CN104862304B - A kind of emulsion-based PCR system - Google Patents
A kind of emulsion-based PCR system Download PDFInfo
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- CN104862304B CN104862304B CN201510299698.6A CN201510299698A CN104862304B CN 104862304 B CN104862304 B CN 104862304B CN 201510299698 A CN201510299698 A CN 201510299698A CN 104862304 B CN104862304 B CN 104862304B
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Abstract
The invention belongs to biological technical field, be specifically related to a kind of emulsion-based PCR system. This emulsion-based PCR system is made up of aqueous phase, oil phase, surfactant and cosurfactant. The emPCR system optimization provided by the invention system environment of nucleic acid sequencing, reagent choose the stability adding emulsion system, make the sequencing result more accurate, homogeneous and stable. Emulsion system Heat stability is good prepared by the program, microsphere is evenly distributed in drop, and monoclonal ratio is high.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of emulsion-based PCR system.
Background technology
Emulsion-based PCR (emulsionPCR) technology utilizes Water-In-Oil structure as the PCR microreactor reacted, and carries out pcr amplification. The maximum feature of emulsion-based PCR is that the independent reaction space that can form huge number is to carry out pcr amplification. Its key technology is " water filling is to oily ", and basic process is before PCR reacts, and the aqueous solution comprising all of reacted constituent of PCR is injected into the oil phase surface of high speed rotating, and aqueous solution moment forms ten hundreds of the droplets wrapped up by oil phase. These droplets are the formation of PCR and reflect space. Due to the PCR reaction that the P1 primer of the P2 primer in aqueous phase and magnetic bead surfaces mediates, the number of copies of this DNA profiling exponentially expands, after PCR reaction terminates, P1 magnetic bead surfaces is just fixed with the homologous dna amplified production of copy huge amount, producing millions of identical copies after each fragment amplification, these copies are also integrated on magnetic bead. The DNA in whole library.
Propose the scheme of an amplification complex DNA mixture based on water in oil emulsion system, target molecule is divided in small emulsion droplet and expands respectively, and this method can carry out when low concentration intentional DNA and substantial amounts of PCR cycle. Each independent fragment carries out independent amplification in the microreactor of oneself, without the impact of other competitiveness or contaminative sequence. The amplification of whole frag-ment libraries is parallel to be carried out. Subsequently, emulsion mixture is broken, and the fragment of amplification is still incorporated on magnetic bead.
Summary of the invention
It is an object of the invention to a kind of emulsion-based PCR system, described PCR system is emulsion system, including aqueous phase, oil phase, surfactant and cosurfactant.
The volume ratio of described aqueous phase and oil phase is 4:9-4:7.
Described oil phase is DC5225C, PGFE(Tripolyglycerol monostearates), in DC749 and mineral oil a kind or several, it is preferred to PGFE and DC749.
Also including protective agent in described system, protective agent is BSA(bovine serum albumin), one or more in human albumin and Polyethylene Glycol, it is preferred to BSA.
Described BSA is through acetylizad, the other BSA of molecular biology grade.
Described surfactant is one or more in span80, TritonX-100, tween80, tween60 and tween20, it is preferred to 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 span802.5-5%, TritonX-1000.4-1.0%, tween800.2-0.5%, tween600.3-0.5%, tween200.2-0.8% that described surfactant accounts for the volume ratio of system; It is preferably span802.5-3.5%, TritonX-1000.7-0.9%, tween800.3-0.4%, tween600.4-0.5% and tween200.4-0.7%.
Described cosurfactant is one or more in Polyethylene Glycol, sorbitol and glycerol.
Containing emPCR auxiliary agent in described aqueous phase; Possibly together with Buffer, primer, archaeal dna polymerase, dNTP, Mg2+And water.
Also including pyrophosphatase in described aqueous phase, pyrophosphatase is thermally-stabilised pyrophosphatase (thermostablepyrophosphatase).
Described emPCR auxiliary agent includes betaine(glycine betaine), D-(+) trehalose(dextrorotation trehalose), L-carnitine(L-carnitine), NP-40(Nonidet P40), one or more in DTT (dithiothreitol, DTT) and DMSO (dimethyl sulfoxide), it is preferred to betaine0.4-1.0M, D-(+) trehalose0.2-0.5M, L-carnitine0.1-0.4M, NP-400.2-0.6%, DTT1-2.5mM and DMSO1-2.5%(percentage composition therein be volume ratio).
Described water is nuclease-freewater (NFW).
All reagent employing analytical pure and above purity.
The emPCR system optimization provided by the invention system environment of nucleic acid sequencing, reagent choose the stability adding microemulsion system, oil-water ratio is suitable for, and makes the sequencing result more accurate, homogeneous and stable. Microemulsion system Heat stability is good prepared by the program, microsphere is evenly distributed in drop, and monoclonal ratio is high. Surfactant and the solubilising degree of auxiliary agent that the present invention chooses are big, and nucleation efficiencies is high, it is appreciated that in the reaction rate improving order-checking.
Detailed description of the invention
Embodiment 1
(1) oil phase, mixes PGFE and DC749 totally 400 �� L.
(2) aqueous phase,
Wherein, emPCR auxiliary agent be Betaine0.4M, D-(+) trehalose0.5M, L-carnitine0.1M, NP-400.2%, DTT2.5mM, DMSO1%.
(3) by the volume ratio accounting for system, span802.5%, tween800.4%, tween200.7% and Polyethylene Glycol 0.2 �� being dissolved in oil phase, TritonX-1000.4% is soluble in the aqueous phase, mixing, emulsifying.
Embodiment 2
(1) oil phase, mixes DC749200 �� L and DC5225C150 �� L.
(2) aqueous phase, adds
Wherein, emPCR auxiliary agent be Betaine1.0M, D-(+) trehalose0.2M, L-carnitine0.4M, NP-400.6%, DTT1mM, DMSO2.5%.
(3) span803.5%, tween800.3%, tween600.4% and glycerol 0.6 �� being dissolved in oil phase, TritonX-1000.7% is soluble in the aqueous phase, mixing, emulsifying.
Embodiment 3
(1) oil phase, mixing DC5225C60 �� L, PGFE30 �� L, DC749180 �� L and mineral oil 90 �� L.
(2) aqueous phase, adds
Wherein, emPCR auxiliary agent be Betaine0.45M, D-(+) trehalose0.4M, L-carnitine0.15M, NP-400.3%, DTT2.3mM, DMSO2.3%.
(3) by the volume ratio accounting for system, by molten to span805%, tween800.2%, tween600.3%, tween200.2% and sorbitol 0.4 �� in oil phase, TritonX-1000.9% is soluble in the aqueous phase, mixing, emulsifying.
Embodiment 4
(1) oil phase, mixing DC5225C100 �� L, PGFE100 �� L and DC749200 �� L.
(2) aqueous phase, adds
Wherein, emPCR auxiliary agent is Betaine0.90M, L-carnitine0.35M, NP-400.5%.
(3) by the volume ratio accounting for system, Polyethylene Glycol, glycerol, tween800.4% and tween200.8% being dissolved in oil phase, TritonX-1001.0% is soluble in the aqueous phase, mixing, emulsifying.
Embodiment 5
(1) oil phase, measures mineral oil 500 �� L.
(2) aqueous phase, adds
Wherein, emPCR auxiliary agent is Betaine1M.
(3) by the volume ratio accounting for system, Polyethylene Glycol 0.1 ��, sorbitol 0.3 ��, glycerol 0.15 ��, span805% and tween600.5% are dissolved in oil phase, oil phase and aqueous phase are mixed, emulsifying.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and this embodiment is also not used to limit the scope of the claims of the present invention, and all equivalences done without departing from the present invention are implemented or change, and are intended to be limited solely by the scope of technical solution of the present invention.
Claims (8)
1. an emulsion-based PCR system, it is characterised in that described PCR system is emulsion system, including aqueous phase, oil phase, surfactant and cosurfactant; Wherein aqueous phase is 23:40,4:7 or 9:16 with the volume ratio of oil phase, and oil phase is two or three in DC5225C, PGFE and DC749; Wherein containing emPCR auxiliary agent in aqueous phase, emPCR auxiliary agent be betaine, D-(+) trehalose, L-carnitine, NP-40, DTT and DMSO mixture, or the mixture of betaine, L-carnitine and NP-40.
2. emulsion-based PCR system as claimed in claim 1, it is characterised in that also include protective agent in described system.
3. emulsion-based PCR system as claimed in claim 1, it is characterised in that described surfactant is one or more in span80, TritonX-100, tween80, tween60 and tween20; When prepared by microemulsion, span80, tween80, tween60 and tween20 are dissolved in oil phase, and TritonX-100 is soluble in the aqueous phase.
4. emulsion-based PCR system as claimed in claim 3, it is characterised in that described surfactant is span80, TritonX-100, tween80 and tween60.
5. emulsion-based PCR system as claimed in claim 1, it is characterised in that it is span802.5-5%, TritonX-1000.4-1.0%, tween800.2-0.5%, tween600.3-0.5%, tween200.2-0.8% that described surfactant accounts for the ratio of system.
6. emulsion-based PCR system as claimed in claim 5, it is characterised in that it is span802.5-3.5%, TritonX-1000.7-0.9%, tween800.3-0.4%, tween600.4-0.5% and tween200.4-0.7% that described surfactant accounts for the ratio of system.
7. emulsion-based PCR system as claimed in claim 1, it is characterised in that described cosurfactant is one or more in Polyethylene Glycol, sorbitol and glycerol.
8. emulsion-based PCR system as claimed in claim 1, it is characterised in that in system, all reagent adopt analytical pure and purity above.
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| CN101413034A (en) * | 2008-11-21 | 2009-04-22 | 东南大学 | Method for preparing molecular cloning chip for high-throughput cloning of nucleic acid molecule |
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