CN104928368A - Microdroplet preparation method suitable for microdroplet digital PCR by using silicone oil - Google Patents
Microdroplet preparation method suitable for microdroplet digital PCR by using silicone oil Download PDFInfo
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a microdroplet preparation method suitable for microdroplet digital PCR by using silicone oil. The preparation method comprises the following steps: preparing a PCR water phase, uniformly mixing an upstream primer, a downstream primer, a PCR template and a PCR mix in sequence, and dropping double distilled water to package a solution system into three pipes, wherein the volumes of all the pipes are the same, and the pipes are used as reaction water phases for later use; preparing a PCR oil phase, and taking dimethyl siloxane as reaction oil phase for later use; adding the water phase PCR into the oil phase, and performing vortex oscillation to fully emulsify the oil phase and the water phase to obtain water-in-oil droplets. According to the preparation method provided by the invention, the water-in-oil droplets with uniform particles and stable thermodynamic property can be conveniently prepared, and the operation of a microdroplet digital PCR experiment is simplified and facilitated.
Description
Technical field
The present invention relates to molecular diagnosis field, be mainly used in digital droplet PCR droplet preparation or other PCR in.
Background technology
Nano microsphere particle diameter prepared by traditional reverse microemulsion process is generally at 10-100 ran, and not only granularity is little, and hollow.More it is applied to and carries out chemically modified on its surface.The invention provides a kind of foundation of novel method, when the basis of the reverse micro emulsion method improved enters oil by water, the immiscible character of profit forms monodispersed water-in-oil droplet one by one, particle diameter is greatly between 10-100 μm, due to interior aqueous solution, can as micro-container of many biochemical reactions.
Traditional PCR method is carried out in the PCR aqueous solution, between template, can produce homologous base complementary and cause the amplification of the non-specific band of PCR, often add false-positive generation between primer.The present invention is on basis prepared by droplet, and template when being reacted by PCR, primer, the mode that Taq enzyme and PCR mix enter oil by water enters the droplet being formed and comprise PCR reaction product, and performing PCR of going forward side by side reacts.Because involved PCR reaction product is reacted in single independently miostagmin reaction device, reduce the generation of nonspecific reaction.
Summary of the invention
The object of this invention is to provide water-in-oil droplet and preparation method thereof in a kind of PCR experiment, be mainly used in the parcel of PCR reaction solution in current digital droplet PCR experiment.By the proportioning of different aqueous phases, oil-phase component, different operational conditions, prepares easily and a kind ofly has the stable water-in-oil reaction droplet of uniform particles, thermomechanical property, the easy operation of PCR experiment, and the accuracy of experimental result.The present invention directly uses silicone oil, and comparatively simple in preparation technology, formulation operations is very convenient.Be different from and make reagent with promoting agent, the cost of silicone oil is low, then more stable in result; Be different from simultaneously and make reagent with promoting agent, silicone oil not easily layering is also more stable.
The object of the invention is to be achieved through the following technical solutions, a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil, comprises the steps:
(1). preparation PCR aqueous phase, PCR aqueous phase comprises upstream primer, downstream primer, pcr template, PCRmix and distilled water; By above-mentioned medicine with this Homogeneous phase mixing, for subsequent use as reaction aqueous phase, it is (0.8-1) that the mixing ratio of above-mentioned formation is closed: (0.8-1): (1-2): (20-25): (19-23); By upstream primer, downstream primer, pcr template, PCRmix successively Homogeneous phase mixing, make solution system packing 3 pipe after dripping distilled water, often the solution system volume of pipe is identical and for subsequent use as reaction aqueous phase;
(2). preparation PCR oil phase, get dimethyl siloxane for subsequent use as reaction oil phase;
(3). get 1 volume PCR oil phase in centrifuge tube, drip 1 volume PCR aqueous phase with liquid-transfering gun, vortex shakes, and obtains water-in-oil droplet;
The particle diameter of described water-in-oil droplet is 10-100 micron.
Dimethyl silica 38 DEG C (100 in described oil phase.F) under, its viscosity versus temperature coefficient is 0.6.
Described aqueous phase dropwise adds oil phase, vortex concussion afterwards.
The volume ratio of described aqueous phase and oil phase is 1:1.
The described vortex concussion time is 4-6 minute.
The concussion of present method mesoscale eddies is conducive to generating even, the stable droplet of size.
Accompanying drawing explanation
Fig. 1 is that the droplet utilizing present method to carry out testing generates result figure mono-.
Fig. 2 is that the droplet utilizing present method to carry out testing generates result figure bis-.
Fig. 3 is the PCR electrophoretic band result figure tri-utilizing present method to carry out testing.
Concrete enforcement
The preparation of PCR oil phase: get 75 μ L dimethyl siloxanes, 38 DEG C (100.F) under, its viscosity versus temperature coefficient is 0.6.
The preparation of PCR aqueous phase: add the upstream primer of 1.5 μ L 10 μm and downstream primer and PCRmix 37.5 μ L in super clean bench in centrifuge tube, then centrifuge tube is transferred to pcr template and add the template that district adds 3 μ L, 31.5 μ L distilled water to 75 μ L are dripped in super clean bench, abundant mixing, packing 3 pipe, often pipe 25 μ L, for subsequent use as reaction aqueous phase.
The preparation of droplet: get 25 μ LPCR oil phases in centrifuge tube, drip 25 μ LPCR aqueous phases with liquid-transfering gun, fully mixes and vortex shakes 5 minutes, makes profit reaction system fully emulsified.
PCR reacts: setting PCR reaction conditions: 95 DEG C of denaturations 7 minutes, 1 circulation, 95 DEG C of sex change 30 seconds, 62 DEG C of renaturation 20 seconds, and 72 DEG C extend 1 minute, wherein sex change, renaturation and extend totally 40 circulations, 72 DEG C of extensions, 1 circulation in 5 minutes.
Take out reacted PCR solution, if there is the faint demixing phenomenon then vortex concussion several seconds.Experimental result as shown in Figure 3.
Contain PC template 600ng in 75 μ L pcr template solution in present method, upstream and downstream primer concentration is 1.5 μ L/10 μm, is conducive to experiment and carries out smoothly.
Claims (7)
1. utilize the droplet preparation method being applicable to droplet digital pcr of silicone oil, it is characterized in that: comprise the steps,
(1). preparation PCR aqueous phase, PCR aqueous phase comprises upstream primer, downstream primer, pcr template, PCRmix and distilled water; By above-mentioned medicine with this Homogeneous phase mixing, for subsequent use as reaction aqueous phase, it is 0.8-1:0.8-1:1-2:20-25:19-23 that the mixing ratio of above-mentioned formation is closed; By upstream primer, downstream primer, pcr template, PCRmix successively Homogeneous phase mixing, make solution system packing 3 pipe after dripping distilled water, often the solution system volume of pipe is identical and for subsequent use as reaction aqueous phase;
(2). preparation PCR oil phase, get dimethyl siloxane for subsequent use as reaction oil phase;
(3). get 1 volume PCR oil phase in centrifuge tube, drip 1 volume PCR aqueous phase with liquid-transfering gun, vortex shakes, and obtains water-in-oil droplet.
2. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: the particle diameter of described water-in-oil droplet is 10-100 micron.
3. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: in described oil phase, at dimethyl silica 38 DEG C, its viscosity versus temperature coefficient is 0.6.
4. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: described aqueous phase dropwise adds oil phase, vortex concussion afterwards.
5. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: the volume ratio of described aqueous phase and oil phase is 1:1.
6. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: the described vortex concussion time is 4-6 minute.
7. a kind of droplet preparation method being applicable to droplet digital pcr utilizing silicone oil according to claim 1, is characterized in that: comprise the steps,
The preparation of PCR oil phase: get 75 μ L dimethyl siloxanes, at 38 DEG C, its viscosity versus temperature coefficient is 0.6;
The preparation of PCR aqueous phase: add the upstream primer of 1.5 μ L 10 μm and downstream primer and PCRmix 37.5 μ L in super clean bench in centrifuge tube, then centrifuge tube is transferred to pcr template and add the template that district adds 3 μ L, 31.5 μ L distilled water to 75 μ L are dripped in super clean bench, abundant mixing, packing 3 pipe, often pipe 25 μ L, for subsequent use as reaction aqueous phase;
The preparation of droplet: get 25 μ LPCR oil phases in centrifuge tube, drip 25 μ LPCR aqueous phases with liquid-transfering gun, fully mixes and vortex shakes 5 minutes, makes profit reaction system fully emulsified;
PCR reacts: setting PCR reaction conditions: 95 DEG C of denaturations 7 minutes, 1 circulation, 95 DEG C of sex change 30 seconds, 62 DEG C of renaturation 20 seconds, and 72 DEG C extend 1 minute, wherein sex change, renaturation and extend totally 40 circulations, 72 DEG C of extensions, 1 circulation in 5 minutes;
Containing PC template 600ng in 75 μ L pcr template solution, upstream and downstream primer concentration is 1.5 μ L/10 μm, is conducive to experiment and carries out smoothly.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103764681A (en) * | 2011-08-23 | 2014-04-30 | 罗切格利卡特公司 | Bispecific antigen binding molecules |
| CN105255874A (en) * | 2015-11-09 | 2016-01-20 | 北京出入境检验检疫局检验检疫技术中心 | Kit for accurately and quantitatively detecting transgenic maize line T25 and detection method thereof |
| CN105331713A (en) * | 2015-11-10 | 2016-02-17 | 中国农业科学院植物保护研究所 | Method for detecting TCK (trichloroK) winter spores in soil by microdroplet digital PCR (ddPCR) |
| CN106635777A (en) * | 2016-11-15 | 2017-05-10 | 杭州凯基科技有限公司 | Gene micro-drop granule chip making device and gene micro-drop granule chip making method |
| CN109457052A (en) * | 2018-12-18 | 2019-03-12 | 苏州德思普生物科技有限公司 | Detect primer, probe, kit and the detection method of human immunodeficiency virus nucleic acid |
| CN110042151A (en) * | 2019-05-16 | 2019-07-23 | 北京达微生物科技有限公司 | A kind of oil phase composition being used to prepare digital pcr droplet and its application |
Citations (3)
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| EP0905516A1 (en) * | 1997-07-31 | 1999-03-31 | Sumitomo Pharmaceuticals Company, Limited | Circulation thin layer liquid phase assay |
| CN101034061A (en) * | 2006-03-09 | 2007-09-12 | 陕西西大北美基因股份有限公司 | Method for detecting mononucleotide polymorphism with biochip |
| CN101275164A (en) * | 2007-03-30 | 2008-10-01 | 株式会社日立制作所 | Method and apparatus for sample preparation |
-
2015
- 2015-05-24 CN CN201510267908.3A patent/CN104928368A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0905516A1 (en) * | 1997-07-31 | 1999-03-31 | Sumitomo Pharmaceuticals Company, Limited | Circulation thin layer liquid phase assay |
| CN101034061A (en) * | 2006-03-09 | 2007-09-12 | 陕西西大北美基因股份有限公司 | Method for detecting mononucleotide polymorphism with biochip |
| CN101275164A (en) * | 2007-03-30 | 2008-10-01 | 株式会社日立制作所 | Method and apparatus for sample preparation |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103764681A (en) * | 2011-08-23 | 2014-04-30 | 罗切格利卡特公司 | Bispecific antigen binding molecules |
| CN105255874A (en) * | 2015-11-09 | 2016-01-20 | 北京出入境检验检疫局检验检疫技术中心 | Kit for accurately and quantitatively detecting transgenic maize line T25 and detection method thereof |
| CN105331713A (en) * | 2015-11-10 | 2016-02-17 | 中国农业科学院植物保护研究所 | Method for detecting TCK (trichloroK) winter spores in soil by microdroplet digital PCR (ddPCR) |
| CN106635777A (en) * | 2016-11-15 | 2017-05-10 | 杭州凯基科技有限公司 | Gene micro-drop granule chip making device and gene micro-drop granule chip making method |
| CN106635777B (en) * | 2016-11-15 | 2019-05-21 | 杭州凯基科技有限公司 | Gene droplet particle chip manufacturing device and method |
| CN109457052A (en) * | 2018-12-18 | 2019-03-12 | 苏州德思普生物科技有限公司 | Detect primer, probe, kit and the detection method of human immunodeficiency virus nucleic acid |
| CN110042151A (en) * | 2019-05-16 | 2019-07-23 | 北京达微生物科技有限公司 | A kind of oil phase composition being used to prepare digital pcr droplet and its application |
| CN110042151B (en) * | 2019-05-16 | 2021-01-26 | 北京达微生物科技有限公司 | Oil phase composition for preparing digital PCR microdroplets and application thereof |
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