CN104946625B - A kind of preparation method of sequencing template based on emulsion-based PCR - Google Patents
A kind of preparation method of sequencing template based on emulsion-based PCR Download PDFInfo
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- CN104946625B CN104946625B CN201510300255.4A CN201510300255A CN104946625B CN 104946625 B CN104946625 B CN 104946625B CN 201510300255 A CN201510300255 A CN 201510300255A CN 104946625 B CN104946625 B CN 104946625B
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Abstract
The invention belongs to biological technical field, be specifically related to the preparation method of a kind of sequencing template based on emulsion-based PCR.Including the acquisition in library, emulsion-based PCR, breakdown of emulsion and the enriching and recovering of library microsphere.Emulsion-based PCR is in amplification procedure, and annealing time is more than 4 minutes, and breakdown of emulsion is selected from two butanol, isopropanol and normal hexane.Initiator is azodiisobutyronitrile and ferrous sulfate.Cosurfactant is selected from Polyethylene Glycol and glycerol.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the preparation method of a kind of sequencing template based on emulsion-based PCR.
Background technology
PCR is the method for the interior DNA rapid amplifying replicated of a kind of external reduced condition Imitating DNA body.Emulsion-based PCR technology
Utilizing the microreactor that Water-In-Oil structure is reacted as PCR, carry out PCR amplification, the feature of emulsion-based PCR maximum is to form number
The huge independent reaction space of mesh is to carry out PCR amplification, and its key technology is before PCR reacts, and will include all of reaction of PCR
The aqueous solution of composition is injected into the oil phase surface of high speed rotating, forms countless little water droplets.
Utilize the feature of emulsion-based PCR, make each microsphere reaction drop comprises only a molecule in emulsion preparation or city
Sequencing library and expand component accordingly, once obtain efficient unimolecule multicopy sequencing template microsphere, for next step
Sequencing procedure obtains more preferable order-checking signal and data provide guarantee.Based on water in oil emulsion system, one amplification is proposed
The scheme of complex DNA mixture, target molecule is divided in small emulsion droplet and expands respectively, this method energy
Enough carry out under the conditions of low concentration intentional DNA and substantial amounts of PCR cycle.Prior art exists some problem, is first emulsion
System is unstable, if emulsion system is unstable, not only reduces emulsion and expands successful probability, add experimentation cost simultaneously,
Waste the time.Next to that in emulsion system drop, monoclonal content ratio is relatively low, increases monoclonal ratio and not only can improve
The preparation efficiency of template, also can shorten the time simultaneously, reduces cost.The bioaccumulation efficiency being finally magnetic bead is relatively low.So being badly in need of building
Vertical a kind of new sequencing template.
Summary of the invention
It is an object of the invention to the preparation method of a kind of sequencing template based on emulsion-based PCR, comprise the following steps:
(1) acquisition of DNA library；
(2) emulsion-based PCR；
(3) breakdown of emulsion；
(4) enriching and recovering of DNA library microsphere.
Described emulsion-based PCR in amplification procedure, annealing time more than 3.5 minutes, preferably more than 4 minutes.
Described breakdown of emulsion uses one or more in two butanol, isopropanol and normal hexane.
The reaction system of described emulsion-based PCR includes aqueous phase, oil phase, initiator, surfactant and cosurfactant.
Described aqueous phase is 4:9-4:7, preferably 8:17 with the volume ratio of oil phase.
Described oil phase is a kind in DC5225C, PGFE, DC193, DC556 and mineral oil or several, is preferably
DC5225C, DC193 and DC556.
Described initiator is azodiisobutyronitrile and ferrous sulfate.
Described surfactant is the one in span80, TritonX-100, tween80, tween60 and tween20
Or several, preferably span80, TritonX-100, tween80 and tween60；When prepared by microemulsion, span80,
Tween80, tween60 and tween20 are dissolved in oil phase, and TritonX-100 is soluble in the aqueous phase.
It is span80 2.5-5% that described surfactant accounts for the volume ratio of system, TritonX-100 0.4-1.0%,
Tween80 0.2-0.5%, tween60 0.3-0.5%, tween20 0.2-0.8%；It is preferably span80 2.5-3.5%,
TritonX-100 0.7-0.9%, tween80 0.3-0.4%, tween60 0.4-0.5% and tween20 0.4-0.7%.
Described cosurfactant is one or more in Polyethylene Glycol, sorbitol and glycerol.
Described aqueous phase includes that emPCR auxiliary agent, amplification liquid, primer, archaeal dna polymerase, dNTP, PPiase(peptide prolyl are suitable
Anti-isomerase), MgCl2And water；Described emPCR auxiliary agent includes betaine(glycine betaine), D-(+) trehalose(dextrorotation Sargassum
Sugar), L-carnitine(L-carnitine), NP-40(Nonidet P40), DTT (dithiothreitol, DTT), formamide
One or more in (Methanamide) and DMSO (dimethyl sulfoxide), preferably betaine, D-(+) trehalose, L-
Carnitine, NP-40, DTT, formamide and DMSO；The ratio accounting for system is Betaine 0.4-1.0M, D-(+)
Trehalose 0.2-0.5M, L-carnitine 0.1-0.4M, NP-40 0.2-0.6%, DTT 1-2.5mM, formamide
0.1-0.2%, DMSO 1-2.5%(percentage composition therein is volume ratio).
The preparation method of the present invention is simple to operate, is very beneficial for large-scale application.Have the advantage that use oil phase,
Aqueous phase surfactant and cosurfactant make the emulsion system of preparation highly stable, and the efficiency of emulsion amplification is high, mispairing
Rate is low, improves the success rate of emulsion amplification, reduces experimental cost, save manpower and materials cost.In emulsion system, drop
Monoclonal proportional amount high, the efficiency having increased to improve emulsion amplification notable of monoclonal ratio.Meanwhile, magnetic microsphere
Bioaccumulation efficiency is high.The selection of initiator further improves the emulsifying effectiveness of emulsion.
Detailed description of the invention
Embodiment 1
The acquisition in 1.DNA library
(1) genomic DNA is carried out fragmentation, jointing, after process, obtain single-stranded DNA banks；
(2) the microsphere subpackage after cleaning is in 2ml EP pipe；
(3) in the EP pipe that subpackage is good, add the single-stranded DNA banks 10 μ L after dilution, vortex concussion 10s, obtain library and mix
Close liquid.
2. emulsion-based PCR
(1) preparation oil phase, mixes DC5225C 550 μ L, DC193 500 μ L and DC556 1200 μ L.
(2) preparation aqueous phase, adds
10x expands liquid 100 μ L
10 μMs of primer 1 120 μ L
25 μMs of primer 2 40 μ L
Archaeal dna polymerase 80 μ L
dNTP 120μL
PPiase 2μL
MgCl2(1M) 30μL
EmPCR auxiliary agent 350 μ L
Double steaming sterilized water surpluses
Amount to 1000 μ L
Wherein, emPCR auxiliary agent is Betaine 0.4M, D-(+) trehalose 0.5M, L-carnitine 0.1M, NP-
40 0.2%, DTT 2.5mM, DMSO 1%.
(3) by accounting for the volume ratio of system, by span80 2.5%, tween80 0.4%, tween20 0.7% and Polyethylene Glycol
0.4 ‰ are dissolved in oil phase, and TritonX-100 0.4% is soluble in the aqueous phase, mixing.
(4) add library mixed liquor, azodiisobutyronitrile and ferrous sulfate, be placed in TissueLyser and carry out emulsifying.
(5) PCR amplification.
3. breakdown of emulsion
(1) two butanol and normal hexane breakdown of emulsion are used.
4. the enriching and recovering of DNA library microsphere
(1) microsphere enrichment prepares
A) microsphere suspensions is centrifugal abandons supernatant, adds 0.15M NaOH vibration mixing, incubated at room 3min.
B) centrifugal supernatant is abandoned, by 500 l annealing buffer eccentric cleaning twice.
C) with the 30 resuspended microspheres of l annealing buffer, enriching primer 3.75 l, vibration mixing are added.
D) annealing: 53 DEG C hatch 5min after be immediately placed in 2min on ice.
E) by 500 l phosphate buffer eccentric cleaning twice, 500 l phosphate buffers are resuspended.
(2) microsphere enrichment
A) take microsphere 20 l, magnetic frame absorption 2min, abandon supernatant.
B) clean twice with 500 l phosphate buffer magnetic frames, use NaCl solution to clean microsphere.
C) using 20 l phosphate buffers resuspended, after vibration mixing, room temperature is centrifuged 5min.
D) it is placed in 2min on magnetic frame, observes.
Embodiment 2
The acquisition in 1.DNA library
(1) use Capture Bead Wash Buffer TW that magnetic microsphere vortex is shaken, be repeated twice, 5000rpm
Centrifugal, remove supernatant；
(2) the microsphere subpackage after cleaning is in 2ml EP pipe；
(3) in the EP pipe that subpackage is good, add the single-stranded DNA banks 10 μ L after dilution, vortex concussion 10s, obtain library and mix
Close liquid.
2. emulsion-based PCR
(1) preparation oil phase, mixes PGPE 450 μ L and DC556 1300 μ L.
(2) preparation aqueous phase, adds
10x expands liquid 100 μ L
10 μMs of primer 1 120 μ L
25 μMs of primer 2 40 μ L
Archaeal dna polymerase 80 μ L
dNTP 120μL
PPiase 2μL
MgCl2(1M) 30μL
EmPCR auxiliary agent 350 μ L
Double steaming sterilized water surpluses
Amount to 1000 μ L
Wherein, emPCR auxiliary agent is Betaine 1.0M, D-(+) trehalose 0.2M, L-carnitine 0.4M, NP-
40 0.6%, DTT 1mM, DMSO 2.5%.
(3) by span80 3.5%, tween80 0.3%, tween60 0.4% and glycerol 0.35 ‰ are dissolved in oil phase,
TritonX-100 0.7% is soluble in the aqueous phase, mixing.
(4) add library mixed liquor, azodiisobutyronitrile and ferrous sulfate, be placed in TissueLyser and carry out emulsifying.
(5) PCR amplification.
3. breakdown of emulsion
(1) collect emulsion, use normal hexane breakdown of emulsion.
4. the enriching and recovering of DNA library microsphere
(1) microsphere enrichment prepares
A) microsphere suspensions is centrifugal abandons supernatant, adds 0.1M NaOH vibration mixing, incubated at room 3min.
B) centrifugal supernatant is abandoned, by 500 l annealing buffer eccentric cleaning twice.
C) with the 30 resuspended microspheres of l annealing buffer, enriching primer 3.75 l, vibration mixing are added.
D) annealing: 53 DEG C hatch 6min after be immediately placed in 2min on ice.
E) by 500 l phosphate buffer eccentric cleaning twice, 500 l phosphate buffers are resuspended.
(2) microsphere enrichment
A) take microsphere 20 l, magnetic frame absorption 2min, abandon supernatant.
B) clean twice with 500 l phosphate buffer magnetic frames, use washed with saline solution microsphere.
C) using 20 l phosphate buffers resuspended, after vibration mixing, room temperature is centrifuged 5min.
D) it is placed in 2min on magnetic frame, observes.
Embodiment 3
The acquisition in 1.DNA library
(1) genomic DNA is carried out fragmentation, jointing, after process, obtain single-stranded DNA banks；
(2) the microsphere subpackage after cleaning is in 2ml EP pipe；
(3) in the EP pipe that subpackage is good, add the single-stranded DNA banks 10 μ L after dilution, vortex concussion 10s, obtain library and mix
Close liquid.
2. emulsion-based PCR
(1) preparation oil phase, mixing DC5225C 300 μ L, PGFE 200 μ L, DC193 800 μ L, DC556 300 μ L and ore deposit
Thing oil 400 μ L.
(2) preparation aqueous phase, adds
10x expands liquid 100 μ L
10 μMs of primer 1 120 μ L
25 μMs of primer 2 40 μ L
Archaeal dna polymerase 80 μ L
dNTP 120μL
PPiase 2μL
MgCl2(1M) 30μL
EmPCR auxiliary agent 350 μ L
Double steaming sterilized water surpluses
Amount to 1000 μ L
Wherein, emPCR auxiliary agent is Betaine 0.45M, D-(+) trehalose 0.4M, L-carnitine 0.15M,
NP-40 0.3%, DTT 2.3mM, formamide 0.1%, DMSO 2.3%.
(3) by accounting for the volume ratio of system, by span80 5%, tween80 0.2%, tween60 0.5%, tween20
0.2% and sorbitol 0.4 ‰ be dissolved in oil phase, TritonX-100 0.9% is soluble in the aqueous phase, mixing.
(4) add library mixed liquor, be placed in TissueLyser and carry out emulsifying.
(5) PCR amplification, response procedures is as follows:
1. 98 DEG C, 6min；
2. 98 DEG C, 30s；
3. 50 DEG C, 3.5min；
4. 69 DEG C, 35s；
Step is 2. to the most totally 50 circulations；
5. 4 DEG C of preservations.
3. breakdown of emulsion
(1) collect emulsion, use two butanol breakdowns of emulsion.
4. the enriching and recovering of DNA library microsphere
(1) microsphere enrichment prepares
A) microsphere suspensions is centrifugal abandons supernatant, adds 0.1M NaOH vibration mixing, incubated at room 3min.
B) centrifugal supernatant is abandoned, by 500 l annealing buffer eccentric cleaning twice.
C) with the 30 resuspended microspheres of l annealing buffer, enriching primer 3.75 l, vibration mixing are added.
D) annealing: 54 DEG C hatch 5.5min after be immediately placed in 2min on ice.
E) by 500 l phosphate buffer eccentric cleaning twice, 500 l phosphate buffers are resuspended.
(2) microsphere enrichment
A) take microsphere 20 l, magnetic frame absorption 2min, abandon supernatant.
B) clean twice with 500 l phosphate buffer magnetic frames, use KCl solution to clean microsphere.
C) using 20 l phosphate buffers resuspended, after vibration mixing, room temperature is centrifuged 5min.
D) it is placed in 2min on magnetic frame, observes.
Embodiment 4
The acquisition in 1.DNA library
(1) use Capture Bead Wash Buffer TW to microsphere vortex shake, in triplicate, 4000rpm from
The heart, removes supernatant；
(2) the microsphere subpackage after cleaning is in 2ml EP pipe；
(3) in the EP pipe that subpackage is good, add the single-stranded DNA banks 10 μ L after dilution, vortex concussion 10s, obtain library and mix
Close liquid.
2. emulsion-based PCR
(1) preparation oil phase, mixes DC5225C 925 μ L and DC193 1200 μ L.
(2) preparation aqueous phase, adds
10x expands liquid 100 μ L
10 μMs of primer 1 120 μ L
25 μMs of primer 2 40 μ L
Archaeal dna polymerase 80 μ L
dNTP 120μL
PPiase 2μL
MgCl2(1M) 30μL
EmPCR auxiliary agent 350 μ L
Double steaming sterilized water surpluses
Amount to 1000 μ L
Wherein, emPCR auxiliary agent is Betaine 0.90M, L-carnitine 0.35M, NP-40 0.5%, formamide
0.2%。
(3) by accounting for the volume ratio of system, Polyethylene Glycol, glycerol, tween80 0.4% and tween20 0.8% are dissolved in
Oil phase, TritonX-100 1.0% is soluble in the aqueous phase, mixing.
(4) add library mixed liquor, azodiisobutyronitrile and ferrous sulfate, be placed in TissueLyser and carry out emulsifying.
(5) PCR amplification, response procedures is as follows:
1. 94 DEG C, 6min；
2. 94 DEG C, 30s；
3. 50 DEG C, 4min；
4. 69 DEG C, 35s；
Step is 2. to the most totally 50 circulations；
5. 4 DEG C of preservations.
3. breakdown of emulsion
(1) two butanol and isopropanol breakdown of emulsion are used.
4. the enriching and recovering of DNA library microsphere
(1) microsphere enrichment prepares
A) microsphere suspensions is centrifugal abandons supernatant, adds 0.15M NaOH vibration mixing, incubated at room 3min.
B) centrifugal supernatant is abandoned, by 500 l annealing buffer eccentric cleaning twice.
C) with the 30 resuspended microspheres of l annealing buffer, enriching primer 3.75 l, vibration mixing are added.
D) annealing: 57 DEG C hatch 5min after be immediately placed in 2min on ice.
E) by 500 l phosphate buffer eccentric cleaning twice, 500 l phosphate buffers are resuspended.
(2) microsphere enrichment
A) take microsphere 20 l, magnetic frame absorption 2min, abandon supernatant.
B) clean twice with 500 l phosphate buffer magnetic frames, use NaCl solution to clean microsphere.
C) using 20 l phosphate buffers resuspended, after vibration mixing, room temperature is centrifuged 5min.
D) it is placed in 2min on magnetic frame, observes.
Embodiment 5
The acquisition in 1.DNA library
(1) genomic DNA is carried out fragmentation, jointing, after process, obtain single-stranded DNA banks；
(2) the microsphere subpackage after cleaning is in 2ml EP pipe；
(3) in the EP pipe that subpackage is good, add the single-stranded DNA banks 10 μ L after dilution, vortex concussion 10s, obtain library and mix
Close liquid.
2. emulsion-based PCR
(1) preparation oil phase, measures mineral oil 2125 μ L.
(2) preparation aqueous phase, adds
10x expands liquid 100 μ L
10 μMs of primer 1 120 μ L
25 μMs of primer 2 40 μ L
Archaeal dna polymerase 80 μ L
dNTP 120μL
PPiase 2μL
MgCl2(1M) 30μL
EmPCR auxiliary agent 350 μ L
Double steaming sterilized water surpluses
Amount to 1000 μ L
Wherein, emPCR auxiliary agent is Betaine 1M.
(3) by accounting for the volume ratio of system, by span80 5%, tween60 0.3%, Polyethylene Glycol 0.3 ‰, sorbitol
0.1 ‰ and glycerol 0.2 ‰ be dissolved in oil phase, oil phase and aqueous phase are mixed.
(4) add library mixed liquor, be placed in TissueLyser and carry out emulsifying.
(5) PCR amplification, response procedures is as follows:
1. 95 DEG C, 4min；
2. 95 DEG C, 30s；
3. 54 DEG C, 4min；
4. 68 DEG C, 30s；
Step is 2. to the most totally 20 circulations；
5. 95 DEG C, 30s；
6. 54 DEG C, 20s；
7. 68 DEG C, 60s；
Step is 5. to the most totally 10 circulations；
8. 10 DEG C of preservations.
3. breakdown of emulsion
(1) two butanol and normal hexane breakdown of emulsion are used.
4. the enriching and recovering of DNA library microsphere
(1) microsphere enrichment prepares
A) microsphere suspensions is centrifugal abandons supernatant, adds 0.15M NaOH vibration mixing, incubated at room 3min.
B) centrifugal supernatant is abandoned, by 500 l annealing buffer eccentric cleaning twice.
C) with the 30 resuspended microspheres of l annealing buffer, enriching primer 3.75 l, vibration mixing are added.
D) annealing: 56 DEG C hatch 5min after be immediately placed in 2min on ice.
E) by 500 l phosphate buffer eccentric cleaning twice, 500 l NaCl solution are resuspended.
(2) microsphere enrichment
A) take microsphere 20 l, magnetic frame absorption 2min, abandon supernatant.
B) clean twice with 500 l phosphate buffer magnetic frames, use Tris-HCl to clean microsphere.
C) using 20 l phosphate buffers resuspended, after vibration mixing, room temperature is centrifuged 5min.
D) it is placed in 2min on magnetic frame, observes.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and this embodiment is also not used to
Limiting the scope of the claims of the present invention, all equivalences done without departing from the present invention are implemented or change, are intended to be limited solely by the technology of the present invention
In the range of scheme.
Claims (13)
1. the preparation method of a sequencing template based on emulsion-based PCR, it is characterised in that comprise the following steps:
(1) acquisition of DNA library；
(2) emulsion-based PCR；
(3) breakdown of emulsion；
(4) enriching and recovering of DNA library microsphere；
The reaction system of described emulsion-based PCR includes aqueous phase, oil phase, initiator, surfactant and cosurfactant；
Described oil phase is the one in following four: (1) DC5225C, DC193 and DC556；(2) PGPE and DC556；(3)
DC5225C, PGFE, DC193, DC556 and mineral oil；(4) DC5225C and DC193.
2. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described emulsion-based PCR
In amplification procedure, annealing time is more than 3.5 minutes.
3. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 2, it is characterised in that described emulsion-based PCR
In amplification procedure, annealing time is more than 4 minutes.
4. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described breakdown of emulsion is adopted
By one or more in two butanol, isopropanol and normal hexane.
5. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described aqueous phase with
The volume ratio of oil phase is 4:9-4:7.
6. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 5, it is characterised in that described aqueous phase with
The volume ratio of oil phase is 8:17.
7. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described oil phase is
DC5225C, DC193 and DC556.
8. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described initiator
For azodiisobutyronitrile and ferrous sulfate.
9. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that live in described surface
Property agent is one or more in span80, TritonX-100, tween80, tween60 and tween20；Prepare at microemulsion
Time, span80, tween80, tween60 and tween20 be dissolved in oil phase, and TritonX-100 is soluble in the aqueous phase.
10. the preparation method of sequencing template based on emulsion-based PCR as claimed in claim 9, it is characterised in that live in described surface
Property agent is span80, TritonX-100, tween80 and tween60.
The preparation method of 11. sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that described in help surface
Activating agent is one or more in Polyethylene Glycol, sorbitol and glycerol.
The preparation method of 12. sequencing template based on emulsion-based PCR as claimed in claim 1, it is characterised in that in described aqueous phase
Including emPCR auxiliary agent, amplification liquid, primer, archaeal dna polymerase, dNTP, PPiase, MgCl2And water；Described emPCR auxiliary agent includes
Betaine, D-(+) one or more in trehalose, L-carnitine, NP-40, DTT, formamide and DMSO.
The preparation method of 13. sequencing template based on emulsion-based PCR as claimed in claim 12, it is characterised in that described emPCR
Auxiliary agent be betaine, D-(+) trehalose, L-carnitine, NP-40, DTT and formamide.
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