RU2451747C2 - Method for homogenising biological samples containing magnetic nanoparticles when extracting dna during automatic sample preparation for pcr analysis - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to molecular biology and chemistry. Disclosed is a method for homogenising a solution of magnetic particles on which DNA is absorbed. The present invention is used in laboratory diagnosis when preparing samples for a polymerase chain reaction, for extracting DNA from biopreparations and removing or neutralising extraneous impurities.

EFFECT: obtaining DNA with purity which is suitable for amplification reaction.

6 dwg, 1 ex

Description

1. The technical field

The invention relates to the field of molecular biology and chemistry. This invention is used in laboratory diagnostics to prepare a sample for PCR, in the extraction (extraction) of DNA from biological products and in the removal or neutralization of impurities to obtain DNA with a purity suitable for the amplification reaction. The invention provides a method for grinding concentration inhomogeneities formed when a blood sample is mixed in a lysing solution with magnetic iron oxide nanoparticles coated with an SiO ₂ adsorbent.

2. The level of technology

The use of magnetic particles is no different in principle from the use of other types of sorbents (latexes, polymer sorbents, glass), but allows the use of magnetic separation instead of centrifugation and filtration. The magnetic particles are based on the obtained iron oxide nanoparticles with superparamagnetic properties. Particles based on iron oxide with superparamagnetic properties have a number of advantages, the most important of which are as follows:

- such particles have a practically zero degree of remanent magnetization, that is, after removing an external magnetic field (transfer from a magnetic tripod to an ordinary one), these particles do not stick together and can be easily resuspended;

- such particles are chemically inert and can be stored for a long time in aqueous solutions, do not inhibit enzymatic reactions, they can be used in experiments in vitro and in vivo without the risk of undesirable consequences;

- such particles offer great opportunities for automating the processes of nucleic acid, protein, and cell isolation.

The prior art following methods for producing nucleic acids using magnetic particles:

A. Cleaning Method Using Magnetic Particles (Patent No. WO 00/42432 of 07.20.2000)

This method of purification of a substance consists in the fact that a material containing a substance and magnetic particles coated and treated with a reagent that binds the particles to the substance is placed in the first medium, which creates the possibility of a binding reaction in which the substance binds to the particles and is introduced into the medium is a magnetic probe, as a result of which the particles adhere to the probe, and the probe, together with the particles and the substance associated with them, is transferred to the second medium and, if necessary, isolated from the second medium and transferred to the third medium, characterized in that in several media, preferably in at least the first medium, and most preferably in all media, a surface tension reducing agent is placed before the probe and particles are transferred from the corresponding medium.

The main disadvantage of this cleaning method is the need to transfer a magnetic probe with magnetic particles adhering to it between different media, which sharply increases the risk of contamination of the test sample.

B. Magnetic polymer particles based on polyvinyl alcohol, a method for their preparation (patent No. WO 97/04862 of 02/13/1997)

The invention relates to granular magnetic carriers, which are obtained by suspending polyvinyl alcohol-containing magnetic colloids — a polymer phase in an organic phase that contains a special mixture of emulsifiers. Get particles with a particle size of 1-8 μm, which can chemically bind ligands. Carriers can be used to isolate and detect biomolecules, cells, antibodies, and nucleic acids.

B. Method for detecting an analyte using colloidal magnetic particles (patent No. WO 03/04453 of 05/30/2003)

This method of detection and quantification is characterized in that it uses magnetic colloidal particles, functionalized on the surface by one specific ligand, which is to be detected or quantified of the analyte. For magnetic separation, strong magnets based on rare-earth elements are used. To completely collect magnetic particles in a magnetic tripod, it usually takes from a few seconds to several minutes. The separation time depends on the volume and viscosity of the fluid from which the magnetic particles are collected.

The problem that arises when using magnetic particles (after adsorption — DNA “sticking” on the surface of magnetic particles and collecting magnetic particles by a magnetic field on one of the walls of the tube) is the formation of a “coil” of magnetic particles “entangled” with DNA, which makes it difficult to conduct further washing steps and excludes the possibility of real-time polymerase chain reaction for DNA analysis.

A task

An object of the present invention is to provide a method for homogenizing and resuspending a solution of DNA adsorbed on magnetic particles by partially defragmenting it and reducing supercoiling, efficient washing and elution - separation of DNA from magnetic particles without transferring a magnetic probe, with the risk of contamination, between different volumes, and ensuring the most favorable conditions for fluorescence analysis of isolated and purified DNA with PCR RealTime devices and, thus, automation of the entire cycle of pr DNA analysis.

3. Disclosure of invention

The task of creating a homogeneous solution of magnetic nanoparticles with DNA samples adsorbed on them, isolated from blood cells, is solved by a new method of partial defragmentation of DNA molecules, namely by grinding any inhomogeneities in the concentration of magnetic particles clamped by a strong inhomogeneous magnetic field between the inner surface of the tube and rotating around its axis magnetic probe. The rotation of the magnetic probe with its simultaneous pressing against the inner wall of the tube was carried out by rotating magnetic (1.2-1.3 T) balls of the tripod.

In a test tube, standardly used for DNA isolation, a volume of 1.5 ml was introduced:

1) 5 μl of magnetic particles at a concentration of 5 mg / ml;

2) 50 μl of blood for DNA isolation;

3) 150 μl of lyse solution;

4) a magnetic cylindrical probe.

The rotation of the magnetic probe around its axis, the collinear axis of the tube and the creation of microturbulent flows contributed to the most complete adsorption - “sticking” of DNA to the surface of magnetic particles. After stopping the rotational motion of the probe and collecting magnetic nanoparticles with DNA with a strong inhomogeneous magnetic field of the tripod balls on the inner surface of the tube and, partially, on the surface of the magnetic probe, magnetic particles “entangled” DNA form a “ball”, which is a very elastic porous medium with a high tensile strength, which was the main obstacle to using the standard method of DNA extraction using magnetic particles and a magnetic tripod.

Various unsuccessful attempts have been made to eliminate (break) clumping of magnetic particles, such as repeated pipetting, cutting the mixer with knives at revolutions of more than 50 Hz, breaking with larger ferromagnetic particles according to the principle of operation of ball mills. Thus, it became impossible to carry out the further standard stages of sample preparation — three washes and elution of DNA from magnetic particles, and, consequently, the inability to isolate pure DNA samples suitable for PCR analysis.

A positive result - the creation of a homogeneous solution of magnetic particles with DNA - was obtained only using the principle of grinding microinhomogeneities sandwiched between two surfaces. Microinhomogeneities with magnetic particles - “lumps” are drawn into the region of a high magnetic field, clinging to the walls of the tube, as is realized in a standard magnetic tripod with a stationary magnetic field. We use magnetic (Nd-Fe-B) balls with a diameter of 5 mm (B _residual ≈1.2-1.3 T) located on the outside of the tube and able to rotate freely around any of its axes as tripod magnets. The second pressing surface in our proposed method is a magnetic probe introduced into the test tube - a cylinder with a diameter of 1.5-2.0 mm and a length of 6-8 mm, magnetized across the cylinder axis (parallel to the tube axis) and having a _residual B of 0.2-0.3 T on the surface. As a material for the manufacture of a magnetic cylinder, a magnetoplast obtained by casting (silicone with the addition of 80% rapidly quenched magnetic powder of neodymium alloy) was used, followed by magnetization using an inductor.

A magnetic cylinder probe, attracted by a strong magnet, clamped the “lumps” between itself and the inner wall of the tube. Rotating around its axis and always staying pressed against the inner wall of the test tube, the magnetic probe effectively rubs the “lumps” of magnetic particles of “entangled” DNA, partially (as shown by subsequent electrophoresis) defragmenting the DNA itself.

In the test tube, a suspension of magnetic particles with DNA adsorbed on them is formed.

4. The implementation of the invention

4.1 Description of the design of the model of the device with which the tests were carried out.

16 test tubes with disposable magnetic cylindrical probes inside were evenly spaced around the circumference in a rack. Between each of 8 pairs of test tubes, 8 super-magnetic (1.2-1.3 T) balls with a diameter of 5 mm were placed in cylindrical cavities (5.1 mm in diameter) of the tripod and were able to rotate freely around any axis. View of the model without tubes is shown in figure 1. The rotation of the eight supermagnetic balls of the tripod was carried out by creating a rotating in the horizontal plane of the magnetic field induction vector in the plane of the circle of their location. The creation of a magnetic field rotating at a frequency of 10–30 Hz in our model was ensured by rotation in the radial plane of one rod magnet located along the diameter of the tube circumference.

Figure 2, shows a bottom view of the model, which shows the location of the bar magnet that creates a rotating magnetic field. The drive was an electric motor powered by batteries (3-6 V), with a rotation frequency of 30-50 Hz. The test tube wells were laid with a flexible heating element (nichrome in a carbon tape, power about 10 W), for heating to 50-60 ° C after the third stage of washing and drying (5 minutes), and also for heating to 60-65 ° C ( 10 minutes) in an elution solution to separate DNA from the sorbent on the surface of magnetic particles. A flexible heating carbon tape with two nichrome threads is also visible in figure 1 and figure 2.

The final stage of sample preparation is the collection of magnetic particles on the tube wall and magnetic probe. Isolated and purified DNA remains in solution.

Steps for isolating DNA from a biological sample using the invention

DNA is isolated from blood samples using a standard set of reagents. For one 1.5 ml tube, use:

Stage 1

- 50 μl of blood + 150 μl of lyse solution + 5 μl of standard concentration of magnetic particles;

- Flushing;

- Retention of magnetic particles on the wall of the tube;

- Removal of the liquid phase from the test tube;

2 stage

- 400 μl of standard wash solution No. 1;

- Flushing;

- Retention of magnetic particles on the wall of the tube;

- Removal of the liquid phase from the test tube;

3 stage

- 200 μl of standard wash solution No. 2;

- Flushing;

- Retention of magnetic particles on the wall of the tube;

- Removal of the liquid phase from the test tube;

4th stage

- 200 μl of standard wash solution No. 3;

- Flushing;

- Retention of magnetic particles on the wall of the tube;

- Removal of the liquid phase from the test tube;

- Drying the contents of the test tube for 5 minutes at a temperature of 50-65 ° C;

5 stage

- 100 μl of standard elution solution for 10 minutes at a temperature of 50-65 ° C;

- Retention of magnetic particles on the wall of the tube;

- Collection of isolated and purified DNA from the tube for subsequent analysis.

Concrete example

To isolate DNA from blood cells, 16 1.5 ml tubes were installed in the model of the device. One tube was used to control the temperature at 4 and 5 stages of sample preparation. For this, a digital temperature sensor was introduced into the sixteenth test tube. The remaining 15 tubes contained a magnetic cylindrical probe.

Stage 1

After introducing into the test tube a solution containing 50 μl of blood + 150 μl of lysis solution, 5 μl of the standard concentration of magnetic particles was added to it. By turning on the rotating magnetic field for 5-10 seconds, due to the rotation of the magnetic probe, uniform mixing of the magnetic particles in the solution and creation of optimal conditions for the adsorption of DNA on their surface were achieved. As experiments have shown, the rotation frequency of the magnetic probe should not exceed 50 Hz. The contrary leads to foaming of the solution, as well as to its spraying on the walls of the tube.

After the rotation stopped, the super-magnetic tripod balls collected magnetic particles on the tube wall at a height of about 100 μl. At the same time, a magnetic cylindrical probe was pressed against the wall of the tube. Part of the magnetic particles also deposited on the surface of the magnetic probe. The drift of magnetic particles from the solution and their deposition on the walls of the tube and the surface of the probe lasted about 1 minute. Then the liquid phase was removed from the test tube with a standard pipette with disposable spouts.

2 stage

After 400 μl of Wash Solution No. 1 was introduced into the tube, the instrument model was turned on for approximately 1 minute. A rotating magnetic probe, being pressed against the tube wall by a supermagnetic tripod ball, rubbed any inhomogeneities containing magnetic particles against the inner tube wall, creating a suspension of magnetic particles. Microturbulent rotational movement of fluid in a test tube facilitated a thorough washing of the DNA.

After the rotation stopped, the super-magnetic tripod balls collected magnetic particles on the tube wall at a height of about 100 μl. At the same time, a magnetic cylindrical probe was pressed against the wall of the tube. Part of the magnetic particles settled, also on the surface of the magnetic probe. The drift of magnetic particles from the solution and their sedimentation on the walls of the tube and the surface of the probe lasts about 1 minute. Then the liquid phase was removed from the test tube with a standard pipette with disposable spouts.

3 stage

After 200 μl of Wash Solution No. 2 was introduced into the tube, the step was carried out similarly.

4th stage

After 200 μl of Wash Solution No. 3 was introduced into the tube, the step was carried out similarly. However, after removal of the liquid phase, the contents of the tube should be drained for 5 minutes at a temperature of 50-65 ° C. For this, a flexible heating element was included, encircling all 16 test tubes in their seats in a rack. The heating element was powered from a standard HY3005C power source with a voltage of about 15 V. Temperature was controlled by a digital temperature sensor located in the sixteenth test tube. The drying procedure should be carried out carefully, as washing solution No. 3 blocks the DNA amplification reaction.

5 stage

After the introduction of 100 μl of the elution solution separating the DNA from the magnetic particles into the tube, a flexible heating element was also turned on, and the temperature was set at 50-65 ° C for 10 minutes. During this time, the rotation of the magnetic probe was carried out periodically for 10-15 seconds every minute. This allowed homogenizing the solution, on the one hand, and did not allow excessive defragmentation of DNA molecules.

After turning off the device, magnetic particles were collected for a minute on the side walls of the tube and a disposable magnetic probe under the influence of an inhomogeneous magnetic field of the balls.

Purified DNA remained in solution, ready for further analysis.

To compare the amount of DNA extracted by the standard method and using the model of the device, including in different tubes, amplification reactions were carried out with samples of each species. Amplification was carried out using the DT-96 detecting amplifier (Registration Certificate of the Federal Service for Supervision of Health and Social Development No. FSR 2007/01250 dated November 20, 2007). The results of the amplification are presented in Fig.6. The characteristic cycle number of the onset of the reaction in both cases is 27.

The degree of DNA defragmentation highlighted by the model of the device can be analyzed by the electrophoregram of Fig.7.

5. Brief Description of the Drawings

The type of model of the device with which the tests were carried out is presented in figure 1. 8 super-magnetic balls and a flexible heating element are visible. Figure 2, depicting a bottom view of the model, shows the location of the bar magnet, creating a rotating magnetic field. The drive was an electric motor powered by batteries (3-6 V), with a rotation frequency of 30-50 Hz. The test tube wells were laid with a flexible heating element (nichrome in a carbon tape), for heating to 50-60 ° C after the third stage of washing and drying (5 minutes), and also for heating to 60-65 ° C (10 minutes) in an eluting a solution for separating DNA from the sorbent on the surface of magnetic particles. A flexible heating carbon tape with two nichrome threads is also visible in figure 1 and figure 2.

Figure 3 presents a General view of the model during testing.

Figure 4 presents the magnetic particles with adsorbed DNA on them after 2 washing solutions.

Figure 5 depicts a method for grinding inhomogeneities sandwiched between a magnetic probe rotating around its axis and the tube wall.

6 depicts the comparative results of high-quality PCR analysis for a manual standard DNA extraction method and a method using a model of a sample preparation instrument. The coincidence of the curves indicates a good model. 7 shows the comparative results of a qualitative PNR analysis of DNA obtained using standard sample preparation, and DNA analysis obtained by the method of homogenization using a model of the device for sample preparation. The results of electrophoresis demonstrate some DNA defragmentation, highlighted by the model of the device (first 6 columns) compared to the standard manual method (7 and 8 columns).

Claims (1)

A method of homogenizing a solution of magnetic particles with DNA adsorbed on them, which consists in grinding a non-uniform concentration of magnetic particles clamped by a non-uniform magnetic field between the inner surface of the tube and a magnetic probe rotating around its axis with a frequency of 20-50 Hz and representing a magnetoplastic cylinder with a diameter of 1 , 5-2.0 mm and a length of 6-8 mm, magnetized across the axis of the cylinder and having a residual 0.2-0.3 T on the surface, while the rotation of the magnetic probe with its simultaneous pressure against the inner wall the tubes provide magnetic balls with a diameter of 5 mm, with a value of B residual 1.2-1.3 T on the surface located on the outside of the tube and are able to freely rotate around their axes under the action of a rod magnet constantly rotating in the radial plane.

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