CN110305941A - A method of single sample hereditary information is obtained based on microflow control technique - Google Patents
A method of single sample hereditary information is obtained based on microflow control technique Download PDFInfo
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- CN110305941A CN110305941A CN201910541379.XA CN201910541379A CN110305941A CN 110305941 A CN110305941 A CN 110305941A CN 201910541379 A CN201910541379 A CN 201910541379A CN 110305941 A CN110305941 A CN 110305941A
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- microballoon
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention provides a kind of methods for obtaining single sample hereditary information based on microflow control technique, and oily phase reagent and three kinds of water phase reagents are added in micro-fluidic chip；Oily phase reagent is wrapped up to mixed water phase by flow control instrument, forms water-in-oil microemulsion drop；The micro emulsion drop of acquisition is gone in centrifuge tube, cracking and reverse transcription reaction are carried out；Demulsification oil is added into the microlayer model of reaction, separates the supernatant water phase containing microballoon in another centrifuge tube；Supernatant water phase containing microballoon is wrapped up with PCR reaction system with oily phase reagent, the water-in-oil microemulsion drop containing PCR amplification system is formed；The drop of acquisition is subjected to pcr amplification reaction.The present invention is based on microflow control technique cooperation Water-In-Oils to encapsulate system, realize the single package by a large amount of cells or bacterium or virus, reverse transcription, pcr amplification reaction are successively carried out, individual cells or bacterium or virus etc. respectively unique hereditary information is obtained, ensure that the reservation of effective information and use.
Description
Technical field
The invention belongs to microfluidic arts, and in particular to one kind obtains single sample hereditary information based on microflow control technique
Method.
Background technique
Hereditary information (genetic information) refers to biology to replicate thing identical with oneself, being transmitted by parental generation
Pass to the information of cell when dividing every time to filial generation or each cell by cell, i.e. base-pair puts in order, or refers to nucleotide
Put in order, the ribonucleotide in deoxynucleotide, RNA in DNA puts in order.Traditional acquisition hereditary information
Method uses the experiment carried out using the RNA of acquisition or DNA as template, but existing method can only obtain comprehensive hereditary information,
Individual cells, bacterium or viral exclusive hereditary information can not accurately be obtained, it is possible to cause to flood the hereditary information of needs.
Meanwhile current existing single cell technology flux is low, reagent cost is high.
Summary of the invention
In order to solve the deficiencies in the prior art, the present invention provides one kind to obtain single sample heredity letter based on microflow control technique
The method of breath.
The purpose of the present invention is achieved through the following technical solutions:
A method of single sample hereditary information is obtained based on microflow control technique, is included the following steps:
S1, by oily phase reagent and cracking and reverse transcription liquid, the sample suspensions liquid containing single sample and oligo dT is carried
Three kinds of water phase reagents of microballoon liquid of microballoon are added in micro-fluidic chip；
S2, using oil phase liquid and the incompatible characteristic of aqueous phase liquid, by flow control instrument make oily phase reagent to it is mixed its
He wraps up at three kinds of water phases, is formed containing cracking and reverse transcription system water-in-oil microemulsion drop；
S3, water-in-oil microemulsion drop obtained in S2 is transferred in centrifuge tube, carries out cracking and reverse transcription reaction；
S4, demulsification oil is added into the microlayer model after reaction, separates the supernatant water phase containing microballoon in another centrifuge tube, uses
In progress pcr amplification reaction；
S5, again by S4 containing microballoon supernatant water phase and PCR reaction system by micro-fluidic chip with oily phase reagent
Package forms the water-in-oil microemulsion drop containing PCR amplification system；
S6, the drop obtained in S5 is subjected to pcr amplification reaction.
Preferably, the water-in-oil microemulsion drop in the S2 include micro emulsion drop only containing an oligo dT microballoon,
Micro emulsion drop only containing a sample, the micro emulsion drop containing an oligo dT microballoon and a sample, containing multiple
The micro emulsion drop of oligo dT microballoon or/and multiple samples had both been free of oligo dT microballoon or had been free of the micro emulsion drop of sample.
Preferably, as the 1-100% that number of samples is total number of drops, and the microballoon of oligo dT is total number of drops 10-
When 100%, the drop number in the water-in-oil microemulsion drop simultaneously containing an oligo dT microballoon and a sample is total
The 0.1-100% of number of drops, only the number of drops containing oligo dT microballoon but without containing sample is the 0-99% of total number of drops, together
When the empty drop without containing oligo dT microballoon and sample number of drops be total number of drops 0-89.1%.
Preferably, the sample include but is not limited to be cell, bacterium or virus.
Preferably, the carrying oligo dT microsphere diameter is 1-200 μm.
Preferably, the microsphere surface material include but is not limited to be polystyrene, polymethyl methacrylate, polypropylene
Amide, hydrogel.
Preferably, the oligo dT microballoon is oligo dT (25) microballoon.
The beneficial effects of the present invention are embodied in: the present invention is based on microflow control techniques, cooperate Water-In-Oil to encapsulate system, realize
A large amount of cell, bacterium or virus are subjected to single package, reverse transcription and PCR are carried out respectively, to obtain individual cells, bacterium
Or respectively unique hereditary information, guarantee effective information can retain and use virus.It is (cell, thin that sample is also improved simultaneously
Bacterium or virus) flux, reduce reagent, consumables cost.The present invention carries out the drop of cell cracking, reverse transcription reaction by substep
Wrap up and react, the drop of pcr amplification reaction package and reaction, preferably carried out reagent optimization and mono-/multi- weight primer it is excellent
Change, so that the practical application of this method is more extensive.Have for the research in the directions such as monocell expressing, single-chain antibody preparation positive
Facilitation.
Detailed description of the invention
Fig. 1: the reverse transcription system schematic illustration in the present invention.
Fig. 2: the channel distributed architecture schematic diagram for the micro-fluidic chip that the present invention is applied to.
Fig. 3: the combination principle schematic diagram of each reagent of the invention in the chip.
Fig. 4: QuantaSoftTMFor the analysis schematic diagram of positive number of drops and total number of drops.Fig. 5: QuantaSoftTM
For the analysis schematic diagram of drop signal distributions.
Specific embodiment
It is (thin to single sample present invention discloses a kind of method for obtaining single sample hereditary information based on microflow control technique
Born of the same parents, bacterium or virus) be packaged, crack, reverse transcription and PCR, to obtain target sequence, meet downstream research and development or
The needs of production.It is elaborated below in conjunction with specific embodiment.
Relate generally in method of the invention: Water-In-Oil list sample encapsulates system, sample cracking and reverse transcription system and PCR
Amplification system.
Wherein, Water-In-Oil encapsulates system: using the incompatible characteristic of oil phase liquid and aqueous phase liquid, mutually trying with water phase oily
Surfactant is added in agent, forms water-in-oil microemulsion drop, and optimize to aqueous phase system, it is micro- to further enhance Water-In-Oil
Emulsion droplets system stability.
Single sample reverse transcription encapsulates system: (including but not limited to thin containing the 1-100% that number of samples is total number of drops
Born of the same parents, bacterium or virus) suspension, with the microballoon containing the carrying oligo dT (25) that number is total number of drops 10-100%
Microballoon liquid, and cracking and reverse transcription liquid are mixed into water phase, are wrapped up using oily phase reagent by micro-fluidic chip, guarantee every
Comprising being no more than a sample and a microballoon in a drop.Drop number simultaneously containing a microballoon and a sample is total
The 1-10% of number of drops.Wherein only containing microballoon but without containing sample number of drops be total number of drops 0-99%.Empty drop is (no
Contain microballoon and sample) number of drops be total number of drops 0-89.1%.
PCR encapsulation: PCR system and the microballoon containing cDNA after reverse transcription are mixed into complete PCR system, utilize oily phase
Reagent is wrapped up by micro-fluidic chip, is guaranteed in each drop comprising being no more than a microballoon.Contain a microballoon simultaneously
Drop number be total number of drops 1-10%.The number of drops of empty drop (without containing microballoon and sample) is the 90- of total number of drops
99%.
Water-In-Oil encapsulates system rupture: demulsification oil being added into water-in-oil system, makes the droplet break of encapsulation, water phase and oil
Mutually separate.Product after single sample reverse transcription product or PCR amplification is in water phase.
Cracking and reverse transcription system: the system includes three parts: a. contains the 1-100% sample that number is total number of drops
The suspension of (including but not limited to cell, bacterium or virus)；B. a carrying for being total number of drops 10-100% containing number
The microballoon liquid of oligo dT (25) microballoon.Wherein, oligo dT (25) microballoon can be in the micron order pipeline of micro-fluidic chip
For deformable (deformable), or the diameter of indeformable (non-deformable), microballoon can be at 1-200 μm；
C. cracking and reverse transcription liquid.
By taking 10000 drops as an example:
A. sample suspensions liquid is to separate single sample (including but not limited to cell, bacterium or virus), using containing
The PBS buffer solution or HEPES buffer solution of Optiprep is quantification of to contain 100-10000 sample by sample suspensions.
B. the microsphere surface material in microballoon liquid can be polystyrene (centre can be superparamagnetic material) or polymethyl
Sour methyl esters or polyacrylamide or other high polymers, and it is connected with Oligo dT (25).Using contain 5-150mM Tris-HCl
Buffer or PBS buffer solution or HEPES buffer solution, 10-175mM KCl solution, 2-120mM MgCl2Solution, 0.5-5M
Betaine solution, 0.01-0.5mM EDTA, 0.1-5% lytic reagent, the stable reagent of 0.5-15% and ultrapure water it is mixed
Liquid suspended microspheres are closed, it is made to contain the microballoon liquid of 1000-10000 carrying oligo dT (25) microballoon.
C. cracking and reverse transcription liquid provide cracking and reverse transcription ingredient.Wherein comprising 5-500mM Tris-HCl buffer or
PBS buffer solution or HEPES buffer solution, 10-500mM KCl solution, 4-240mM MgCl2Solution, 0.5-5M Betaine solution,
0.5-5mM dNTP solution, 0.5-10mM DTT, 0.1-5% lytic reagent, the stable reagent of 5-50%, the suppression of 5-100U RNA enzyme
The mixed liquor of preparation, 5-100U reverse transcriptase and ultrapure water
D.PCR amplification system: the system includes PCR amplification liquid and the microballoon for reversing the carrying cDNA recorded.PCR amplification liquid
Including 5-500mM Tris-HCl buffer or PBS buffer solution or HEPES buffer solution, 10-500mM KCl solution, 5-500mM
(NH4)2SO4Solution, 0.2-120mM MgCl2Solution, 2-20mM dNTP solution, the stable reagent of 5-50%, 0.1-3U high are protected
True amplification enzyme, substance or multi-primers and ultrapure water.
Embodiment 1: unicellular reverse transcription and the experiment of PCR are carried out by micro-fluidic chip
The present embodiment designs with the method mentioned in the invention and carries out unicellular reverse transcription and the experiment of PCR.
Firstly, corresponding reagent is added according to table 1 using the micro-fluidic chip of Suzhou Rui Xun Biotechnology Co., Ltd:
Table 1: the reagent type and volume being added in micro-fluidic chip
| Channel | Reagent | Volume |
| #1 | Oily phase reagent | 150μl |
| #2 | Cell cracking and reverse transcription liquid | 30μl |
| #3 | HEK 293F cell suspending liquid | 30μl |
| #4 | Oligo dT polyacrylamide microsphere liquid | 45μl |
Channel distributed architecture such as Fig. 2 institute of the micro-fluidic chip of above-described Suzhou Rui Xun Biotechnology Co., Ltd
Show.
Apply different pressures respectively to #1, #2, #3 and #4 using the biological flow control instrument of the sharp news in Suzhou according to table 2, so that #2, #3
And wrapped up after the water phase reagent mixing in the channel #4 by the oily phase reagent in the channel #1, about 63 μm of diameter of drop is formed, and be expelled to #
In 5 channels.
Table 2: flow control instrument pressure sets table
| Channel | Reagent | Pressure |
| #1 | Oily phase reagent | 2.2Psi |
| #2 | Cell cracking and reverse transcription liquid | 0.6Psi |
| #3 | Cell suspending liquid | 0.66Psi |
| #4 | Oligo dT polyacrylamide microsphere liquid | 1.0Psi |
Combination signal between the above specific reagent each in the chip is as shown in Figure 3, wherein polyacrylamide microsphere is in miniflow
Deformation and water phase, oily phase reagent, sample are formed together drop in keyholed back plate road.The drop eventually formed is 1) empty drop 2) it contains only
Have a microballoon 3) containing a microballoon and sample 4) contain multiple microballoons or sample (not shown).
Then, it by all agent transfers in the channel #5 into 0.2ml centrifuge tube, is cracked according to 3 thermal cycle conditions of table
With reverse transcription.
Table 3: reverse transcription thermal cycle conditions
| Temperature | Time |
| 50℃ | 120 minutes |
| 70℃ | 15 minutes |
| 4℃ | ∞ |
Then, demulsification oil is added into the droplet after reaction, separates supernatant water phase (including microballoon) in new centrifuge tube.
Using 200 μ L Low-TE buffer solution for cleaning microballoons, using the liquid of cleaning as cleaning solution, save as negative template.It uses again
200 μ L Low-TE buffer suspended microspheres, as positive template.Using the microballoon after cleaning solution and cleaning respectively as template,
PCR reaction system is configured according to table 4, wherein primer, the probe according to the design of GAPDH cDNA sequence are shown in Table 5.
Table 4:PCR system configurations
Table 5: amplimer, probe design
As similarly corresponding reagent is added into the micro-fluidic chip of Suzhou Rui Xun Biotechnology Co., Ltd in table 6.And it presses
Apply different pressures respectively to #1, #2, #3 and #4 using flow control instrument according to table 7, so that the reagent in the channel #4 is by the channel #1, #2, #3
Oily phase reagent package, form the drop of diameter about 63um, and be expelled in the channel #5.
Table 6: the reagent list being added in micro-fluidic chip
| Channel | Reagent | Volume |
| #1 | Oily phase reagent | 150μl |
| #2 | Oily phase reagent | 150μl |
| #3 | Oily phase reagent | 150μl |
| #4 | PCR reaction system (containing the microballoon after cleaning solution or cleaning) | 40μl |
Table 7: flow control instrument pressure setting
| Channel | Reagent | Volume |
| #1 | Oily phase reagent | 2.0Psi |
| #2 | Oily phase reagent | 0.9Psi |
| #3 | Oily phase reagent | 0.9Psi |
| #4 | PCR reaction system | 0.7Psi |
Then all agent transfers in the channel #5 are cracked into 0.2ml PCR plate according to the thermal cycle conditions of table 8
With reverse transcription.
Table 8:PCR thermal cycle conditions
Bio Rad QX 200 is used after reactionTMReader reads drop signal, and uses QuantaSoftTMData analysis is soft
Part analyzes data, and in conjunction with shown in Fig. 4, Fig. 5, cleaning solution amounts to 13930 drops, all feminine gender drops as template；Cleaning
Microballoon afterwards amounts to 13209 drops as template, and it is 70 that wherein FAM positive number of drops, which is 28, HEX positive number of drops, remaining
For negative drop.
Test sample in the present embodiment is the cell of HEK293F cell line.Cleaning solution is as template, and experimental result is in double
It is negative, it was demonstrated that there is no cDNA residual in cleaning solution；Microballoon after cleaning is as template, and experimental result occurs positive, it was demonstrated that cDNA
It is present on microballoon.The result and experiment are expected consistent.Principle according to the invention, mRNA information all reverse transcriptions of individual cells
On the same microballoon, single microballoon can be dispensed by PCR system, to obtain the expressing information of mRNA in individual cells.
Method in the present invention can guarantee that effective information can be retained and use, at the same compared to used at present 96 or
The operation of 384 orifice plate of person can handle the cell of thousands of to tens of thousands of quantity in a few minutes, largely improve sample (packet
Include but be not limited to cell, bacterium or virus) flux；And it is anti-in single cell technology 96 orifice plates or 384 orifice plates that use at present
Answering volume is microlitre rank, and the method provided according to the present invention, reaction volume can be reduced to a nanoliter rank, individual cells section
The amount of reagent of 3 orders of magnitude has been saved, reagent, consumables cost are reduced.The present invention carries out sample cracking, reverse transcription by substep
The drop of reaction wraps up and reacts, the drop of pcr amplification reaction package and reaction, can easier progress reagent optimization and
The optimization of mono-/multi- weight primer, is suitble to wider array of practical application.Research for directions such as monocell expressing, single-chain antibody preparations
With positive facilitation.
Certainly still there are many specific embodiments by the present invention, are just not listed one by one herein.It is all using equivalent replacement or
Equivalent transformation and all technical solutions formed, all fall within the scope of protection of present invention.
Claims (7)
1. a kind of method for obtaining single sample hereditary information based on microflow control technique, characterized by the following steps:
S1, by oily phase reagent and cracking and reverse transcription liquid, the sample suspensions liquid containing single sample and oligo dT microballoon is carried
Three kinds of water phase reagents of microballoon liquid be added in micro-fluidic chip；
S2, using oil phase liquid and the incompatible characteristic of aqueous phase liquid, by flow control instrument make oily phase reagent to it is mixed other three
Kind water phase is wrapped up, and is formed containing cracking and reverse transcription system water-in-oil microemulsion drop；
S3, water-in-oil microemulsion drop obtained in S2 is transferred in centrifuge tube, carries out cracking and reverse transcription reaction；
S4, into the microlayer model after reaction be added demulsification oil, separate the supernatant water phase containing microballoon in another centrifuge tube, for into
Row pcr amplification reaction；
S5, again by S4 containing microballoon supernatant water phase and PCR reaction system wrapped up with oily phase reagent by micro-fluidic chip,
Form the water-in-oil microemulsion drop containing PCR amplification system；
S6, the drop obtained in S5 is subjected to pcr amplification reaction.
2. a kind of method for obtaining single sample hereditary information based on microflow control technique as described in claim 1, it is characterised in that:
Water-in-oil microemulsion drop in the S2 includes micro emulsion drop only containing an oligo dT microballoon, only containing a sample
Micro emulsion drop, contains multiple oligo dT microballoons or/and more at the micro emulsion drop containing an oligo dT microballoon and a sample
The micro emulsion drop of a sample had both been free of oligo dT microballoon or had been free of the micro emulsion drop of sample.
3. a kind of method for obtaining single sample hereditary information based on microflow control technique as claimed in claim 2, it is characterised in that:
When number of samples is the 1-100% of total number of drops, and the microballoon of oligo dT is total number of drops 10-100%, the Water-In-Oil
Drop number in micro emulsion drop simultaneously containing an oligo dT microballoon and a sample is the 0.1-100% of total number of drops, only
Number of drops containing oligo dT microballoon but without containing sample is the 0-99% of total number of drops, while not containing oligo dT microballoon
Number of drops with the empty drop of sample is the 0-89.1% of total number of drops.
4. a kind of method for obtaining single sample hereditary information based on microflow control technique as claimed in claim 2, it is characterised in that:
The sample include but is not limited to be cell, bacterium or virus.
5. a kind of method for obtaining single sample hereditary information based on microflow control technique as described in claim 1, it is characterised in that:
The carrying oligo dT microsphere diameter is 1-200 μm.
6. a kind of method for obtaining single sample hereditary information based on microflow control technique as claimed in claim 5, it is characterised in that:
The microsphere surface material include but is not limited to be polystyrene, polymethyl methacrylate, polyacrylamide, hydrogel.
7. a kind of method for obtaining single sample hereditary information based on microflow control technique as described in claim 1, it is characterised in that:
The oligo dT microballoon is oligo dT(25) microballoon.
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