CN102220599B - Quasi-interpenetrating polymer network used as capillary gel electrophoresis sieving medium and preparation method and use thereof - Google Patents
Quasi-interpenetrating polymer network used as capillary gel electrophoresis sieving medium and preparation method and use thereof Download PDFInfo
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Abstract
The invention relates to a sieving medium for capillary gel electrophoresis and methods for preparing and using the sieving medium. More particularly, the invention provides a quasi-interpenetrating polymer network used as capillary gel electrophoresis sieving medium and the preparation method and the use thereof.
Description
Technical field
The present invention relates to the sieving medium for capillary gel electrophoresis, and the method for preparing and use this sieving medium.More specifically, the invention provides a kind of accurate interpenetrating polymer networks as the capillary gel electrophoresis sieving medium, and the Preparation method and use of this accurate interpenetrating polymer networks.
Background technology
Capillary electrophoresis is a kind of separation means rapidly and efficiently.Charge species moves to the other end from an end capillaceous under effect of electric field.
Capillary gel electrophoresis is to separate one of macromolecular substance most effectual way, as biomacromolecule protein, and polypeptide, RNA and DNA etc.Capillary gel electrophoresis uses quartz capillary usually, and inside diameter ranges is at 50 microns to 200 microns, and length is from 10cm to 100cm.For the protection assay is not adsorbed, eliminate electroosmotic flow capillaceous simultaneously, capillary tube inner wall scribbles a protective layer usually.The most frequently used coating is the polyacrylamide coating.Capillary gel electrophoresis separating medium character determines the resolution of macromolecular substance.For example, polyacrylamide gel is better to the separating effect of dna fragmentation.
High molecular weight polyacrylamide is the best medium of DNA resolution in present all polymkeric substance.Can the DNA isolation fragment reach 1200 base numbers.But polyacrylamide does not have from the coating ability.Must add coating at capillary tube inner wall, eliminate capillary tube inner wall to absorption and the electroosmotic flow of DNA, effectively the DNA isolation fragment.Utilize polyacrylamide as the major advantage of sieving medium to be: good reproducibility, separating power is strong, and chemical property is stable.But polyacrylamide also has a lot of weak points.For example it is not from the coating ability, and the kapillary of use must have coating protection.In addition, higher by the gel viscosity of high molecular weight polyacrylamide preparation, need high pressure gel could be poured in the kapillary.
Poly-(N,N-DMAA) also has the ability of very strong DNA isolation fragment, and capillary tube inner wall does not need the coating layer protection when using poly-(N,N-DMAA).Poly-(N,N-DMAA) to the separating effect of dna fragmentation a little less than polyacrylamide.But poly-(N,N-DMAA) gel has very strong coating ability certainly, can eliminate the absorption of DNA and reduce electroosmotic flow.The viscosity of poly-(N,N-DMAA) gel is well below polyacrylamide gel.
If these two kinds of polymer organic ground are combined, just can obtain desirable DNA separating medium.But because polyacrylamide is different with poly-(N,N-DMAA) both polarity, these two kinds of polymkeric substance are by the simple mechanically mixing body that can not be uniformly mixed, and can present the microcosmic two-phase phenomenon of not dissolving each other.Barbier V etc., Electrophoresis 2002, and 23:1441-1449 discloses and organically the polymer scale of these two kinds of different in kinds has been combined together.Being main chain with the polyacrylamide, be that side chain synthesizes comb shaped polymer with poly-(N,N-DMAA).This comb shaped polymer can suppress electroosmotic flow effectively, need not add any coating at capillary tube inner wall, just can effective DNA isolation fragment.But this synthetic method more complicated.
Desirable sieving medium needs the viscosity of very strong wetting ability, appropriateness, also will have from applying function.Synthetic at present homopolymer can't satisfy above-mentioned condition simultaneously.Random copolymers is the way commonly used that two kinds or two or more polymer organic are combined.Multipolymer has possessed the feature of two kinds of polymkeric substance simultaneously.Therefore, the polymkeric substance that sieving capacity is arranged and the polymkeric substance that has oneself to apply function are synthesized multipolymer, just can become a kind of novel capillary electrophoresis separation sieving medium that satisfies above condition, in particular for the separation of DNA.When acrylamide and DMAA copolymerization formation random copolymers, just simultaneously DNA is had good separating power and has capillary tube inner wall from the coating ability.Song L etc., Electrophoresis, 2001,22:729-736, disclose and utilized the outstanding sieving capacity of polyacrylamide and poly-(N,N-DMAA) good coating function certainly, passed through free radical polymerisation process, acrylamide and N,N-DMAA with different ratios 3: 1,2: 1 and 1: 1 synthetic random copolymers that obtains.Can be under the condition that does not have the Additional Protection layer DNA isolation fragment.
But work as both scale not simultaneously, random copolymers also is not quite similar to the dna fragmentation separating effect.Experiment finds that for the multipolymer of acrylamide and N,N-DMAA, when the content of acrylamide was higher, this multipolymer was better to the separating effect of DNA.But when the N,N-DMAA content in this multipolymer reduced to a certain degree, the ratio of N,N-DMAA was too low, and the coating function certainly of random copolymers can be affected, and separating effect also decreases.
In the forming process of random copolymers N,N-DMAA by stochastic distribution to the acrylamide polymerization chain.This random distribution can cause each synthetic multipolymer incomplete same.Cause the synthetic multipolymer of different batches different from applying aspect the ability at kapillary, thereby influence the repeatability of dna fragmentation separating effect.
Inierpeneirating network structure is the way commonly used that two kinds or two or more polymer organic are combined.For example; Song L etc.; Electrophoresis; 2001,22:3688-3698 discloses by LPA (linear polyacrylamide) and with the accurate inierpeneirating network structure that PVP (Polyvinylpyrolidone (PVP)) forms dsDNA has been separated, DMA (N; the N-DMAA) is dissolved in the PVP solution; free radical polymerization under nitrogen protection obtains the network structure of LPA and PVP IPN, just can be used for the separation of dsDNA.
" interpenetrating polymer networks (Interpenetrating Polymer Networks; IPN) " be defined as two or more polymkeric substance with the latticed form combination, wherein at least a polymkeric substance synthesizes in the presence of another polymkeric substance is direct or is crosslinked.If it all is crosslinked constituting two kinds of polymeric constituents of IPN, then be called " full interpenetrating polymer networks ", that is, " entirely-IPN " (Full-IPN); If only a kind of crosslinked polymer is arranged, another kind of polymkeric substance is line style, then is called " semiinterpenetrating polymer network ", that is, " partly-IPN " (Semi-IPN).The English of " accurate interpenetrating polymer networks " is Quasi-IPN.Two kinds of (or more kinds of) polymkeric substance of accurate interpenetrating polymer networks all are line styles, constitute accurate interpenetrating(polymer)networks formula structure mutually.As do not add cross-linking reagent (cross-linking agent) in preparation process, interpenetrating polymer networks then is as the criterion.As in preparation process, adding cross-linking reagent, it then is interpenetrating polymer networks.Under the same molecular amount, the flowability of accurate interpenetrating polymer networks is mobile littler than linear molecule, and flowability complete, semiinterpenetrating polymer network is poorer than the flowability of accurate interpenetrating polymer networks.
Two or more the cross-linked polymer that forms inierpeneirating network structure runs through mutually and forms polymer network.Crosslinked can be that physical crosslinking can be chemically crosslinked also, but does not have covalently cross-linked effect between the polymkeric substance that runs through mutually.The cross-linked network of polymer formation can reduce macroface to be separated, and therefore the network structure that forms is more more stable than the network that polymer blend method forms.
When two kinds of polymkeric substance form cross-linked polymer by inierpeneirating network structure, its stability depends on consistency and the intermolecular power of attracting each other of two kinds of polymkeric substance.When the polarity of two kinds of polymkeric substance differed big, consistency reduced, and the stability of inierpeneirating network structure will reduce.
Summary of the invention
Be to solve the defective that exists in the prior art, the invention provides and a kind ofly have that stability is high, resolving power is high, the order-checking is effective, the accurate interpenetrating polymer networks as the polymer chain of capillary gel electrophoresis sieving medium of long service life.
The present inventor finds unexpectedly: at synthesis of acrylamide and N, during the random copolymers of N-DMAA, suitably add the poly-(N of linearity that molecular weight varies in size, the N-DMAA), make described random copolymers and poly-(N, the N-DMAA) forms accurate interpenetrating polymer networks structure, thereby guarantee mutual solubility, stability and the circulation ratio of whole gel; Can realize high resolving power and excellent order-checking effect when simultaneously, described accurate interpenetrating polymer networks structure is as the capillary gel electrophoresis sieving medium.
According to an aspect of the present invention, the invention provides a kind of accurate interpenetrating polymer networks of polymer chain, described polymer chain comprises:
(a) poly-(N,N-DMAA) chain of long-chain linear, its viscosity-average molecular weight is in the scope of 800kDa～1200kDa;
(b) linear poly-(N,N-DMAA) chain of short chain, its viscosity-average molecular weight is in the scope of 20kDa～60kDa; With
(c) acrylamide and N, the random copolymers chain of N-DMAA, described random copolymers is at the poly-(N of described long-chain linear, the N-DMAA) (a) gathers (N with described short chain linearity, the N-DMAA) passes through acrylamide monomer and N under the existence (b), N-DMAA monomer carries out redox radical polymerization preparation, and the molecular weight size of described random copolymers is in the scope of 1MDa～10MDa
Wherein, described long-chain, the linear poly-(N of short chain, the N-DMAA) chain and described acrylamide and N, the random copolymers chain of N-DMAA tangles each other, IPN, form the accurate interpenetrating polymer networks of polymer chain, and wherein, the accurate interpenetrating polymer networks of described polymer chain does not have chemically crosslinked.
The viscosity-average molecular weight of described long-chain linear poly-(N,N-DMAA) is preferably 1000kDa.
The viscosity-average molecular weight of described short chain linear poly-(N,N-DMAA) is preferably 36kDa.
In the random copolymers chain of described acrylamide and N,N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 90: 10～99.9: 0.1, is preferably 90: 10～99: 1, more preferably 95: 5.
The molecular weight of random copolymerization of the present invention (acrylamide-N,N-DMAA) is preferably 6MDa.
Random copolymerization (acrylamide-N of the present invention, the N-DMAA) ratio with long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of short chain is 50: 50～99: 1, be preferably 80: 20～95: 5, most preferably be 10: 1 (weight ratio).
The ratio of long-chain of the present invention poly-(N,N-DMAA) and short chain poly-(N,N-DMAA) is from 5: 1～1: 1, is preferably 3: 2 (weight ratio).
Accurate interpenetrating polymer networks of the present invention, compare polyacrylamide, random copolymerization wherein (acrylamide-N,N-DMAA) easier and linear poly-(N,N-DMAA) (PDMA) forms more stable accurate interpenetrating polymer networks.The present invention is with the poly-(N of the linearity of two kinds of different molecular weight sizes, the N-DMAA) with acrylamide and N, when the random copolymers of N-DMAA forms accurate interpenetrating polymer networks, long-chain linear gathers (N, the N-DMAA) can increase for capillaceous from the coating ability, poly-(the N of short chain linearity, the N-DMAA) can increase mixing ability between polymkeric substance, make accurate interpenetrating polymer reticulated structure more stable, also guaranteed mutual solubility and the circulation ratio of whole gel.
The present invention combines the outstanding sieving capacity of polyacrylamide and poly-N, the coating function certainly that the N-DMAA is good, by at poly-(N, the N-DMAA) synthetic random copolymerization (acrylamide-N in the solution of Cun Zaiing, N-dimethylpropyl alkene acid amides), form a kind of novel accurate interpenetrating polymer networks.The sieving medium that this novel accurate interpenetrating polymer networks can be used as the gel capillary electrophoresis separation is for separating of biomacromolecule, especially DNA isolation, RNA fragment.It is high that the accurate interpenetrating polymer networks of this random copolymerization has stability, characteristics such as DNA, RNA resolving power height.
According to another aspect of the present invention, the invention provides the method for the accurate interpenetrating polymer networks of preparation polymer chain of the present invention:
At the poly-(N of long-chain linear, the N-DMAA) the linear poly-(N of chain and short chain, the N-DMAA) under the existence of chain with acrylamide monomer and N, N-DMAA monomer carries out redox radical polymerization to obtain the poly-(N of described long-chain linear, the N-DMAA) chain, linear poly-(the N of described short chain, the N-DMAA) chain and random copolymerization (acrylamide-N, the N-DMAA) accurate interpenetrating polymer networks, wherein, described long-chain linear gathers (N, the N-DMAA) viscosity-average molecular weight is in 800kDa～1200kDa scope, preferred 1000kDa, linear poly-(the N of described short chain, the N-DMAA) viscosity-average molecular weight is in 20kDa～60kDa scope, preferred 36kDa, described random copolymerization (acrylamide-N, the N-DMAA) molecular weight size is at 1MDa～10MDa, preferred 6MDa;
Wherein, described long-chain, the linear poly-(N of short chain, the N-DMAA) chain and described random copolymerization (acrylamide-N, the N-DMAA) chain tangle each other, IPN, form the accurate interpenetrating polymer networks of polymer chain, and wherein, the accurate interpenetrating polymer networks of described polymer chain does not have chemically crosslinked.
Wherein, the long-chain linear that molecular weight of the present invention varies in size poly-(N,N-DMAA) and short chain linear poly-(N,N-DMAA) can adopt the ordinary method preparation of this area, as long as meet described molecular weight requirement.
For example, the molecular weight size is in 20kDa～60kDa scope, be preferably the linear poly-(N of short chain of 36kDa, the N-DMAA) be in containing the aqueous solution of Virahol, to cause N,N-DMAA (DMA) polymerization by ammonium persulphate (APS)/Tetramethyl Ethylene Diamine (TEMED) to obtain PDMA.The molecular weight size is in 800kDa～1200kDa scope, be preferably the poly-(N of long-chain linear of 1000kDa, the N-DMAA) causing N,N-DMAA by ammonium persulphate (APS)/Tetramethyl Ethylene Diamine (TEMED) in containing methanol in water is polymerized.
Random copolymerization (acrylamide-N of the present invention; the N-DMAA) be at described long-chain, the linear poly-(N of short chain; have down the N-DMAA), in containing the TTE buffered soln of 7M urea, reduce by the initiated oxidation under nitrogen protection of ammonium persulphate (APS)/Tetramethyl Ethylene Diamine (TEMED) that radical polymerization obtains; thereby obtain acrylamide and N; the random copolymers of N-DMAA and linear accurate interpenetrating polymer networks structure of gathering (N,N-DMAA).Wherein also can use other redox catalysts for radical polymerization that can be used for polypropylene acid amides and N,N-DMAA known in the art except ammonium persulphate/Tetramethyl Ethylene Diamine.
The accurate interpenetrating polymer networks synthetic method of polymer chain of the present invention is simple, and experimental repeatability is good, is prepared from by the redox free radical polymerisation process.Do not need further separation and purification after the reaction, can be directly used in the biomacromolecule material, for example separation of DNA, RNA fragment,
Molecular weight described in the context of the invention is viscosity-average molecular weight.The measuring method of described viscosity-average molecular weight is as described below:
The intrinsic viscosity of polymkeric substance (η) is measured with " dark type viscometer " in NaCl solution, and measuring temperature is 30 ℃.The viscosity-average molecular weight of polymkeric substance (Mv) is calculated with the Mark-Houwink formula
[1,2]
[η]＝9.33×10
-3Mv
0.75
Table 1 illustrated the linear poly-(N of operable short chain among the present invention, the N-DMAA), the poly-(N of long-chain linear, the N-DMAA) and intrinsic viscosity (cP) and the viscosity-average molecular weight Mv (kDa) of random copolymerization (acrylamide-N, N-dimethylpropyl alkene acid amides).
Table 1
| Polymkeric substance | Intrinsic viscosity (cP) | Viscosity-average molecular weight Mv (kDa) |
| Short chain linear poly-(N,N-DMAA) | 21 | 30 |
| Long-chain linear gathers (N,N-DMAA) | 295 | 1000 |
| Random copolymerization (acrylamide-N, N-dimethylpropyl alkene acid amides) | 1130 | 6000 |
[0042] [1]FRANCOIS J，SARAZIN D，SCHWARTZ T，et al.Polyacrylamidein water：molecular weight dependence of<R2>and[η]and theproblem of the excluded volume exponent[J]，Polymer，1979，20：969-975。
[2]SCHWARTZ T，FRANCOIS J，WEILL G.Dynamic dimensions inthe polyacrylamide-water system[J]，Polymer，1980，21：247-249。
According to another aspect of the present invention, the invention provides a kind of method of using capillary gel electrophoresis separating bio macromolecular substance, wherein use the accurate interpenetrating polymer networks of polymer chain of the present invention as sieving medium.In particular, the accurate interpenetrating polymer networks of polymer chain of the present invention can be used at capillary gel electrophoresis separating bio macromolecular substance, and is concrete as DNA, RNA fragment.As MegaBACE1000 and 4000 sequenator DNA isolation or the RNA fragment of using Pharmacia company to produce, average separation length can reach 750bp.
Use the accurate interpenetrating polymer networks of polymer chain of the present invention as sieving medium, it is effective to check order, long service life.Effective being mainly reflected on order-checking length improves of checking order.Gel of the present invention forms protective layer to capillary tube inner wall, belongs to dynamic coating.Coating is even, good reproducibility, and use traditional commercialization polyacrylamide gel order-checking, as the MegaBACE Long Read Matrix sequencing gel of Pharmacia company, capillary tube inner wall must have fixing protective layer.The general MegaBACE length that on average checks order can reach more than the 700bp.In use, the damage that coating can be progressively.Along with the passing of duration of service, the DNA separating effect is variation progressively.And sequencing gel of the present invention has and dynamically is coated with layer function, and capillary tube inner wall does not need the additional coatings protection.Capillary energy is reused.The order-checking mean length can reach 750bp.The problem that does not exist the DNA resolution to change in time.
The accompanying drawing summary
Fig. 1 is the peak figure by MegaBACE 1000DNA Sequencer acquisition from glue of the present invention, is labeled as " MegaBACE is from glue ".
Fig. 2 is the peak figure that is obtained by MegaBACE 1000DNA Sequencer of the commercially available glue that is purchased, is labeled as " MegaBACE commercially available glue ".
Embodiment
All reagent are all available from Aldrich company.
Embodiment 1: linear poly-(N,N-DMAA) preparation (PDMA) of short chain
Remove ionized water 275ml, DMA25ml, IPA (Virahol) 9.5mL in there-necked flask. vacuumized then 15 minutes.Led to nitrogen 1 hour while stirring then.Add 10% APS solution 120 microlitres, TEMED30 microlitre subsequently.Reaction is one hour in 50 degrees centigrade water-bath.Reacted solution was put into the dialysis tubing dialysis two days.The molecular weight cutting of dialysis bag is 12,000.Obtain white solid by freezing and sublimation.Productive rate 80%.The product viscosity-average molecular weight is about 36kDa.
Embodiment 2: the preparation of long-chain linear poly-(N,N-DMAA)
Remove ionized water 460ml, DMA63ml, methyl alcohol 16mL is in there-necked flask.Vacuumized then 15 minutes.Led to nitrogen 1 hour while stirring then.1 as a child added 10% APS 300 microlitres, TEMED 30 microlitres to solution.Reaction is one hour in 50 degrees centigrade water-bath.Reacted solution was put into the dialysis tubing dialysis two days.The molecular weight cutting of dialysis bag is 12,000.Obtain white solid by freezing and sublimation.Productive rate 80%.The product viscosity-average molecular weight is about 1000kDa.
Embodiment 3: the preparation of random copolymerization (acrylamide-N,N-DMAA) and poly-(N,N-DMAA) accurate interpenetrating net polymer gel
Add 175 milliliters of 7M urea in the there-necked flask, add acrylamide 5.50g, add DMA 52 microlitres (i.e. 0.05 gram), add the PDMA glue 0.2g of embodiment 1 gained short chain linearity, add the PDMA glue 0.3g of embodiment 2 gained long-chain linears.Fully mix.Vacuumized then 15 minutes.Lead to nitrogen then while stirring more than 1 hour.Carry out under condition of ice bath, add 10% APS 40 microlitres, TEMED 8 microlitres begin reaction.Being reflected at 4-8 hour can finish.Add 20ml 10XTTE damping fluid (prescription sees Table 2).Gentleness was rocked 12 hours on the shaking table, and resulting gel can be directly used in order-checking.
Embodiment 4: the preparation of random copolymerization (acrylamide-N, N-dimethylpropyl alkene acid amides) and poly-(N,N-DMAA) accurate interpenetrating net polymer gel
Add 175 milliliters of 7M urea in the there-necked flask, add acrylamide 4.95g, add DMA 572 microlitres (i.e. 0.56 gram), add the PDMA gel 0.2g of embodiment 1 gained short chain linearity, add the PDMA gel 0.3g of embodiment 2 gained long-chain linears.Fully mix.Vacuumized then 15 minutes.Lead to nitrogen then while stirring more than 1 hour.Carry out under condition of ice bath, add 10% APS 40 microlitres, TEMED 8 microlitres begin reaction.Being reflected at 4-8 hour can finish.Add 20ml 10XTTE damping fluid (prescription sees Table 2).Gentleness was rocked 12 hours on the shaking table, and resulting gel can be directly used in order-checking.
Embodiment 5: the preparation of random copolymerization (acrylamide-N, N-dimethylpropyl alkene acid amides) and poly-(N,N-DMAA) accurate interpenetrating net polymer gel
Add 175 milliliters of 7M urea in the there-necked flask, add acrylamide 4.94g, add DMA 270 microlitres (i.e. 0.26 gram), add the PDMA gel 0.2g of embodiment 1 gained short chain linearity, add the PDMA gel 0.3g of embodiment 2 gained long-chain linears.Fully mix.Vacuumized then 15 minutes.Lead to nitrogen then while stirring more than 1 hour.Carry out under condition of ice bath, add 10% APS 40 microlitres, TEMED 8 microlitres begin reaction.Add 20ml 10XTTE damping fluid (prescription sees Table 2).Gentleness was rocked 12 hours on the shaking table, and being reflected at 4-8 hour can finish.Resulting gel can be directly used in order-checking.
Table 2
| 10X TTE damping fluid |
| 0.5M Tris-base (Tutofusin tris) 60.55g 0.5M TAPS-acid (N-three (methylol) methyl-3-aminopropanesulfonicacid acid) 121.65g (Sigma part number T9659) 10mM EDTA (ethylenediamine tetraacetic acid (EDTA)) 3.72g |
Embodiment 6: with gained gel separation RNA fragment, carry out macromolecular substance order-checking
1. the details of specimen in use and source
Sample: (Shenzhen provides small cabbage moth RNA, is used for small cabbage moth (Plutella xylostella) the sensitive strain sample in construction cDNA library, comprises worm's ovum, larva, worm pupa and adult.System is gathered in Xin Dian field, Fuzhou City, Fujian Province in July, 2004 by Fujian Academy of Agricultural Sciences.Fuzhou City, Fujian Province Fujian Institute Of Applied Ecological Of Agriculture and Forestry University in beginning in 2006 at indoor feeding so far).
Library construction: full-length cDNA library
Host bacterium: intestinal bacteria
Resistance: paraxin
Carrier: pDNR-LIB 4.2k
Primer: T7:5 '-ACATCCACTTTGCCTTTCTC-3 '
Insert fragment: 1-3k
Plasmid concentration: 50ng/ microlitre
2. instrument:
MegaBACE 1000DNA Sequencer is available from Pharmacia.
3. set factors:
Sample injection voltage: 2KV
Trial voltage: 7.5KV
Sample inject time: 70 seconds
Test period: 145 minutes
4. testing sequence:
Pour into the gel of embodiment 5 gained according to MegaBACE 1000DNA Sequencer instrument (available from Pharmacia) working specification, will contrast the commercial MegaBASE glue (MegaBACE of usefulness simultaneously
TMLong-read Matrix (being the MegaBACE commercially available glue) is available from AmershamBioscience) also be circulated in the other MegaBASE instrument and compare experiment.With the small cabbage moth RNA preparation (construction cDNA library) of checking order, the sample of finally handling is joined respectively in above-mentioned two instruments, check order.Gained the results are shown in accompanying drawing 1 and accompanying drawing 2.
5. test-results:
I. overall contrast:
Table 3.MegaBACE is from the glue test-results
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | 459/85 | 578/93 | 488/91 | 786/95 | 577/93 | 789/94 | 457/94 | 286/88 | 621/95 | 694/96 | 749/94 | 721/93 |
| B | 484/95 | 460/91 | 727/93 | 493/97 | 128/89 | 761/95 | 400/87 | 710/95 | 643/96 | 724/94 | 629/95 | 793/94 |
| C | 469/88 | 409/88 | 502/88 | 666/95 | 650/93 | 721/97 | 4/64 | 643/96 | 547/93 | 611/95 | 767/94 | 224/89 |
| D | 337/94 | 593/89 | 532/94 | 269/87 | 367/84 | 398/91 | 592/91 | 688/95 | 304/91 | 133/94 | 482/88 | |
| E | 130/91 | 132/87 | 465/89 | 821/96 | 727/96 | 139/94 | 165/85 | 735/91 | 548/91 | 562/91 | 643/92 | 549/90 |
| F | 76/77 | 581/90 | 414/88 | 328/96 | 330/83 | 813/94 | 797/92 | 782/92 | 416/86 | 669/95 | 768/93 | 677/92 |
| G | 359/89 | 70/80 | 698/90 | 545/89 | 170/85 | 729/90 | 654/90 | 213/83 | 141/88 | 578/87 | 595/91 | 283/87 |
| H | 2/53 | 441/97 | 819/93 | 114/84 | 477/94 | 732/96 | 755/95 | 765/94 | 516/87 | 782/92 | 129/82 | 629/88 |
Table 4.MegaBACE commercially available glue test-results
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | 434/84 | 488/88 | 209/87 | 410/88 | 475/89 | 271/91 | 467/94 | 142/94 | 486/92 | 240/92 | 377/89 | 446/89 |
| B | 433/92 | 532/89 | 313/90 | 469/90 | 252/86 | 341/88 | 522/89 | 570/92 | 658/91 | 423/87 | 467/89 | 613/88 |
| C | 470/91 | 480/90 | 280/91 | 306/87 | 203/91 | 413/91 | 74/83 | 376/88 | 243/86 | 288/89 | 232/87 | |
| D | 427/90 | 286/91 | 218/86 | 194/82 | 261/90 | 443/89 | 504/88 | 200/88 | 133/91 | 302/86 | ||
| E | 131/94 | 335/86 | 274/87 | 469/88 | 248/91 | 26/78 | 587/92 | 325/87 | 425/90 | 352/87 | ||
| F | 154/81 | 509/86 | 248/89 | 320/92 | 217/86 | 460/92 | 480/93 | 438/88 | 365/91 | 467/89 | 279/93 | 338/85 |
| G | 330/88 | 163/80 | 305/88 | 221/89 | 287/84 | 405/92 | 134/92 | 451/90 | 287/92 | 154/93 | ||
| H | 5/67 | 387/92 | 469/88 | 134/91 | 412/90 | 590/93 | 706/88 | 272/89 | 543/92 | 260/91 | 314/87 |
[0087]Illustrate:
(1) have in the grid of data in above-mentioned table 3 and the table 4: have underscore for the order-checking effect the poorest, italic for the order-checking effect relatively poor, all the other for the order-checking effective;
(2) in above-mentioned table 3 and the table 4: horizontal, ordinate zou is represented hole count, specifically is 96 orifice plates.
(3) in above-mentioned table 3 and the table 4: data are example with 434/84, wherein 434 be the base number, namely read long; 84 is success ratio, is the instrument data presented.
The length of always reading in the table 5 is counted the total alkali radix of addition for the base of above-mentioned 96 orifice plates, that is, long for always reading.
Table 5
| Always read long | Success ratio | |
| MegaBACE is from glue | 48528 | 79％ |
| The MegaBACE commercially available glue | 30647 | 55％ |
The contrast of I I. kapillary number of times:
MegaBACE is from glue
Checking order needs to change kapillary after 867 times.
The MegaBACE commercially available glue
Checking order needs to change kapillary after 538 times.
III. peak figure contrast:
See also accompanying drawing 1-2.
IV. discussion of results:
At first, aggregate analysis, MegaBACE of the present invention is from glue no matter peak figure, peak type, or on work-ing life all above the MegaBACE commercially available glue.
Particularly:
(1) by overall contrast, MegaBACE from glue always read long, on average read long and success ratio all is higher than the MegaBACE commercially available glue.The data number of representative " it is effective to check order " is out and away more than the data number that represents " it is effective to check order " in the table 4 in the table 3.
(2) by peak figure contrast, the MegaBACE commercially available glue can not clearly be read base about 300bp, and peak separation is not right.
Yet, still can clearly read base behind the MegaBACE glue 300bp of the present invention, meet the requirements.
(3) observe by the kapillary access times, use MegaBACE to be longer than the MegaBACE commercially available glue work-ing life far away from the kapillary of glue.
Claims (24)
1. the accurate interpenetrating polymer networks of a polymer chain, described polymer chain comprises:
(a) poly-(N,N-DMAA) chain of long-chain linear, its viscosity-average molecular weight is in the scope of 800kDa～1200kDa;
(b) linear poly-(N,N-DMAA) chain of short chain, its viscosity-average molecular weight is in the scope of 20kDa～60kDa; With
(c) acrylamide and N, the random copolymers chain of N-DMAA, described random copolymers is at the poly-(N of described long-chain linear, the N-DMAA) the linear poly-(N of chain (a) and described short chain, the N-DMAA) passes through acrylamide monomer and N under the existence of chain (b), N-DMAA monomer carries out redox radical polymerization preparation, the molecular weight size of described random copolymers is in the scope of 1MDa～10MDa, wherein at described acrylamide and N, in the random copolymers chain of N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 90: 10～99.9: 0.1;
Wherein, described long-chain, the linear poly-(N of short chain, the N-DMAA) and described acrylamide and N, the random copolymers chain of N-DMAA tangles each other, IPN, form the accurate interpenetrating polymer networks of polymer chain, and wherein, the accurate interpenetrating polymer networks of described polymer chain do not have chemically crosslinked and
Wherein said random copolymerization (acrylamide-N,N-DMAA) is 50: 50～99: 1 with the weight ratio of described long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of described short chain.
2. the accurate interpenetrating polymer networks of the polymer chain of claim 1, the viscosity-average molecular weight of wherein said long-chain linear poly-(N,N-DMAA) is 1000kDa.
3. the accurate interpenetrating polymer networks of the polymer chain of claim 1, the viscosity-average molecular weight of wherein said short chain linear poly-(N,N-DMAA) is 36kDa.
4. the accurate interpenetrating polymer networks of the polymer chain of claim 1, the molecular weight of wherein said random copolymers is 6MDa.
5. the accurate interpenetrating polymer networks of the polymer chain of claim 1, wherein at described acrylamide and N, in the random copolymers chain of N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 90: 10～99: 1.
6. the accurate interpenetrating polymer networks of the polymer chain of claim 5, wherein in the random copolymers chain of described acrylamide and N,N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 95: 5.
7. the accurate interpenetrating polymer networks of the polymer chain of claim 1, wherein said random copolymerization (acrylamide-N, the N-DMAA) with the poly-(N of described long-chain, the N-DMAA) and the weight ratio of poly-(N,N-DMAA) both sums of described short chain be 80: 20～95: 5.
8. the accurate interpenetrating polymer networks of the polymer chain of claim 7, wherein said random copolymerization (acrylamide-N, the N-DMAA) weight ratio with described long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of described short chain is 10: 1.
9. each the accurate interpenetrating polymer networks of polymer chain of claim 1-8, the weight ratio that wherein said long-chain poly-(N,N-DMAA) and described short chain gather (N,N-DMAA) is 5: 1～1: 1.
10. the accurate interpenetrating polymer networks of the polymer chain of claim 9, the weight ratio that wherein said long-chain poly-(N,N-DMAA) and described short chain gather (N,N-DMAA) is 3: 2.
11. the method for the accurate interpenetrating polymer networks of the polymer chain of preparation claim 1:
At the poly-(N of long-chain linear, the N-DMAA) the linear poly-(N of chain and short chain, the N-DMAA) under the existence of chain with acrylamide monomer and N, N-DMAA monomer carries out redox radical polymerization to obtain the poly-(N of described long-chain linear, the N-DMAA) chain, linear poly-(the N of described short chain, the N-DMAA) chain and random copolymerization (acrylamide-N, the N-DMAA) accurate interpenetrating polymer networks, wherein at described acrylamide and N, in the random copolymers chain of N-DMAA, described monomer acrylamide and described monomer N, the weight ratio of N-DMAA is 90: 10～99.9: 0.1
Wherein, described long-chain linear gathers (N, the N-DMAA) viscosity-average molecular weight is in 800kDa～1200kDa scope, linear poly-(the N of described short chain, the N-DMAA) viscosity-average molecular weight is in 20kDa～60kDa scope, the molecular weight size of described random copolymerization (acrylamide-N,N-DMAA) is at 1MDa～10MDa;
Wherein, described long-chain, the linear poly-(N of short chain, the N-DMAA) chain and described random copolymerization (acrylamide-N, the N-DMAA) chain tangle each other, IPN, form the accurate interpenetrating polymer networks of polymer chain, and wherein, the accurate interpenetrating polymer networks of described polymer chain do not have chemically crosslinked and
Wherein said random copolymerization (acrylamide-N,N-DMAA) is 50: 50～99: 1 with the weight ratio of described long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of described short chain.
12. the method for claim 11, the viscosity-average molecular weight of wherein said long-chain linear poly-(N,N-DMAA) is 1000kDa.
13. the method for claim 11, the viscosity-average molecular weight of wherein said short chain linear poly-(N,N-DMAA) is 36kDa.
14. the method for claim 11, the molecular weight of wherein said random copolymerization (acrylamide-N,N-DMAA) is 6MDa.
15. the method for claim 11, wherein in the random copolymers chain of described acrylamide and N,N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 90: 10～99: 1.
16. the method for claim 15, wherein in the random copolymers chain of described acrylamide and N,N-DMAA, the weight ratio of described monomer acrylamide and described monomer N,N-DMAA is 95: 5.
17. the method for claim 11, wherein said random copolymerization (acrylamide-N, the N-DMAA) weight ratio with described long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of described short chain is 80: 20～95: 5.
18. the method for claim 17, wherein said random copolymerization (acrylamide-N,N-DMAA) is 10: 1 with the weight ratio of described long-chain poly-(N,N-DMAA) and poly-(N,N-DMAA) both sums of described short chain.
19. each method of claim 11-18, the weight ratio that wherein said long-chain poly-(N,N-DMAA) and described short chain gather (N,N-DMAA) is 5: 1～1: 1.
20. the method for claim 19, the weight ratio of wherein said long-chain poly-(N,N-DMAA) and described short chain poly-(N,N-DMAA) is 3: 2.
21. by each the accurate interpenetrating polymer networks of polymer chain of method preparation of aforementioned claim 11-20.
22. a method of using capillary gel electrophoresis separating bio macromolecular substance wherein uses the accurate interpenetrating polymer networks of each polymer chain in aforementioned claim 1-10 and 21 as sieving medium.
23. the method for claim 22, wherein said biomacromolecule material is DNA.
24. the method for claim 22, wherein said biomacromolecule material is RNA.
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