CN104844742B - Preparation method for the polystyrene microsphere of gene order-checking nucleic acid amplification - Google Patents

Preparation method for the polystyrene microsphere of gene order-checking nucleic acid amplification Download PDF

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CN104844742B
CN104844742B CN201510204426.3A CN201510204426A CN104844742B CN 104844742 B CN104844742 B CN 104844742B CN 201510204426 A CN201510204426 A CN 201510204426A CN 104844742 B CN104844742 B CN 104844742B
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preparation
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microsphere
room temperature
gene order
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CN104844742A (en
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蔡亦梅
高静
徐潇
吴超
张睿
王者馥
王绪敏
殷金龙
任鲁风
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Beijing Zhongkezixin Technology Co Ltd
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Abstract

The invention belongs to gene order-checking field, the preparation method being specifically related to a kind of polystyrene microsphere being applied to gene order-checking nucleic acid amplification. Described microspherulite diameter is 25 microns, concretely comprises the following steps: dispersant is added in reaction medium by (1), passes into nitrogen; Stir to homogeneous system; (2) styrene monomer and initiator are added in reaction system; Reaction system is heated up; Then isothermal reaction is carried out under agitation; It is cooled to room temperature, washing, dries. The present invention adopts dispersion copolymerization method successfully to prepare Monodisperse Polystyrene Microspheres, particle diameter be 25 microns and the coefficient of dispersion low.

Description

Preparation method for the polystyrene microsphere of gene order-checking nucleic acid amplification
Technical field
The invention belongs to gene order-checking field, the preparation method being specifically related to a kind of polystyrene microsphere being applied to gene order-checking nucleic acid amplification.
Background technology
As one of most important invention of life science in last century, DNA sequencing technology profoundly changes people to the understanding of life quintessence and control ability, has greatly promoted the development that global life science is studied. Were it not for sequencing technologies, gene order just cannot be determined, the investigative technique such as enzyme action, clone, reverse transcription, cDNA, PCR, SNP, RNAi is not just known where to begin at all yet, and life science does not have the flourish of today. The life science GenBank that All the world knows, and the Human Genome Project that the global cooperation lasting 13 years costs 300,000,000 dollars completes, be based upon on the basis of order-checking invariably.
Having invented from Sanger in 1977 since make use of double; two deoxidations of archaeal dna polymerase to terminate the technology that principle measures nucleotide sequence, sequencing technologies is updated, and new method is come out one after another, and order-checking scale also constantly expands. The simple operation of sequencing technologies, with low costization and the developing direction that high flux chemical conversion is sequencing technologies. SOLiD, 454 and Solexa etc. be representative high throughput sequencing technologies of new generation, divide the world of sequencing equally with the attitude of three state's tripartite confrontations. Three kinds of technology are each has something to recommend him, and the speed that development updates is with rapid changepl. never-ending changes and improvements. But the process that presently, prepared by its sequencing library is all still complex.
The preparation of sequencing library as first in whole sequencing procedure and and an important step, the quality of sequencing result is had conclusive effect. The preparation of sequencing library generally includes following step. The first step is how to be extracted in high quality from sample to be detected by nucleic acid. For different samples, experiment flow and mode selected by people will have bigger difference; Second step is how the nucleic acid of long segment to be carried out small fragment flower to process, RNA equal samples mainly for genomic DNA and long segment, because sequenator is for reading long restriction at present, the random small fragment of preparation can only be checked order, splice again through bioinformatics method, draw the sequence information of full-length genome.Current nucleic acid small fragmentization uses physical action to carry out substantially, and ultrasound wave becomes the universal way smashing DNA due to the various advantages of himself, can be obtained the small pieces segment DNA of different length by adjustment ultrasonic power and ultrasonic time. 3rd step is how the two ends of small fragment nucleic acid to be connected the universal linker sequence that above order-checking is required; 4th step is how the nucleotide sequence being connected to joint sequence to be carried out unimolecule multicopy amplification, includes emulsion-based PCR, bridge-type PCR and rolling circle amplification etc., guarantees the raw information of real reflected sample while expanding order-checking signal intensity. And emulsion-based PCR is generally adopted microsphere and catches a template DNA to bead, utilize emulsion-based PCR to expand single template, same template is expanded on a microsphere up to a million template clones.
In general, the microsphere adopted in emulsion-based PCR is polystyrene (PS) microsphere, and carries out Streptavidin modification at microsphere surface, so that it can catch biotin labeled DNA molecular. At present, the method synthesizing Monodisperse Polystyrene Microspheres has microemulsion polymerization method, emulsion polymerization, dispersion copolymerization method, suspension polymerization and seeded polymerization etc. But existing synthetic method is all be polymerized after cross-linking agent and monomer mixing again, the polymer microballoon polymer of this way synthesis is overall crosslinking, often sphericity is poor, and size tunable is low, thus typically requires sorting and can be only achieved the requirement less than 5% of the particle size distribution deviation. In order to be synthetically derived the microsphere of crosslinking, but do not interfere with sphericity and the monodispersity of microsphere, although the method that Japan Patent JP58-106554 and JP63-191818 proposes seeding polymerization, namely first pass through emulsion polymerization and obtain seed, then increase, expand particle. Disadvantage of this method is that microsphere is in growth course, produce secondary, it is necessary to screening removes, and causes product yield to reduce, complicated operation is less economical. Therefore, will obtaining the micron size of uniform particle diameter, to have the polymer microballoon particle of crosslinking be very difficult.
The polymer microballoon difficulty that synthesis has monodispersity is very big, its severe reaction conditions, complicated process of preparation, and reaction controlling requires abnormal strict. the prior synthesizing method of polymer microballoon has emulsion polymerization and suspension polymerization, emulsion polymerization can only prepare the microsphere less than 500nm, although microsphere particle diameter prepared by suspension polymerization is at 100-1000 ��m, but polydispersity, also it is difficult to obtain monodispersed microsphere even across repeatedly sieving, dispersin polymerization and seeding polymerization that nineteen seventies grows up can prepare particle diameter 1-100 ��m, and there is the polymer microballoon of monodispersity, it it is the better method preparing monodisperse polymer microsphere at present, but diffuse-aggregate reaction medium need to use organic solvent such as ethanol etc., also need to add stabilizer such as polyvinylpyrrolidone etc., purity and the post processing of polymerizate are made troubles by agents useful for same.
Summary of the invention
The preparation method that it is an object of the present invention to provide a kind of polystyrene microsphere, its microsphere prepared can be used for the nucleic acid amplification in the preparation of gene order-checking library, and described microspherulite diameter is 25 microns, specifically comprises the following steps that
(1) dispersant is added in reaction medium, pass into nitrogen; Stir to homogeneous system;
(2) styrene monomer and initiator are added in reaction system; Reaction system is heated up; Then isothermal reaction is carried out under agitation;It is cooled to room temperature, washing, dries.
In one embodiment of the invention, described reaction medium is selected from the one or more kinds of combination in any of water, ethanol, glycol monoethyl ether and methanol. In preferred embodiments, described reaction medium is water and glycol monoethyl ether, and wherein, the percentage by weight of water and glycol monoethyl ether is 10��90% and 10��90%; Can also be 20��40% and 60��80%, it is preferred to 23% and 77%.
Described dispersant is the one or more kinds of combination in any in sodium phosphate, polyvinylpyrrolidone, Polyethylene Glycol, polyacrylic acid, polyvinyl alcohol and hydroxypropyl cellulose. In preferred embodiments, described dispersant is made up of polyvinyl alcohol, hydroxypropyl cellulose and Polyethylene Glycol, and the percentage by weight of polyvinyl alcohol, hydroxypropyl cellulose and Polyethylene Glycol is 10��80%, 10��80% and 10��80%; Can also be 20��40%, 30��50% and 20��50%, it is preferred to 25%, 35% and 40%.
Initiator is selected from one or both the arbitrary proportion combination of azodiisobutyronitrile (AIBN) or benzoyl peroxide (BPO). In preferred embodiments, described initiator is azodiisobutyronitrile.
In another embodiment of the invention, in final reaction system, the percentage by weight of styrene monomer, dispersant, initiator and reaction medium is followed successively by the styrene monomer of 5��40%, the dispersant of 0.1��1%, the initiator of 0.025��0.125%, reaction medium surplus; It is preferably the styrene monomer of 32%, the dispersant of 0.3%, the initiator of 0.05%, reaction medium surplus.
The present invention further carries out in scheme, described step (2) reaction system is warming up to 10-90 DEG C, it is preferred to 75 DEG C; Mixing speed controls at 80��120 revs/min, it is preferred to 100 revs/min.
Preparation of Monodispersed Polystyrene Microspheres by Dispersion Polymerization is affected by 4 conditions such as monomer, initiator, dispersant and reaction mediums, and wherein monomer and dispersant are two principal elements affecting particle size distribution. Under certain reaction condition, along with the increase of monomer consumption and concentration, the particle diameter of polystyrene microsphere increases; Along with the increase of stabilizing agent dosage, the particle diameter of polystyrene microsphere reduces.
In gene order-checking, the particle diameter for the PS microsphere of DNA library amplifying nucleic acid amplification is generally 25 microns, and requires that monodispersity is good. But in PS method for preparing microsphere, PS microsphere monodispersity prepared by dispersion copolymerization method is good, but the particle diameter of microsphere is less, although the PS microspherulite diameter of seeded polymerization and suspension polymerization preparation is relatively big, but the monodispersity of microsphere is poor, and the coefficient of dispersion is high. Therefore in the art, prepare the Monodisperse Polystyrene Microspheres that particle diameter is 20 to 50 microns and there are the problems referred to above always. The present invention is groped by lot of experiments and has been surprisingly found that, under the specific reaction condition of one, adopt dispersion copolymerization method successfully prepare Monodisperse Polystyrene Microspheres, particle diameter be 25 microns and the coefficient of dispersion low.
Detailed description of the invention
The illustration present invention further below. It is pointed out that following description is only the illustration to claimed technical scheme, the not any restriction to these technical schemes. The content that protection scope of the present invention is recorded with appended claims is as the criterion.
The preparation of embodiment 1 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes.It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 2 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.0g, the hydroxypropyl cellulose of 1.59g and 1.81g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.81g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 12 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 3 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the hydroxypropyl cellulose of 2.64g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 4 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropyl cellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 5 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropyl cellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.6g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 6 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.2g, the hydroxypropyl cellulose of 1.74g and 1.86g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.85g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 7 polystyrene microsphere
The Polyethylene Glycol of the water of 200g, the glycol monoethyl ether of 800g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 8 polystyrene microsphere
The Polyethylene Glycol of the water of 260g, the glycol monoethyl ether of 740g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes.It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 9 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 450g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres.
The preparation of embodiment 10 polystyrene microsphere
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection; and at room temperature stirring is subsequently adding styrene monomer 500g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes. It is warming up to 75 DEG C, isothermal reaction 8 hours. Reaction terminates, and is cooled to room temperature, and product is centrifuged, and uses ethanol cyclic washing, dry, obtains Monodisperse Polystyrene Microspheres, and particle diameter is 25.0 ��m, the coefficient of dispersion 1.7%.
The sign of polystyrene microsphere prepared by embodiment 1-10
Adopt JSM-6700F scanning electron microscope that particle diameter and the particle size distribution of obtained PS microsphere are measured. With 100 microspheres for benchmark, calculating mean diameter and the particle size distribution of microsphere, formula is as follows:
In formula: �� is standard variance; diDiameter for single particle; D is the average diameter of particle; N is number of particles, fsFor the coefficient of dispersion, concrete outcome is as follows:
Present invention merely illustrates some claimed specific embodiments; technical characteristic described in one of them or more technical scheme can be combined with arbitrary one or more technical schemes; the technical scheme that these are combined and obtain is also in the application protection domain, technical scheme that is combined just as these and that obtain specifically has been recorded in the disclosure of invention.

Claims (1)

1. a preparation method for polystyrene microsphere, its microsphere prepared can be used for the nucleic acid amplification in the preparation of gene order-checking library, and described microspherulite diameter is 25 microns, specific as follows:
The Polyethylene Glycol of the water of 230g, the glycol monoethyl ether of 770g, the polyvinyl alcohol of 1.1g, the hydroxypropyl cellulose of 1.54g and 1.76g is added in reaction vessel; pass into nitrogen protection, and at room temperature stirring is subsequently adding styrene monomer 473g and azodiisobutyronitrile 0.73g to homogeneous system in 20 minutes; it is warming up to 75 DEG C; isothermal reaction 8 hours, reaction terminates, and is cooled to room temperature; product is centrifuged; and use ethanol cyclic washing, and dry, obtain Monodisperse Polystyrene Microspheres.
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