CN109694807A - A kind of DNA extraction element - Google Patents
A kind of DNA extraction element Download PDFInfo
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- CN109694807A CN109694807A CN201711001932.8A CN201711001932A CN109694807A CN 109694807 A CN109694807 A CN 109694807A CN 201711001932 A CN201711001932 A CN 201711001932A CN 109694807 A CN109694807 A CN 109694807A
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- 238000007400 DNA extraction Methods 0.000 title claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 104
- 238000001914 filtration Methods 0.000 claims abstract description 49
- 239000000284 extract Substances 0.000 claims abstract description 37
- 239000000463 material Substances 0.000 claims abstract description 20
- 239000003463 adsorbent Substances 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims description 17
- 239000008280 blood Substances 0.000 claims description 9
- 210000003743 erythrocyte Anatomy 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 5
- 229920003023 plastic Polymers 0.000 claims description 4
- 239000004033 plastic Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 2
- 210000000601 blood cell Anatomy 0.000 claims 1
- 239000013592 cell lysate Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 19
- 238000010521 absorption reaction Methods 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 96
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 21
- 229910052710 silicon Inorganic materials 0.000 description 21
- 239000010703 silicon Substances 0.000 description 21
- 239000000243 solution Substances 0.000 description 14
- 239000000872 buffer Substances 0.000 description 9
- 230000005291 magnetic effect Effects 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 239000011324 bead Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000036571 hydration Effects 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000000197 pyrolysis Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- RZYKUPXRYIOEME-UHFFFAOYSA-N CCCCCCCCCCCC[S] Chemical compound CCCCCCCCCCCC[S] RZYKUPXRYIOEME-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- -1 salt ion Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
Classifications
-
- 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
Abstract
The present invention relates to a kind of DNA extraction elements.The DNA extraction element includes centrifuge and DNA extracts centrifugal pan, and the DNA extracts centrifugal pan and is mounted on the centrifuge, and it includes at least one DNA extraction unit that the DNA, which extracts centrifugal pan,；The front of the DNA extraction unit is equipped with the first inlet, the second inlet, third inlet, interim liquid storage area and filtering area, and the back side is equipped with the first liquid storage area and the second liquid storage area；DNA adsorbent material is equipped in filtering area；First inlet, the second inlet pass through runner respectively and are connected to filtering area；Third inlet is connected to by runner with interim liquid storage area, and interim liquid storage area is connected to by runner with filtering area；Second liquid storage area is connected to by runner with the first liquid storage area, and the first liquid storage area is connected to by runner with filtering area.The DNA adsorbent material is preferably pellosil.The reaction process of pellosil absorption method is integrated on centrifugal pan by the present invention, can efficiently realize that DNA is extracted.
Description
Technical field
The invention belongs to biotechnologys, DNA extractive technique field, and in particular to a kind of DNA extraction element can facilitate height
Realize that DNA is extracted in effect ground.
Background technique
Existing DNA extraction method mainly include the following types:
1) classical phenol-chloroform method.Isometric phenol chloroform mixed liquor is added after generally referring to cell cracking, according to DNA
It is soluble easily in water, be not readily dissolved in the characteristic of organic solvent, under organic solvent environment, protein by denaturation precipitating and centrifugation after,
Organic solvent is present in test tube bottom, and DNA is present in upper water, and protein precipitation is present between two kinds of liquid, finally uses
DNA is separated from the water by ethyl alcohol, and precipitating recycles after centrifugation.The centrifugation behaviour of multiple complexity is needed in phenol-chloroform extraction process
Make, time-consuming, be easy to cause the cross contamination of sample, so that DNA output is on the low side, is not suitable for high-volume and extracts.
2) paramagnetic particle method.Due to magnetic bead under given conditions can specifically in conjunction with nucleic acid without with the egg in sample
The impurity such as white, carbohydrate and esters combine thus be widely used in nucleic acid extraction, pathogen detection etc..Magnetic bead is added to
By cracking after mixed liquor in, in the buffer of slant acidity (PH 5.0), from cell the DNA molecular of separate out quickly by
It is adsorbed on the magnetic bead with positive charge.The magnetic bead for being adsorbed with DNA can be directed to reunite in the side of test tube under magnetic fields.This
When, liquid in pipe to be drawn and abandoned, cleaning solution buffer is added, test tube removes magnetic field, and repeated flushing places into magnetic field afterwards several times,
Magnetic bead can reunite in tube wall again.The buffer and eluent that meta-alkalescence (PH 8.0) is added can say that DNA is eluted from magnetic bead
Get off, thus the Genomic DNA solution purified.Paramagnetic particle method extraction DNA is complex for operation step and DNA purity compares pellosil
Absorption method is low, the expensive receiving model for having exceeded hospital and health clinics in towns and townships from far-off regions of full automatic instrument for extracting nucleic acid
It encloses.
3) pellosil absorption method: this method uses nucleic acid absorption pellosil, is a kind of special silicon matrix adsorbent material, energy
Enough specific adsorption DNA, and RNA is passed through with protein.Under normal circumstances, the surface DNA covers one layer and is made of hydrone
Hydrophilic film, with maintain its water solubility.The addition of high salt concentration ion destroys orderly arranging relatively for DNA surface hydrophilic film
Column, form hydrophobic environment.In the environment, DNA can be combined effectively with pellosil, and protein, metabolite and other dirts
Dye object cannot then combine.In high ionic strength buffers solution, the negative electrical charge of the silicon face on pellosil is gradually decreased, to make
The repulsive force that the negative electrical charge and silicon face that the phosphate group of exposure has after DNA molecular dehydration generate is reduced, and is formd a large amount of
The hydration levels of hydrated ion, DNA molecular reduce and are forced to be aggregated on the surface of silicon.Cell pyrolysis liquid can also interfere with simultaneously
Hydrogen bond between double chain DNA molecule forms and generates single-stranded DNA molecular, and single strand dna and silicon face form hydrogen bond, and this
The effect of kind hydrogen bond is much larger than the electrostatic repulsion forces of DNA molecular and silicon face.If the pH value of high ionic strength buffers solution is low
In the PKa value of silicon face, this electrostatic repulsion forces can become smaller, this has been considerably improved silicon face to the adsorption capacity of DNA molecular,
But when the pH value of buffer solution is higher than the PKa value of silicon face, the DNA molecular of silicon face absorption will be eluted down by buffer
Come.
Summary of the invention
The present invention, can be by the reaction process collection of pellosil absorption method in view of the above-mentioned problems, provide a kind of DNA extraction element
At on the centrifugal pan, efficiently realizing that DNA is extracted.
The technical solution adopted by the invention is as follows:
A kind of DNA extraction element, including centrifuge and DNA extract centrifugal pan, and the DNA extraction centrifugal pan is mounted on described
On centrifuge, it includes at least one DNA extraction unit that the DNA, which extracts centrifugal pan,；The front of the DNA extraction unit is equipped with the
One inlet, the second inlet, third inlet, interim liquid storage area and filtering area, the back side are equipped with the first liquid storage area and the second storage
Liquid zone；DNA adsorbent material is equipped in filtering area；First inlet, the second inlet pass through runner respectively and are connected to filtering area；The
Three inlets are connected to by runner with interim liquid storage area, and interim liquid storage area is connected to by runner with filtering area；Second liquid storage area is logical
It crosses runner to be connected to the first liquid storage area, the first liquid storage area is connected to by runner with filtering area.
Further, the DNA adsorbent material is pellosil.
Further, the front and back of the DNA extraction unit covers film.
Further, the first inlet is for being added blood and erythrocyte cracked liquid；Second inlet is highly concentrated for being added
Spend salt ion solution；Third inlet is for being added DNA eluent；Interim liquid storage area is for temporarily storing in third inlet
DNA eluent, and cooperate centrifuge revolving speed be arranged realize third inlet in liquid after in the first inlet and second into
Liquid mouth enters filtering area；Filtering area is used to realize that the filtering of DNA is extracted using DNA adsorbent material；First liquid storage area and the second storage
Liquid zone for realizing DNA collection.
Further, the first inlet, the second inlet extract the center of centrifugal pan than filtering area closer to DNA；Third
Inlet, interim liquid storage area extract the center of centrifugal pan than filtering area closer to DNA；Third inlet is more leaned on than interim liquid storage area
Nearly DNA extracts the center of centrifugal pan；Filtering area extracts the center of centrifugal pan than the first liquid storage area closer to DNA；First liquid storage area
The center of centrifugal pan is extracted closer to DNA than the second liquid storage area.
Further, when centrifuge is rotated using revolving speed 1, the solution in the first inlet, the second inlet entered
In filter area and mix；When centrifuge is rotated using revolving speed 2, the DNA in solution in filtering area is adhered to DNA adsorbent material
On, remaining impurity enters the first liquid storage area, while the eluent under the revolving speed in third inlet enters interim liquid storage area；?
When centrifuge is rotated using revolving speed 3, the DNA eluent in interim liquid storage area enters filtering area, and miscellaneous in the first liquid storage area
Matter enters the second liquid storage area；When centrifuge is rotated using revolving speed 4, the DNA under eluting enters the first liquid storage area.
Further, it is plastics that the DNA, which extracts the material of centrifugal pan,.
The reaction process by pellosil absorption method of DNA extraction element novelty of the invention is integrated into one piece of centrifugal pan
On, it has the advantages that
1) it realizes and is once loaded onto result output, greatly simplify DNA and extract process；
2) whole process that DNA is extracted is automatically performed in the centrifugal pan of sealing, low to environmental requirement；
3) whole-course automation reduces the skill requirement to laboratory technician；
4) centrifugal pan is medical disposable material (materials such as disposable plastic can be used), and being finished can cancel, and save experiment
Time；
5) corollary equipment of the reaction process only needs a compact centrifuge, significantly reduces cost, while the equipment
It is convenient for carrying, solves the medical care problem of extreme environment and remote districts.
Detailed description of the invention
Fig. 1 is the 3D front perspective view that DNA extracts centrifugal pan.
Fig. 2 is the positive 2D figure that DNA extracts centrifugal pan.
Fig. 3 is the back side 2D figure that DNA extracts centrifugal pan.
Fig. 4 is the structural schematic diagram for the DNA extraction unit that DNA is extracted on centrifugal pan.
Fig. 5 is that DNA extracts schematic diagram of the filtering area of centrifugal pan before being embedded in pellosil.
Fig. 6 is that DNA extracts schematic diagram of the filtering area of centrifugal pan after being embedded in pellosil.
Figure label explanation:
100-DNA extracts centrifugal pan；110-DNA extraction unit；The first inlet of 111-；The second inlet of 112-；113-
Third inlet；The interim liquid storage area of 114-；115- filtering area；The first liquid storage area of 116-；The second liquid storage area of 117-；118-DNA inhales
Enclosure material.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, below by specific embodiment and
Attached drawing is described in further details the present invention.
One .DNA extraction element
DNA extraction element includes centrifuge and DNA extracts centrifugal pan, and the DNA extracts centrifugal pan and is mounted on the centrifugation
On machine.As shown in Figure 1, the DNA of the present embodiment, which extracts centrifugal pan 100, is equipped with several circumferentially equally distributed DNA extractions
Unit 110.Wherein bold portion indicates to be located at centrifugal pan front, and dotted portion indicates to be located at the centrifugal pan back side.Fig. 2 individually illustrates
The positive structure of centrifugal pan, Fig. 3 individually illustrate the structure at the centrifugal pan back side.The centre of the centrifugal pan is equipped with mounting hole 111,
As shown in Figure 1, the centrifugal pan can be mounted on centrifuge by the mounting hole, to constitute a kind of DNA extraction element.
Fig. 4 is the schematic diagram of an individual DNA extraction unit 110.As shown in the drawing, a DNA extraction unit is being just
Face is equipped with the first inlet 111, the second inlet 112, third inlet 113, interim liquid storage area 114 and filtering area 115, the back side
Equipped with the first liquid storage area 116 and the second liquid storage area 117.It is equipped with DNA adsorbent material 118 (as shown in Figure 5) in filtering area 115, this
The DNA adsorbent material uses Silicon moulds in embodiment.First inlet 111, the second inlet 112 pass through runner and filtering respectively
Area 115 is connected to；Third inlet 113 is connected to by runner with interim liquid storage area 114, and interim liquid storage area 114 passes through runner and mistake
Area 115 is filtered to be connected to；Second liquid storage area 117 be connected to by runner with the first liquid storage area 116, the first liquid storage area 116 pass through runner and
Filtering area 115 is connected to.
Fig. 5 is that filtering area 115 is embedded in the DNA adsorbent material 118 i.e. schematic diagram of Silicon moulds.Wherein Fig. 5 is insertion silica gel
Schematic diagram before film, Fig. 6 are the schematic diagrames being embedded in after pellosil.
Above-mentioned 111~117 be the aperture in centrifugal pan front or the back side, is connected to therebetween by runner shown in figure.
The front and back sides of centrifugal pan are required to paste one and centrifugal pan film of a size, by whole holes (i.e. 111~117) and stream
Road covering.
For three inlets 111~113, before pasting film, to be corresponded on film convenient for liquid is added thereto
Make corresponding opening (aperture) in the position of three inlets 111~113.In addition, in addition to blood, remaining reagent can also be with
It is embedded into before pasting film in three inlets 111~113, when use only adds blood.
Above-mentioned 111~117 effect is respectively described below:
First inlet 111: for being separately added into blood and erythrocyte cracked liquid.
Second inlet 112: for high salt concentration solion to be added.
Third inlet 113: for DNA eluent (TE buffer) to be added.
Interim liquid storage area 114: for temporarily storing the DNA eluent being added in inlet C, and cooperate the revolving speed of centrifuge
It is arranged (being detailed in following DNA extraction process), enters filtering area in inlet A and inlet B after the liquid in realization inlet C
D。
Filtering area 115: for installing pellosil, realize that the filtering of DNA is extracted.
First liquid storage area 116, the second liquid storage area 117: for realizing the collection (being detailed in following DNA extraction process) of DNA.
Above-mentioned DNA extracts centrifugal pan and preferably uses plastic material, such as PS, PC, PMMA, COC, COP.
Two .DNA extracting methods
1. experiment reagent and instrument
A) experiment reagent: blood, erythrocyte cracked liquid, DNA eluent
B) laboratory apparatus: DNA extracts centrifugal pan, centrifuge, quantitative liquid shifter
2.DNA extracts process
The step process for carrying out DNA extraction using above-mentioned DNA extraction element is as follows:
1) to be separately added into suitable blood, erythrocyte cracked liquid in 111 hole of the first inlet of centrifugal pan (red for cracking
Cell)；Suitable high salt concentration solion is added in second inlet 112 (for lytic cell, released dna)；Third into
Suitable DNA eluent is added in liquid mouth 113.
Erythrocyte cracked liquid can be using Tris-HCl etc..High salt concentration solion can use SDS (dodecyl sulphur
Sour sodium), ammonium acetate, guanidine hydrochloride and Proteinase K mixture.
The formula of DNA eluent in the present embodiment are as follows: Tris-HCl (pH8.0) 10mmol/L, EDTA (pH8.0) 1mmol/
L。
In the present embodiment, blood is 10 μ L, and erythrocyte cracked liquid is 20 μ L, and high salt concentration solion is 30 μ L, and DNA is washed
De- liquid is 50 μ L.
2) by centrifugal pan be packed into centrifuge in, the vibration generated when by being rotated both clockwise and counterclockwise, mix first into
Blood and erythrocyte cracked liquid in liquid mouth 111.It, can also be using equipment such as vortex blending instruments other than this blending manner
It is mixed.
3) centrifuge is rotated using revolving speed 1, and the solution in the first inlet 111 and the second inlet 112 is made to enter filtering
In area 115 and mix.
4) centrifuge is rotated using revolving speed 2, is adhered to the DNA in the solution in filtering area 115 on pellosil, remaining is miscellaneous
Matter enters the first liquid storage area 116, while the DNA eluent under the revolving speed in third inlet 113 enters interim liquid storage area
114。
Under normal circumstances, the surface DNA covers one layer by the molecular hydrophilic film of moisture, to maintain its water solubility.
The addition of high salt concentration solion can destroy the opposite ordered arrangement of DNA surface hydrophilic film, form hydrophobic environment.?
In this environment, DNA can have enough effects to be combined with pellosil, and protein, metabolite and other pollutants then cannot be with pellosils
In conjunction with hence into the first liquid storage area 116.
5) centrifugal pan is rotated using revolving speed 3, and the DNA eluent in interim liquid storage area 114 is made to enter filtering area 115, and
The impurity in the first liquid storage area 116 is set to enter the second liquid storage area 117.
In this high ionic strength buffers solution of DNA eluent, the negative electrical charge of the silicon face on pellosil is gradually decreased,
The repulsive force that negative electrical charge and silicon face to make the phosphate group exposed after DNA molecular dehydration have generate is reduced, and is formd
The hydration levels of a large amount of hydrated ion, DNA molecular reduce and are forced to be aggregated on the surface of silicon.Cell pyrolysis liquid simultaneously
The hydrogen bond between double chain DNA molecule can be interfered to be formed and generate single-stranded DNA molecular, single strand dna and silicon face form hydrogen
Key, and the effect of this hydrogen bond is much larger than the electrostatic repulsion forces of DNA molecular and silicon face.If high ionic strength buffers solution
PH value is lower than the PKa value of silicon face, and this electrostatic repulsion forces can become smaller, this has been considerably improved suction of the silicon face to DNA molecular
Attached ability, but when the pH value of buffer solution is higher than the PKa value of silicon face, the DNA molecular of silicon face absorption will be by buffer
It elutes.
6) centrifuge is rotated using revolving speed 4, and the DNA under elution is made to enter the first liquid storage area 116.
7) film sealed in the first liquid storage area 116 is punctured with quantitative liquid shifter, takes out DNA, to realize that DNA is extracted.
In above-mentioned revolving speed 1~4, the numerical value of revolving speed and the size of centrifugal pan are related, and when specific implementation can be according to centrifugal pan
Size, to aforementioned four revolving speed reasonable value, as long as can be realized the effect of above steps.For example, working as centrifugal pan
When diameter is 120mm, the preferred value range of revolving speed 1~4 are as follows:
Revolving speed 1:500rpm-1000rpm；
Revolving speed 2:10000rpm-20000rpm；
Revolving speed 3:4000rpm-6000rpm；
Revolving speed 4:10000rpm-20000rpm.
The revolving speed of above-mentioned centrifuge can be controlled by computer, or touch screen is arranged on centrifuge and is controlled
Deng.In addition, the function of vortex blending instrument can be also integrated on centrifuge, to realize above-mentioned steps 2) in mixing processing.
In the present invention, the DNA extraction unit that DNA is extracted on centrifugal pan is also possible to other shapes, 111~117 it is opposite
Position is also not necessarily limited to form shown in the drawings, as long as can be realized function recited above.In general, 111~117
Along the distributing order of centrifugal pan radial direction are as follows:
A) the first inlet 111, the second inlet 112 extract the center (circle of centrifugal pan than filtering area 115 closer to DNA
The heart).The relative position of first inlet 111 and the second inlet 112 does not specially require, and the center of centrifugal pan is extracted with DNA
Distance can be equal or approximately equal.
B) third inlet 113, interim liquid storage area 114 extract the center of centrifugal pan than filtering area 115 closer to DNA.The
Three inlets 113 extract the center of centrifugal pan than interim liquid storage area 114 closer to DNA.
C) 115 to the first liquid storage area 116 of filtering area extracts the center of centrifugal pan, the first liquid storage area 116 to the closer to DNA
Two liquid storage areas 117 extract the center of centrifugal pan closer to DNA.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than is limited, the ordinary skill of this field
Personnel can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the spirit and scope of the present invention, this
The protection scope of invention should be subject to described in claims.
Claims (7)
1. a kind of DNA extraction element, which is characterized in that extract centrifugal pan including centrifuge and DNA, the DNA extracts centrifugal pan
It is mounted on the centrifuge, it includes at least one DNA extraction unit that the DNA, which extracts centrifugal pan,；The DNA extraction unit
Front is equipped with the first inlet, the second inlet, third inlet, interim liquid storage area and filtering area, and the back side is equipped with the first liquid storage
Area and the second liquid storage area；DNA adsorbent material is equipped in filtering area；First inlet, the second inlet pass through runner and mistake respectively
Filter area's connection；Third inlet is connected to by runner with interim liquid storage area, and interim liquid storage area is connected to by runner with filtering area；The
Two liquid storage areas are connected to by runner with the first liquid storage area, and the first liquid storage area is connected to by runner with filtering area.
2. DNA extraction element as described in claim 1, which is characterized in that the DNA adsorbent material is pellosil.
3. DNA extraction element as described in claim 1, which is characterized in that the front and back that the DNA extracts centrifugal pan covers
Lid film, the film cover interim liquid storage area, filtering area, the first liquid storage area and the second liquid storage area.
4. DNA extraction element as described in claim 1, which is characterized in that the first inlet is for being added blood and red blood cell
Lysate；Second inlet is for being added high salt concentration solion；Third inlet is for being added DNA eluent；Interim storage
Liquid zone cooperates the revolving speed of centrifuge to be arranged and realizes third inlet for temporarily storing the DNA eluent in third inlet
Enter filtering area in the first inlet and the second inlet after interior liquid；Filtering area is used to realize DNA using DNA adsorbent material
Filtering extract；First liquid storage area and the second liquid storage area for realizing DNA collection.
5. DNA extraction element as described in claim 1, which is characterized in that the first inlet, the second inlet are more than filtering area
The center of centrifugal pan is extracted close to DNA；Third inlet, interim liquid storage area are extracted in centrifugal pan than filtering area closer to DNA
The heart；Third inlet extracts the center of centrifugal pan than interim liquid storage area closer to DNA；Filtering area than the first liquid storage area closer to
The center of DNA extraction centrifugal pan；First liquid storage area extracts the center of centrifugal pan than the second liquid storage area closer to DNA.
6. DNA extraction element as described in claim 1, which is characterized in that centrifuge using revolving speed 1 rotate when, first into
Solution in liquid mouth, the second inlet enters in filtering area and mixes；When centrifuge is rotated using revolving speed 2, in filtering area
DNA in solution is adhered on DNA adsorbent material, remaining impurity enters the first liquid storage area, while the third feed liquor under the revolving speed
Eluent in mouthful enters interim liquid storage area；When centrifuge is rotated using revolving speed 3, DNA eluent in interim liquid storage area into
Enter filtering area, and the impurity in the first liquid storage area enters the second liquid storage area；When centrifuge is rotated using revolving speed 4, under elution
DNA enter the first liquid storage area.
7. DNA extraction element as described in claim 1, which is characterized in that the material that the DNA extracts centrifugal pan is plastics.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109694808A (en) * | 2017-10-24 | 2019-04-30 | 苏州含光微纳科技有限公司 | A kind of DNA extraction centrifugal pan |
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