A DNA extraction centrifugal disk

Abstract

The present invention relates to a DNA extraction centrifugal disk. The DNA extraction centrifugal disk comprises at least one DNA extraction unit; the front of the DNA extraction unit is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet, a temporary liquid storage area and a filtration area, and the back is provided with a first liquid storage area and a second liquid storage area; a DNA adsorption material is provided in the filtration area; the first liquid inlet and the second liquid inlet are respectively connected to the filtration area through a flow channel; the third liquid inlet is connected to the temporary liquid storage area through a flow channel, and the temporary liquid storage area is connected to the filtration area through a flow channel; the second liquid storage area is connected to the first liquid storage area through a flow channel, and the first liquid storage area is connected to the filtration area through a flow channel. The DNA adsorption material is preferably a silica gel membrane. The present invention integrates the reaction process of the silica gel membrane adsorption method into the centrifugal disk, which can efficiently realize DNA extraction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and DNA extraction technology, and in particular relates to a DNA extraction centrifugal disk, which can realize DNA extraction conveniently and efficiently. Background Art

[0002] The existing DNA extraction methods mainly include the following:

[0003] 1) Classic phenol-chloroform method. Generally, it refers to adding an equal volume of phenol-chloroform mixture after cell lysis. According to the characteristics of DNA being easily soluble in water but not soluble in organic solvents, in an organic solvent environment, after protein denaturation, precipitation and centrifugation, the organic solvent exists at the bottom of the test tube, the DNA exists in the upper water layer, and the protein precipitate exists between the two liquids. Finally, ethanol is used to separate the DNA from the water, precipitate it, and recover it after centrifugation. The phenol-chloroform extraction process requires multiple complex centrifugation operations, which is time-consuming and prone to cross-contamination of samples, resulting in a low DNA yield, and is not suitable for large-scale extraction.

[0004] 2) Magnetic bead method. Since magnetic beads can specifically bind to nucleic acids under certain conditions but not to impurities such as proteins, sugars and esters in the sample, they are widely used in nucleic acid extraction, pathogen detection and other aspects. Magnetic beads are added to the lysed mixed solution, and in the acidic (PH 5.0) buffer, the DNA molecules released from the cells are quickly adsorbed to the positively charged magnetic beads. The magnetic beads adsorbed with DNA can be directed to aggregate on the side of the test tube under the action of the magnetic field. At this time, the liquid in the tube is aspirated and discarded, and the washing buffer is added. The test tube is removed from the magnetic field, and after repeated rinsing several times, it is placed in the magnetic field again, and the magnetic beads can aggregate on the tube wall again. Adding alkaline (PH 8.0) buffer and elution solution can elute DNA from the magnetic beads, thereby obtaining a purified genomic DNA solution. The operation steps of the magnetic bead method for extracting DNA are cumbersome and the extraction purity is lower than that of the silica gel membrane adsorption method. The fully automatic nucleic acid extractor is expensive and beyond the affordability of hospitals in remote areas and township health centers.

[0005] 3) Silica gel membrane adsorption method: This method uses nucleic acid adsorption silica gel membrane, which is a special silicon matrix adsorption material that can specifically adsorb DNA, while RNA and protein pass through. Under normal circumstances, the surface of DNA is covered with a hydrophilic film composed of water molecules to maintain its water solubility. The addition of high concentrations of salt ions destroys the relatively orderly arrangement of the hydrophilic film on the surface of DNA, forming a hydrophobic environment. In this environment, DNA can effectively bind to the silica gel membrane, while proteins, metabolites and other pollutants cannot bind. In a high ionic strength buffer solution, the negative charge on the silicon surface of the silica gel membrane gradually decreases, thereby reducing the repulsive force between the negative charge of the phosphate groups exposed after dehydration of the DNA molecules and the silicon surface, forming a large number of hydrated ions, and the degree of hydration of the DNA molecules is reduced and driven to polymerize on the silicon surface. At the same time, the cell lysate will also interfere with the formation of hydrogen bonds between double-stranded DNA molecules to produce single-stranded DNA molecules, which form hydrogen bonds with the silicon surface, and the effect of this hydrogen bond is far greater than the electrostatic repulsion between the DNA molecules and the silicon surface. If the pH value of the high ionic strength buffer solution is lower than the PKa value of the silicon surface, the electrostatic repulsion will become smaller, which greatly improves the adsorption capacity of the silicon surface for DNA molecules. However, when the pH value of the buffer solution is higher than the PKa value of the silicon surface, the DNA molecules adsorbed on the silicon surface will be eluted by the buffer solution. Summary of the invention

[0006] In view of the above problems, the present invention provides a centrifugal disk for DNA extraction, on which the reaction process of the silica gel membrane adsorption method can be integrated, thereby realizing DNA extraction with high efficiency.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A DNA extraction centrifugal disk comprises at least one DNA extraction unit; the front side of the DNA extraction unit is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet, a temporary liquid storage area and a filtering area, and the back side is provided with a first liquid storage area and a second liquid storage area; a DNA adsorption material is arranged in the filtering area; the first liquid inlet and the second liquid inlet are respectively connected with the filtering area through flow channels; the third liquid inlet is connected with the temporary liquid storage area through the flow channel, and the temporary liquid storage area is connected with the filtering area through the flow channel; the second liquid storage area is connected with the first liquid storage area through the flow channel, and the first liquid storage area is connected with the filtering area through the flow channel.

[0009] Furthermore, the DNA adsorption material is a silica gel membrane.

[0010] Furthermore, the front and back sides of the DNA extraction unit are covered with thin films.

[0011] Furthermore, the first liquid inlet is used for adding blood and red blood cell lysate; the second liquid inlet is used for adding high-concentration salt ion solution; the third liquid inlet is used for adding DNA elution solution; the temporary liquid storage area is used to temporarily store the DNA elution solution in the third liquid inlet, and cooperate with the speed setting of the centrifuge to ensure that the liquid in the third liquid inlet enters the filtration area through the first liquid inlet and the second liquid inlet; the filtration area is used to use DNA adsorption material to realize DNA filtration extraction; the first liquid storage area and the second liquid storage area are used to realize DNA collection.

[0012] Furthermore, the first liquid inlet and the second liquid inlet are closer to the center of the DNA extraction centrifugal disk than the filtration area; the third liquid inlet and the temporary liquid storage area are closer to the center of the DNA extraction centrifugal disk than the filtration area; the third liquid inlet is closer to the center of the DNA extraction centrifugal disk than the temporary liquid storage area; the filtration area is closer to the center of the DNA extraction centrifugal disk than the first liquid storage area; and the first liquid storage area is closer to the center of the DNA extraction centrifugal disk than the second liquid storage area.

[0013] Furthermore, when the centrifuge rotates at a speed of 1, the solutions in the first liquid inlet and the second liquid inlet enter the filtration area and mix; when the centrifuge rotates at a speed of 2, the DNA in the solution in the filtration area adheres to the DNA adsorption material, and the remaining impurities enter the first liquid storage area, and at the same time, the elution liquid in the third liquid inlet enters the temporary liquid storage area at this speed; when the centrifuge rotates at a speed of 3, the DNA elution liquid in the temporary liquid storage area enters the filtration area, and the impurities in the first liquid storage area enter the second liquid storage area; when the centrifuge rotates at a speed of 4, the eluted DNA enters the first liquid storage area.

[0014] Furthermore, the material of the DNA extraction centrifugal disk is plastic.

[0015] The present invention innovatively integrates the reaction process of the silica gel membrane adsorption method into a centrifugal disk, which has the following beneficial effects:

[0016] 1) It realizes one-time sample addition to result output, greatly simplifying the DNA extraction process;

[0017] 2) The entire process of DNA extraction is completed automatically in a sealed centrifugal disk, with low environmental requirements;

[0018] 3) The whole process is automated, which reduces the skill requirements for the experimenter;

[0019] 4) The centrifugal disc is a disposable consumable (disposable plastic or other materials can be used), which can be discarded after use, saving experimental time;

[0020] 5) The reaction process only requires a small centrifuge as supporting equipment, which greatly reduces the cost. At the same time, the equipment is easy to carry, solving medical problems in extreme environments and remote areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a 3D front perspective view of a DNA extraction centrifuge disk.

[0022] Figure 2 This is a 2D image of the front side of the DNA extraction centrifuge disk.

[0023] Figure 3 This is a 2D image of the back side of the DNA extraction centrifuge disk.

[0024] Figure 4 It is a schematic diagram of the structure of a DNA extraction unit on a DNA extraction centrifugal disk.

[0025] Figure 5 Schematic diagram of the filtration area of ​​the DNA extraction centrifuge disk before embedding in the silica membrane.

[0026] Figure 6 Schematic diagram of the filtration area of ​​the DNA extraction centrifuge disk after embedding the silica membrane.

[0027] Description of the numbers in the figure:

[0028] 100-DNA extraction centrifugal disk; 110-DNA extraction unit; 111-first liquid inlet; 112-second liquid inlet; 113-third liquid inlet; 114-temporary liquid storage area; 115-filtration area; 116-first liquid storage area; 117-second liquid storage area; 118-DNA adsorption material. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below through specific embodiments and drawings.

[0030] 1. DNA Extraction Centrifuge Plate

[0031] like Figure 1 As shown, the DNA extraction centrifugal disk 100 of this embodiment is provided with a plurality of DNA extraction units 110 evenly distributed along the circumference, wherein the solid line portion indicates the portion located at the front of the centrifugal disk, and the dotted line portion indicates the portion located at the back of the centrifugal disk. Figure 2 The structure of the front side of the centrifugal disc is shown separately. Figure 3 The structure on the back of the centrifugal disc is shown separately. A mounting hole is provided in the middle of the centrifugal disc. Figure 1 As shown, the centrifugal disc can be mounted on a centrifuge through the mounting hole, thereby forming a DNA extraction device.

[0032] Figure 4Schematic diagram of a single DNA extraction unit 110. As shown in the figure, a DNA extraction unit is provided with a first liquid inlet 111, a second liquid inlet 112, a third liquid inlet 113, a temporary liquid storage area 114 and a filtration area 115 on the front side, and a first liquid storage area 116 and a second liquid storage area 117 on the back side. A DNA adsorption material 118 (such as Figure 5 As shown in FIG. 1 , in this embodiment, the DNA adsorption material adopts a silica gel mold. The first liquid inlet 111 and the second liquid inlet 112 are respectively connected to the filtering area 115 through the flow channel; the third liquid inlet 113 is connected to the temporary liquid storage area 114 through the flow channel, and the temporary liquid storage area 114 is connected to the filtering area 115 through the flow channel; the second liquid storage area 117 is connected to the first liquid storage area 116 through the flow channel, and the first liquid storage area 116 is connected to the filtering area 115 through the flow channel.

[0033] Figure 5 Schematic diagram of the DNA adsorption material 118, i.e., the silica gel mold, embedded in the filtration area 115. Figure 5 This is a schematic diagram before embedding in the silicone membrane. Figure 6 This is a schematic diagram after embedding in silicone membrane.

[0034] The above 111-117 are all openings on the front or back of the centrifugal disk, which are connected by the flow channel shown in the figure. A film of the same size as the centrifugal disk needs to be pasted on both the front and back sides of the centrifugal disk to cover all the holes (i.e. 111-117) and the flow channel.

[0035] For the three liquid inlets 111-113, in order to facilitate adding liquid thereto, before applying the film, corresponding openings (small holes) are made on the film at positions corresponding to the three liquid inlets 111-113. In addition, in addition to blood, other reagents can also be pre-buried in the three liquid inlets 111-113 before applying the film, and only blood can be added when using.

[0036] The functions of the above 111 to 117 are respectively described as follows:

[0037] The first liquid inlet 111 is used to add blood and red blood cell lysing solution respectively.

[0038] The second liquid inlet 112 is used for adding high concentration salt ion solution.

[0039] The third liquid inlet 113 is used for adding DNA elution solution (TE buffer).

[0040] Temporary liquid storage area 114: used to temporarily store the DNA elution solution added to the liquid inlet C, and cooperate with the speed setting of the centrifuge (see the DNA extraction process below for details) to achieve that the liquid in the liquid inlet C enters the filtration area D through the liquid inlets A and B.

[0041] Filtration area 115: used to install a silica gel membrane to achieve filtration extraction of DNA.

[0042] The first liquid storage area 116 and the second liquid storage area 117 are used to collect DNA (see the DNA extraction process below for details).

[0043] The above-mentioned DNA extraction centrifugal disk is preferably made of plastic materials, such as PS, PC, PMMA, COC, COP, etc.

[0044] 2. DNA extraction method

[0045] 1. Experimental reagents and instruments

[0046] a) Experimental reagents: blood, red blood cell lysis buffer, DNA elution buffer

[0047] b) Experimental instruments: DNA extraction centrifuge, centrifuge, quantitative pipette

[0048] 2. DNA extraction process

[0049] The steps of DNA extraction using the above DNA extraction centrifugal disk are as follows:

[0050] 1) Add appropriate amounts of blood and red blood cell lysis solution (for lysing red blood cells) into the first liquid inlet 111 of the centrifugal disk; add an appropriate amount of high-concentration salt ion solution (for lysing cells and releasing DNA) into the second liquid inlet 112; and add an appropriate amount of DNA elution solution into the third liquid inlet 113.

[0051] The red blood cell lysing solution can be Tris-HCl, etc. The high concentration salt ion solution can be a mixture of SDS (sodium dodecyl sulfate), ammonium acetate, guanidine hydrochloride and proteinase K.

[0052] The formula of the DNA elution solution in this embodiment is: Tris-HCl (pH 8.0) 10 mmol / L, EDTA (pH 8.0) 1 mmol / L.

[0053] In this embodiment, the blood is 10 μL, the red blood cell lysate is 20 μL, the high concentration salt ion solution is 30 μL, and the DNA elution solution is 50 μL.

[0054] 2) The centrifugal disc is placed in a centrifuge, and the blood and red blood cell lysate in the first liquid inlet 111 are mixed by vibration generated by clockwise and counterclockwise rotation. In addition to this mixing method, a vortex mixer or other equipment can also be used for mixing.

[0055] 3) The centrifuge rotates at a speed of 1, so that the solutions in the first liquid inlet 111 and the second liquid inlet 112 enter the filtration area 115 and are mixed.

[0056] 4) The centrifuge rotates at a speed of 2, so that the DNA in the solution in the filtration area 115 adheres to the silica gel membrane, and the remaining impurities enter the first liquid storage area 116. At the same time, at this speed, the DNA elution solution in the third liquid inlet 113 enters the temporary liquid storage area 114.

[0057] Under normal circumstances, the surface of DNA is covered with a hydrophilic film composed of water molecules to maintain its water solubility. The addition of a high concentration of salt ion solution can destroy the relatively orderly arrangement of the hydrophilic film on the surface of DNA, forming a hydrophobic environment. In this environment, DNA can effectively bind to the silica gel membrane, while proteins, metabolites and other pollutants cannot bind to the silica gel membrane and enter the first liquid storage area 116.

[0058] 5) The centrifugal disk rotates at a speed of 3, so that the DNA elution solution in the temporary liquid storage area 114 enters the filtration area 115, and the impurities in the first liquid storage area 116 enter the second liquid storage area 117.

[0059] In the high ionic strength buffer solution such as DNA elution solution, the negative charge on the silicon surface of the silica gel membrane gradually decreases, thereby reducing the repulsive force between the negative charge of the phosphate groups exposed after dehydration of the DNA molecules and the silicon surface, forming a large number of hydrated ions, and the degree of hydration of the DNA molecules is reduced and driven to polymerize on the silicon surface. At the same time, the cell lysate will also interfere with the formation of hydrogen bonds between double-stranded DNA molecules to produce single-stranded DNA molecules, which form hydrogen bonds with the silicon surface, and the effect of this hydrogen bond is far greater than the electrostatic repulsion between the DNA molecules and the silicon surface. If the pH value of the high ionic strength buffer solution is lower than the PKa value of the silicon surface, this electrostatic repulsion will become smaller, which greatly improves the adsorption capacity of the silicon surface for DNA molecules, but when the pH value of the buffer solution is higher than the PKa value of the silicon surface, the DNA molecules adsorbed on the silicon surface will be eluted by the buffer.

[0060] 6) The centrifuge rotates at a speed of 4, so that the eluted DNA enters the first liquid storage area 116.

[0061] 7) Use a quantitative pipette to pierce the sealed film on the first liquid storage area 116 and take out the DNA, thereby achieving DNA extraction.

[0062] In the above speeds 1 to 4, the speed values ​​are related to the size of the centrifugal disc. In specific implementation, the above four speeds can be reasonably set according to the size of the centrifugal disc, as long as the effects of the above steps can be achieved. For example, when the diameter of the centrifugal disc is 120 mm, the preferred value range of speeds 1 to 4 is:

[0063] Speed ​​1: 500rpm-1000rpm;

[0064] Speed ​​2: 10000rpm-20000rpm;

[0065] Speed ​​3: 4000rpm-6000rpm;

[0066] Speed ​​4: 10000rpm-20000rpm.

[0067] The rotation speed of the centrifuge can be controlled by a computer, or by setting a touch screen on the centrifuge for control, etc. In addition, the centrifuge can also be integrated with a vortex mixer to achieve the mixing process in step 2).

[0068] In the present invention, the DNA extraction unit on the DNA extraction centrifugal disk may also be in other shapes, and the relative positions of 111 to 117 are not limited to the forms shown in the drawings, as long as the functions described above can be achieved. Generally speaking, the arrangement order of 111 to 117 along the radial direction of the centrifugal disk is:

[0069] a) The first liquid inlet 111 and the second liquid inlet 112 are closer to the center (center of the circle) of the DNA extraction centrifugal disk than the filtration area 115. There is no special requirement for the relative position of the first liquid inlet 111 and the second liquid inlet 112, and the distances from the center of the DNA extraction centrifugal disk can be equal or approximately equal.

[0070] b) The third liquid inlet 113 and the temporary liquid storage area 114 are closer to the center of the DNA extraction centrifugal disk than the filtration area 115. The third liquid inlet 113 is closer to the center of the DNA extraction centrifugal disk than the temporary liquid storage area 114.

[0071] c) The filtration area 115 is closer to the center of the DNA extraction centrifugal disk than the first liquid storage area 116 , and the first liquid storage area 116 is closer to the center of the DNA extraction centrifugal disk than the second liquid storage area 117 .

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. A person skilled in the art may modify or make equivalent substitutions for the technical solutions of the present invention without departing from the spirit and scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A DNA extraction centrifugal disk, characterized in that: The invention comprises at least one DNA extraction unit; the front side of the DNA extraction unit is provided with a first liquid inlet, a second liquid inlet, a third liquid inlet, a temporary liquid storage area and a filtering area, and the back side is provided with a first liquid storage area and a second liquid storage area; a DNA adsorption material is arranged in the filtering area; the first liquid inlet and the second liquid inlet are respectively connected with the filtering area through flow channels; the third liquid inlet is connected with the temporary liquid storage area through the flow channel, and the temporary liquid storage area is connected with the filtering area through the flow channel; the second liquid storage area is connected with the first liquid storage area through the flow channel, and the first liquid storage area is connected with the filtering area through the flow channel; the first liquid inlet is used for adding blood and red blood cell lysis solution; the second liquid inlet is used for adding high concentration salt ion solution; the third liquid inlet is used for adding DNA elution solution; the temporary liquid storage area is used for temporarily storing the DNA elution solution in the third liquid inlet, and the liquid in the third liquid inlet enters the filtering area through the first liquid inlet and the second liquid inlet in combination with the speed setting of the centrifuge; the filtering area is used for filtering and extracting DNA by using DNA adsorption material; the first liquid storage area and the second liquid storage area are used for DNA collection is achieved; when the centrifuge rotates at a speed of 1, the solutions in the first liquid inlet and the second liquid inlet enter the filtration area and are mixed; when the centrifuge rotates at a speed of 2, the DNA in the solution in the filtration area adheres to the DNA adsorption material, and the remaining impurities enter the first liquid storage area, and at the same time, the elution liquid in the third liquid inlet enters the temporary liquid storage area at this speed; when the centrifuge rotates at a speed of 3, the DNA elution liquid in the temporary liquid storage area enters the filtration area, and the impurities in the first liquid storage area enter the second liquid storage area; when the centrifuge rotates at a speed of 4, the eluted DNA enters the first liquid storage area; the first liquid inlet and the second liquid inlet are closer to the center of the DNA extraction centrifugal disk than the filtration area; the third liquid inlet and the temporary liquid storage area are closer to the center of the DNA extraction centrifugal disk than the filtration area; the third liquid inlet is closer to the center of the DNA extraction centrifugal disk than the temporary liquid storage area; the filtration area is closer to the center of the DNA extraction centrifugal disk than the first liquid storage area; the first liquid storage area is closer to the center of the DNA extraction centrifugal disk than the second liquid storage area.

2. The DNA extraction centrifugal disk according to claim 1, characterized in that: The DNA adsorption material is a silica gel membrane.

3. The DNA extraction centrifugal disk according to claim 1, characterized in that: The front and back sides of the DNA extraction unit are covered with films, and the films cover the temporary liquid storage area, the filtration area, the first liquid storage area, and the second liquid storage area.

4. The DNA extraction centrifugal disk according to claim 1, characterized in that: The material of the DNA extraction centrifugal disk is plastic.

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