNA kit Protocl extraction of free DNA in breast cancer blood samples, analysis of concentration and purity:
TABLE 2
As shown in table 2: mag-
The concentration of DNA in breast cancer blood samples extracted by cfDNA kit Protocl is 9.6ng/ml, and the extraction concentration is lower than that of the free DNA extraction kit in the embodiment. A260 is the ultraviolet absorption wavelength of DNA, A280 is the ultraviolet absorption wavelength of protein, the ratio of A260/280 is 0.75, the ratio is lower than 2.0, and the purity is lower. The implementation case is that the concentration and purity of free DNA extracted from breast cancer blood sample by the free DNA extraction kit are all superior to those of Mag-
cfDNA kit Protocl。
Effects and effects of the embodiments
According to the free DNA extraction kit related to the embodiment, naked magnetic beads, cholesterol, DOPC, DSPE-PEG-MAL, DOTAP and scientific and reasonable proportions among the naked magnetic beads, the PVP with a proper proportion is added in the preparation process of the liposome magnetic beads, and then the dichloromethane is rotationally evaporated by a rotary evaporator and dialyzed for a reasonable time through a dialysis bag with a proper molecular weight cutoff, so the liposome magnetic beads prepared by the embodiment have the characteristics of small particle size, positive charge, high purity and the like, and are favorable for being efficiently combined with free DNA in plasma.
Because the concentration of the liposome magnetic beads in the liposome magnetic bead solution is proper, when the free DNA extraction kit provided by the embodiment is used for extracting the free DNA, all the free DNA in the plasma sample can be ensured to be adsorbed by the liposome magnetic beads.
Since the liposome magnetic bead solution further contains 3.8M guanidine hydrochloride, 1M NaCl, 1% Triton x-100, 0.5% SDS, 10mM Tris and 1mM EDTA, it is possible to gently lyse cells and completely release free DNA so that the free DNA is adsorbed by the liposome magnetic beads.
Because the free DNA extraction kit that this embodiment provided still has washing liquid I, washing liquid II, eluant and magnetic separation frame, consequently, the free DNA volume that utilizes this kit to extract is many, the purity is high, of high quality, is favorable to follow-up experiment operations such as fluorescence quantitative PCR detection.
The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.
Claims (6)
1. A free DNA extraction kit, comprising:
the preparation method of the liposome magnetic bead solution comprises the following steps:
step 1, mixing a naked magnetic ball solution containing X mg of naked magnetic balls with a dichloromethane solution to obtain a first liquid, mixing a cholesterol/dichloromethane solution containing 0.02X mg-0.15X mg of cholesterol, a dioleoyl lecithin/dichloromethane solution containing 0.05X mg-0.2X mg of dioleoyl lecithin, a phospholipid polyethylene glycol maleimide/dichloromethane solution containing 0.05X mg-0.2X mg of phospholipid polyethylene glycol maleimide, and a (2, 3-dioleoyl-propyl) -trimethylamine/dichloromethane solution containing 0.05X mg-0.2X mg of (2, 3-dioleoyl-propyl) -trimethylamine to obtain a second liquid, and mixing the first liquid with the naked magnetic ball solution containing X mg of naked magnetic balls and the dichloromethane solution to obtain a second liquid according to the following ratio: adding the first liquid into the second liquid according to the volume ratio of 1: 1-1: 5, and ultrasonically mixing uniformly to obtain a third liquid;
step 2, respectively adding 0.05X mg-0.1X mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide solution and 0.05X mg-0.1X mg of N-hydroxysuccinimide solution into the third liquid, and ultrasonically mixing uniformly to obtain primary emulsion;
step 3, adding 0.05-0.1X mg of polyvinylpyrrolidone solution into the colostrum and uniformly mixing to form multiple emulsion;
step 4, removing dichloromethane in the double milk to obtain a fourth liquid; and
step 5, dialyzing the fourth liquid in a dialysis bag for a first time to obtain a suspension containing the liposome magnetic beads,
wherein, in the liposome magnetic bead solution,
the concentration of the liposome magnetic beads is 10-50 mg/ml.
2. The free DNA extraction kit according to claim 1, wherein:
wherein the pH value of the liposome magnetic bead solution is 6.5-6.8,
the liposome magnetic bead solution also contained 3.8M guanidine hydrochloride, 1M NaCl, 1% Triton x-100, 0.5% SDS, 10mM Tris, and 1mM EDTA.
3. The free DNA extraction kit according to claim 1, wherein:
wherein the cut-off molecular weight of the dialysis bag is 8000 kD-12000 kD,
the first time is 24-72 h.
4. The free DNA extraction kit according to claim 1, wherein:
wherein, in the step 4, the dichloromethane in the double milk is removed by rotary evaporation of a rotary evaporator.
5. The free DNA extraction kit according to claim 1, further comprising:
washing liquid I, washing liquid II, eluent and a magnetic separation frame,
the washing solution I is an aqueous solution containing 1M NaCl, 0.5% SDS and 60% ethanol, and is used for washing the free DNA sample and washing off redundant substances;
the washing solution II is 70% ethanol water solution and is used for washing the free DNA sample for the second time and washing off redundant substances again;
the eluent is 10mM Tris-HCl solution with the pH value of 7.0-7.25, and is used for eluting the free DNA sample from the surface of the liposome magnetic bead and entering the eluent;
the magnetic separation frame comprises a separation frame with multiple apertures and an extractable neodymium iron boron permanent magnet with embedded high energy, and is used for efficiently separating liposome magnetic beads, so that the free DNA can be rapidly separated and purified.
6. A method for extracting free DNA in scientific experiments by using the free DNA extraction kit of claim 5, which comprises the following steps:
step A, adding 5ml of sample plasma into a 5ml centrifuge tube, and centrifuging for 10min in a centrifuge at 1500 rpm;
step B, taking 1 ml-2 ml of the middle-upper layer liquid of the centrifuge tube to be placed in a 5ml EP tube, adding 15ul of the washing liquid I, carrying out vortex mixing for 30sec, placing the tube in a water bath kettle for incubation at 60 ℃ for 30min, and carrying out oscillation mixing once every 10min in the incubation process;
step C, after the incubation is finished, standing for 5min on ice;
step D, adding 30ul of the liposome magnetic bead solution, uniformly mixing for 30sec by oscillation, and uniformly mixing for 30min on a 3D uniformly mixing instrument so as to fully combine free DNA with the liposome magnetic beads;
e, placing the EP tube solution subjected to the step D on the magnetic separation frame, standing until the solution becomes clear, and removing a supernatant;
step F, adding 1ml of the washing solution II, uniformly mixing for 1min in a vortex mode, re-suspending the liposome magnetic beads, incubating for 10min at room temperature, placing the solution of the EP tube on the magnetic separation frame after the incubation is finished, standing until the solution becomes clear, and removing supernatant;
and G, adding 1ml of eluent into the EP tube, uniformly blowing the magnetic ball by using a liquid transfer gun, uniformly mixing for 5min by vortex, incubating at room temperature for 30min, placing the EP tube on the magnetic separation frame, standing until the solution becomes clear, and recovering the supernatant after magnetic separation, thereby obtaining the free DNA.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105873569A (en) * | 2013-11-06 | 2016-08-17 | 芝加哥大学 | Nanoscale carriers for the delivery or co-delivery of chemotherapeutics, nucleic acids and photosensitizers |
| CN108031442A (en) * | 2017-12-29 | 2018-05-15 | 猎源（上海）生物医药科技有限公司 | A kind of nano-lipid magnetic ball and preparation method thereof, the kit of separation and Extraction dissociative DNA and its application |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105873569A (en) * | 2013-11-06 | 2016-08-17 | 芝加哥大学 | Nanoscale carriers for the delivery or co-delivery of chemotherapeutics, nucleic acids and photosensitizers |
| CN108031442A (en) * | 2017-12-29 | 2018-05-15 | 猎源（上海）生物医药科技有限公司 | A kind of nano-lipid magnetic ball and preparation method thereof, the kit of separation and Extraction dissociative DNA and its application |
Non-Patent Citations (3)
| Title |
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| Cationic liposomes as gene delivery system: transfection efficiency and new application;F Xiong等;《Pharmazie》;20110331;第66卷(第3期);第158-164页 * |
| Lipid Shell-Enveloped Polymeric Nanoparticles with High Integrity of Lipid Shells Improve Mucus Penetration and Interaction with Cystic Fibrosis-Related Bacterial Biofilms;Feng Wan等;《ACS APPLIED MATERIALS & INTERFACES》;20180223;第10卷(第13期);第10678-10687页 * |
| The effect of cholesterol domains on PEGylated liposomal gene delivery in vitro;Long Xu等;《Ther Deliv.》;20110314;第2卷(第4期);第451-460页 * |
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