CN206927902U - A kind of DNA thickening filtrations device - Google Patents
A kind of DNA thickening filtrations device Download PDFInfo
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- CN206927902U CN206927902U CN201720797874.3U CN201720797874U CN206927902U CN 206927902 U CN206927902 U CN 206927902U CN 201720797874 U CN201720797874 U CN 201720797874U CN 206927902 U CN206927902 U CN 206927902U
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- dna
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- filtrations device
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Abstract
The utility model discloses a kind of DNA thickening filtrations device.DNA thickening filtrations device provided by the utility model includes filter and the waste liquid plate being placed under filter；Some through holes are provided with filter；Filter membrane is provided with through hole；Through-hole side wall and filtering film edge closely connect.CtDNA can be separated from the dissociative DNA of blood plasma using DNA thickening filtrations device provided by the utility model, the ctDNA of separation concentration and purity is satisfied by high-flux sequence requirement for construction data base, and the ctDNA once extracted can be applied all to downstream experiment and analysis, effectively improve detection sensitivity, blood plasma dosage is reduced, reduces patient's pain and blood loss.DNA thickening filtrations device provided by the utility model has important application value, suitable for large-scale popularization application.
Description
Technical field
It the utility model is related to a kind of DNA thickening filtrations device.
Background technology
Circulating tumor DNA (Circulating tumour DNA, ctDNA) is mainly institute after dead tumour cell rupture
Genomic DNA discharge, fragmentation.At present, ctDNA can be as the mark of tumor prognosis, and can use
To predict the effect of drug therapy, this is also that ctDNA applies very important one side as tumor monitoring.
On the tumor development stage, ctDNA contents are generally higher late or in metastatic tumo(u)r, and in early stage or limitation
Content is relatively low in tumour.In different type tumour, there is also difference for ctDNA levels.Because ctDNA content is low, only accounts for and follow
The ratio of ring Cell-free DNA (Cell-Free DNA, cfDNA) very little, about several a ten thousandths to one thousandth, and blood
In dissociative DNA it is seldom, every about in the blood plasma of milliliter only have more than ten nanograms (ng) dissociative DNA.Therefore, in order to improve
CtDNA recall rate and sensitivity, DNA extraction efficiencies need to be improved and carry out thickening filtration.
The plasma free nucleic acid extraction kit of main flow is divided into two classes on the market at present：Cross embrane method and paramagnetic particle method, but no matter
It is excessive which type of plasma free nucleic acid extraction kit all deposits volume of dissolution after extraction, can not be completely used for downstream library
The problem of structure, although the problem can be solved by increasing the system of library construction, cost is higher.
Utility model content
The purpose of this utility model is to provide a kind of DNA thickening filtrations device.
DNA thickening filtrations device provided by the utility model, including filter 1 and the waste liquid plate 2 that is placed under filter；
Some through holes 3 are provided with filter；Filter membrane 4 is provided with through hole；Through-hole side wall and filtering film edge closely connect.
The DNA thickening filtrations device is the device separated according to the clip size of target dna.According to target dna
Clip size determine DNA thickening filtration devices in filter membrane membrane aperture.The target dna concretely ctDNA.Work as target
When DNA is ctDNA, the membrane aperture of filter membrane can be 3K in DNA thickening filtration devices.
The filter and the waste liquid plate are integrally formed.
The waste liquid plate is removably placed under the filter.
The filter and the waste liquid plate can be fixed with fixed component 6.
Some through holes on the filter are equally distributed.It may be provided with the waste liquid plate corresponding with the through hole
Some waste liquid chambers 5.
The through hole can be cylindric.
The aperture of the through hole can be 7mm-9mm.The aperture of the through hole concretely 8mm.Each through hole leads to adjacent
The Edge Distance in hole can be 1mm-3mm.Each through hole and the Edge Distance of adjacent through-holes concretely 2mm.
The vertical range of horizontal plane of the filter membrane away from through hole lower edge can be 0mm-0.1mm.The filter membrane is away from through hole
The vertical range of the horizontal plane of lower edge concretely 0mm.
The vertical range of horizontal plane of the filter away from waste liquid plate upper limb can be 2mm-4mm.The filter is away from waste liquid
The vertical range of the horizontal plane of plate upper limb concretely 2mm.
Some waste liquid chambers 5 corresponding with the through hole are may be provided with the waste liquid plate.
The waste liquid chamber can be cylindrical cavity.
The diameter of the waste liquid chamber can be 7mm-9mm, highly can be 5mm-30mm.The diameter of the waste liquid chamber is concretely
9mm, highly can be 20mm.
The Edge Distance of each waste liquid chamber and adjacent waste liquid chamber can be 1mm-3mm.Each waste liquid chamber and adjacent waste liquid chamber
Edge Distance concretely 1mm.
The vertical range of horizontal plane of the filter membrane away from waste liquid chamber lower edge can be 22mm-24mm.The filter membrane is away from useless
The vertical range of the horizontal plane of sap cavity lower edge concretely 22mm.
The utility model provides a kind of DNA thickening filtrations device.Filled using DNA thickening filtrations provided by the utility model
CtDNA can be separated from the dissociative DNA of blood plasma by putting, and the ctDNA of separation concentration and purity are satisfied by high-flux sequence and build storehouse
It is required that and the ctDNA once extracted can be applied all to downstream experiment and analysis, effectively improve detection sensitivity, reduce
Blood plasma dosage, reduce patient's pain and blood loss.DNA thickening filtrations device provided by the utility model has important application valency
Value.
Brief description of the drawings
Fig. 1 is the longitudinal cutting structure schematic diagram of DNA thickening filtration devices.
Fig. 2 is the top view of DNA thickening filtration devices.
Fig. 3 is the yield of dissociative DNA after the dissolving of different volume of dissolution.
Fig. 4 is dissociative DNA yield after different dissolving number dissolvings.
Embodiment
Following embodiment facilitates a better understanding of the utility model, but does not limit the utility model.Following embodiments
In experimental method, be conventional method unless otherwise specified.Test material used in following embodiments, such as without special theory
It is bright, it is to be commercially available from routine biochemistry reagent shop.Quantitative test in following examples, is respectively provided with and repeats reality three times
Test, results averaged.
The preparation of embodiment 1, DNA thickening filtration devices
Prepare DNA thickening filtration devices.DNA thickening filtrations device is made up of filter, waste liquid plate and fixed component.Waste liquid
It is equally distributed that 96 waste liquid chambers and 96 waste liquid chambers are provided with plate, and waste liquid chamber is cylindrical cavity, each waste liquid chamber
A diameter of 9mm, highly it is 20mm, the Edge Distance of each waste liquid chamber and adjacent waste liquid chamber is 1mm.It is provided with and gives up on filter
96 through holes corresponding to sap cavity, the aperture of each through hole are 8mm, and the Edge Distance of each through hole and adjacent through-holes is 2mm；Each
The filter membrane that membrane aperture is 3K is provided with through hole, through-hole side wall and filtering film edge closely connect, and filter membrane is away under waste liquid chamber
The vertical range of the horizontal plane of edge is 22mm.Filter is connected with waste liquid plate by fixed component.The material of fixed component is gold
Belong to (such as iron, copper).
Embodiment 2, using DNA thickening filtrations device separate ctDNA
Plasma free nucleic acid extraction kit is the product of Qiagen companies.Elution Buffer are plasma free nucleic acid
Component in extracts kit.
First, the extraction of dissociative DNA and its condition optimizing in human plasma
1st, volume of dissolution
(1) 2mL human plasmas are taken, extracting dissociative DNA according to the operating procedure of plasma free nucleic acid extraction kit, (extraction is swum
The step of from DNA, is carried out to dissociative DNA absorption on film).
(2) after completing step (1), to the film for adsorbed dissociative DNA on add 20 μ L, 30 μ L, 60 μ L or 90 μ L and be preheated to
56 DEG C of Elution Buffer, are stored at room temperature 3min, and then 16000rpm centrifuges 1min, collect liquid and detect dissociative DNA
Yield, further count organic efficiency.
Organic efficiency=addition N μ L Elution Buffer dissociative DNA yield (ng)/μ L of addition 90
ElutionBuffer dissociative DNA yield (ng) × 100%；N is 20,30,60 or 90.
The yield of dissociative DNA is shown in Fig. 3 after different volume of dissolution dissolvings.As a result show, when volume of dissolution is respectively 20 μ L, 30
When μ L, 60 μ L and 90 μ L, organic efficiency is followed successively by 50%, 70%, 105% and 100%.Therefore, volume of dissolution is 60 μ L
Improve organic efficiency.
2nd, number is dissolved
(1) with (1) in step 1.
(2) after completing step (1), to the film for adsorbed dissociative DNA on add 30 μ L or 60 μ L are preheated to 56 DEG C
Elution Buffer, 3min is stored at room temperature, then 16000rpm centrifuges 1min, collects liquid 1 and detects in liquid 1 and dissociates
DNA output.
(3) film of step (2) is taken into, liquid 1 is added, is stored at room temperature 3min, then 16000rpm centrifuges 1min, collects
Liquid 2 simultaneously detects DNA output of dissociating in liquid 2.
Dissociative DNA yield is shown in that (dissolving is once the result of step (2) to Fig. 4, and dissolving is secondary to be after difference dissolving number dissolving
The result of step (3)).As a result show, compared with dissolving once, dissolve secondary dissociative DNA output increased about 10%.
The above results show that the optimal dissolution condition that dissociative DNA extracts in human plasma is：To the film for having adsorbed dissociative DNA
60 μ L of upper addition are preheated to 56 DEG C of Elution Buffer, are stored at room temperature 3min, and then 16000rpm centrifuges 1min, collection liquid
Body；Then to the liquid for adding collection on the film of the adsorption of DNA again, 3min is stored at room temperature, then 16000rpm is centrifuged
1min, the liquid of collection are the dissociative DNA extracted.
2nd, ctDNA is separated using DNA thickening filtrations device
The step of separating ctDNA using DNA thickening filtrations device is as follows successively：
1st, take filter, by this without using hole position use ParafilmTM；Take waste liquid plate, by filter without using
The corresponding waste liquid chamber of hole position seal.
2nd, after completing step 1, take in step 12 liquid 2 of (3), add filter hole position (need adherent addition, should not
Touch filter membrane).
3rd, after completing step 2, the filter and waste liquid plate are fitted together (it is required that using hole position using fixed component
With waste liquid chamber correspond), then 4 DEG C, 3000g centrifugation 60min (i.e. centrifugation to use hole position in filter noresidue liquid be
Only, criterion is：As no liquid is reflective on filter membrane, then filter noresidue liquid；As there is liquid reflective on filter membrane, then
Centrifugation should be continued untill filter noresidue liquid).
4th, after completing step 3, filter is taken, adds 17 μ L ddH2O, it is stored at room temperature 5min.
5th, after completing step 4,15 μ L, the ctDNA as separated from dissociative DNA are suctioned out using pumping filter is concentrated in vacuo.
By ctDNA-80 DEG C of preservation of separation.
The above results show that the DNA thickening filtrations device prepared using embodiment 1 separates ctDNA.
The membrane aperture of filter membrane in DNA thickening filtration devices is determined according to the DNA sizes of separation.It is for example, above-mentioned
The ctDNA separated in embodiment clip size is 150-170bp, so the membrane aperture of selection filter membrane is 3K.This practicality is new
The inventor of type uses the filtering UF membrane ctDNA (table 1) of other membrane apertures, and organic efficiency is not as good as the filtering that membrane aperture is 3K
Film.
Table 1
| The membrane aperture of filter membrane | Centrifugal force | Centrifugation time | Organic efficiency |
| 3K | 3000g | 60min | 95% |
| 10K | 3000g | 10min | 50% |
| 30K | 3000g | 3min | 20% |
| 100K | 3000g | 1min | 0% |
Claims (10)
- A kind of 1. DNA thickening filtrations device, it is characterised in that：DNA thickening filtrations device includes filter (1) and is placed on filtering Waste liquid plate (2) under plate；Some through holes (3) are provided with filter；Filter membrane (4) is provided with through hole；Through-hole side wall and mistake Filter membrane edge closely connects.
- 2. DNA thickening filtrations device as claimed in claim 1, it is characterised in that：The filter and the waste liquid plate are one Shaping.
- 3. DNA thickening filtrations device as claimed in claim 1, it is characterised in that：The waste liquid plate is removably placed on described Under filter.
- 4. DNA thickening filtrations device as claimed in claim 3, it is characterised in that：The filter and the waste liquid plate are fixed Part (6) is fixed.
- 5. the DNA thickening filtrations device as described in Claims 1-4 is any, it is characterised in that：Some through holes on the filter It is equally distributed.
- 6. DNA thickening filtrations device as claimed in claim 5, it is characterised in that：The through hole is cylindric.
- 7. the DNA thickening filtrations device as described in Claims 1-4 is any, it is characterised in that：It is provided with the waste liquid plate and institute State some waste liquid chambers (5) corresponding to through hole.
- 8. DNA thickening filtrations device as claimed in claim 5, it is characterised in that：It is provided with the waste liquid plate and the through hole Corresponding some waste liquid chambers (5).
- 9. DNA thickening filtrations device as claimed in claim 8, it is characterised in that：The waste liquid chamber is cylindrical cavity.
- 10. DNA thickening filtrations device as claimed in claim 1, it is characterised in that：The filter membrane is away from the waste liquid chamber lower edge The vertical range of horizontal plane is 22mm-24mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720797874.3U CN206927902U (en) | 2017-07-04 | 2017-07-04 | A kind of DNA thickening filtrations device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720797874.3U CN206927902U (en) | 2017-07-04 | 2017-07-04 | A kind of DNA thickening filtrations device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN206927902U true CN206927902U (en) | 2018-01-26 |
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ID=61353117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201720797874.3U Active CN206927902U (en) | 2017-07-04 | 2017-07-04 | A kind of DNA thickening filtrations device |
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| Country | Link |
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| CN (1) | CN206927902U (en) |
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2017
- 2017-07-04 CN CN201720797874.3U patent/CN206927902U/en active Active
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