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WO1997018463A1 - Supports polymeres dereticules pour electrophorese - Google Patents

Supports polymeres dereticules pour electrophorese Download PDF

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Publication number
WO1997018463A1
WO1997018463A1 PCT/US1996/018113 US9618113W WO9718463A1 WO 1997018463 A1 WO1997018463 A1 WO 1997018463A1 US 9618113 W US9618113 W US 9618113W WO 9718463 A1 WO9718463 A1 WO 9718463A1
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gel
temperature
polymer
electrophoretic
crosslinked
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Herbert H. Hooper
Alexander P. Sassi
David S. Soane
Young Bae
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Monogram Biosciences Inc
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Soane Biosciences Inc
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Priority claimed from US08/589,150 external-priority patent/US5885432A/en
Application filed by Soane Biosciences Inc filed Critical Soane Biosciences Inc
Priority to AU77280/96A priority Critical patent/AU7728096A/en
Publication of WO1997018463A1 publication Critical patent/WO1997018463A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • Electrophoresis is an extremely versatile tool with which to idenufy compounds associated with developments in biotechnology. Electrophoresis is used extensively for the separation, identification and preparaUon of pure samples of nucleic acids, polypeptides, and carbohydrates.
  • Separations may be associated with the detection of biomolecules (polypeptides, polynucleotides, polysaccharides, and combinations thereof, for example, glycosylated proteins, DNA-protein complexes) having different molecular charactenstics, such as numbers of monomers, different sequences, different conformations, different charge/mass ratios, or different hydrophobicities/ hydrophihcities Essential to the success of the gel electrophoresis is the nature of the gel and the manner in which it is prepared
  • the gel medium in electrophoresis serves one or more iunctions
  • the medium can serve as an anti-convective support, a molecular sieve, a gradient of pH, or some other function.
  • two compositions dominate the gel compositions which are generally employed in slab gel formats: polyacrylamide and agarose.
  • the polyacrylamides are normally crosslinked to provide for a sieving structure, where the proportion of crosslinking monomers determines the molecular weight range which may be separated by the gel.
  • the addition polymers are normally formed in situ, where substantial care must be taken in the preparation of the gel to insure uniformity, substantial completion of the polymerization, and reproducibility of the separations achieved on the gel.
  • the source of agarose is a naturally occurring material, so there can be great variation in the quality of the agarose, the nature of the contaminants, and the like. Therefore, there is substantial uncertainty in going from one batch to another batch of agarose whether one is obtaining gels of comparable quality.
  • the agarose contributes mechanical and anticonvective properties, and prevents the un-crosslinked polymer from dissolving into the surrounding buffer.
  • examples include combinations of un-crosslinked polyacrylamide and agarose (Bode et al.) and combinations of hydroxyethylcellulose and agarose (Perlman et al.).
  • compositions to those described above have been suggested such as combinations of agarose and galactomannan (U.S. Patent No. 5,230,832), modified cellulose (Perlman et al. Anal. Biochem. 163:247-254 (1987)), water soluble gums (U.S. Patent No. 4,290,91 1; 4,894,250; and 4,952,686), and 1,3- glucans (WO 93/08200). Hydroxyethylcellulose has been suggested for use in capillary electrophoresis (Barron et al. J. Chromatography A, 652:3-16 (1993)). Entangled polymer solutions for capillary electrophoresis have been described in Heller, J. Chromatography A, V710N2 (1995) 309-321.
  • Electrophoretic device components, methods, and compositions for use in such components are provided, where the compositions comprise un-crosslinked high molecular weight polymers having a reversible temperature responsive viscosity change in polar solutions.
  • the un-crosslinked polymers may be used by themselves or in conjunction with non -temperature sensitive un-crosslinked polymers, cross- linked polymers or gelling agents.
  • the compositions go from a low viscosity, pourable state to a high viscosity, sieving state for electrophoresis.
  • the compositions find use in various electrophoretic gel configurations, such as slab gels, capillaries and microchannels.
  • the methods taught here comprise adjusting the temperature such that the composition is on the low viscosity side of the transition during loading into the device, and then changing the temperature such that the composition is on the high viscosity side of the transition during separation.
  • This method which has not been previously taught to our knowledge, can be practiced with any compositions that undergo a reversible viscosity transition of the type described herein. Exemplary compositions are provided, but the generality of the method is such that it may be practiced with a wide variety of similar or different compositions that exhibit the requisite viscosity responsiveness. General rules for identifying families of such compositions are taught, wherein the method taught here can be applied with these other compositions.
  • Fig. 1 is an electropherogram illustrating the results of the separation of ⁇ X174-H ⁇ e /// DNA fragments in a slab gel composite matrix of agarose, ⁇ PC and ⁇ EC.
  • Fig. 2 is an electropherogram illustrating the results of the separation of ⁇ X174-H ⁇ e /// DNA fragments in a slab gel composite matrix of agarose, linear poly DMA/DEA and ⁇ EC.
  • Fig. 3 is an electropherogram illustrating separation of pBR322/ / DNA fragments by capillary electrophoresis in a composite matrix of HEC and HPC, as described in Example 7.
  • Figs 4a & 4b are electropherograms showing the separation of ⁇ X174-H ⁇ e /// DNA fragments by capillary electrophoresis in a composite matrix of HEC and HPC at 25 and 38 °C, as described in Example 8.
  • Figs. 5a provides a viscosity profile for a media comprising a synthetic, temperature sensitive un-crosslinked polymer
  • Fig. 5b is an electropherogram of M13mpl8 fragments by capillary electrophoresis in the media, as described in Example 9.
  • Preparing gels for electrophoresis is substantially simplified by employing as the sieving composition un-crosslinked high molecular weight polymers, which polymers may be synthetic, naturally occurring, or modified naturally occurring polymers.
  • the polymers may be addition polymers, condensation polymers, homo polymers, co-polymers, block, random, or graft polymers, and the like.
  • the un-crosslinked polymers may be used by themselves or in conjunction with crosslinked polymers or gelling agents.
  • the polymers will, for the most part, provide a viscosity at a temperature in the range of about 20 to 50°C at a weight % concentration of 0.25 to 20%, more usually 0.5 to 10% , of from about 1 to 100,000 cP, usually 1 to 50,000 cP, more usually 5 to 10,000 cP.
  • the media may be used for complete filling of the gel or matrix holder of the device in which the media are employed.
  • the media comprise a continuous fluid phase. In the high viscosity state, the media will have at least a 100% greater viscosity than the media in the low viscosity state.
  • the viscosity in the high viscosity state will be at least about 100 cP, more usually at least about 750 cP, generally at least about 1,000 cP, and the viscosity may be as high as 5 million cP or higher, where the media becomes a solid, elastic gel-like solid mass that is no longer flowable, pourable, or pumpable.
  • the media will comprise a continuous fluid phase, normally comprising a polar solvent, preferably an aqueous polar solvent, where the amount of water would generally range from about 10-100% .
  • the media may comprise various organic polar solvents, such as ethanol, dimethylformamide, hexamethylphosphoramide, acetonitrile, diethylether, dimethyl sulfoxide, etc.
  • the co-solvents will be present in less than about 90 volume percent, more usually less than about 50 volume percent, particularly less than about 25 volume percent.
  • various salts particularly buffering salts, where the concentration of the salts will vary from about 0.01 to 0.5, more usually 0.01 to 0.1 M.
  • the salts may include Tris, phosphate, EDTA, borate, acetate, MOPS, etc.
  • the pH may vary widely, generally being in the range of about 2 to 10, more usually in the range of about 5 to 9.
  • the un-crosslinked polymers which provide for temperature responsive viscosity changes may be varied widely as to their composition.
  • the un-crosslinked polymer may be a natural or synthetic homopolymer, random copolymer, multi- block copolymer, grafted copolymer of a linear, branch or comb-like structure, and the like.
  • a number of thermoreversible un-crosslinked polymers have been reported in the literature.
  • thermoreversible is intended a polymer which in a polar, usually aqueous, medium is able to go from a pourable solution to a high viscosity medium through a narrow, usually less than 20°C, more usually in the range of about 10 to 15°C, temperature change.
  • modified celluloses such as hydroxyalkyl celluloses where alkyl is of from 2 to 4, usually 2 to 3 carbon atoms, which may have been further modified by alkylation of the cellulose with an alkyl group of from 1 to 3 carbon atoms, usually 1 to 2 carbon atoms.
  • Illustrative compositions include ethylhydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc.
  • These derivatized naturally occurring polymers have molecular weight ranges in the range of about 75 to 500 kD.
  • Naturally occurring polysaccharides include tamarind seed polysaccharides, and the like.
  • Various addition polymers may be employed, prepared from a wide variety of derivatized acrylamide (includes methacrylamide) monomers, where the derivatives are present on the amide nitrogen.
  • Substituents on the nitrogen include adamantyl; glycinyl; benzyl; cyclohexyl; diethyl; dodecyl; 3-methoxypropyl; ethoxyethyl; tetrahydrofurfuryl; isobornyl; dimethyl; diacetonyl; 3-
  • (diethylamino)propyl; methyl; and 1-naphthylmethyl will range from about 1 to 12 carbon atoms, more usually from about 1 to 8 carbon atoms, may be aliphatic, alicyclic, aromatic, heterocyclic, or combinations thereof, substituted or unsubstituted, generally be substituted with oxygen or nitrogen, as oxy, oxo or amino.
  • copolymers of N,N-dialkylamides where the alkyl groups are of from 1 to 5, usually 1 to 3 carbon atoms, more particularly where the alkyl groups on a nitrogen atom are the same.
  • Other polymers which may be used include polyvinyl alcohols.
  • the addition polymers may be prepared by any convenient polymerization technique which affords the desired minimum molecular weight of at least about 30 kD, more usually at least about 100 kD, and maybe up to 5,000 kD or more, as a number average molecular weight.
  • free radical polymerization is carried out in appropriate diluent, where the monomer(s) are dissolved in an appropriate media, such as water, aqueous/organic, or organic medium. Polymerization is initiated using initiators appropriate for the monomer and medium, e.g. ammonium persulfate/TMED, ammonium persulfate/ sodium metabisulfate in aqueous media, azoisobutyronitrile in organic media, and the like.
  • the monomers are dissolved in the medium of choice in conjunction with the initiator and stirred.
  • oxygen may be removed from the solution prior to initiation of the reaction by means such as vacuum degassing, bubbling a stream of nitrogen or helium through the medium, or the like.
  • the resultant molecular weight will be influenced by the concentration of initiator, temperature of reaction, choice of diluent and concentration of monomer.
  • the resulting polymers may be purified with extensive dialysis against pure water and subsequently recovered by conventional drying methods, such as freeze-drying, spray drying, film-drying, roll-drying, precipitation and the like, followed by washing.
  • Graft copolymers of two distinct polymers may be employed as the un- crosslinked polymer, where a first polymer forms a scaffolding structure and a second polymer, engrafted onto the first polymer, reversibly changes its structure or orientation with respect to the first polymer in response to an applied stimulus.
  • An exemplary graft polymer would be one in which a first or scaffolding polymer forms a structure of large pores with minimal sieving capability.
  • the second or switchable polymer would be engrafted onto the first polymer so that in the first state prior to application of the stimulus, the engrafted second polymer strands would align adjacent to the first polymer thereby occupying a minimal portion of the pores of the first polymer.
  • the second polymer Upon application of the stimulus, the second polymer changes position so as to occupy a substantial portion of the pores of the first polymer, thereby significantly changing the sieving properties of the graft polymer medium.
  • the first or scaffolding polymer that provides a structure to the media would generally be one which reversibly changes from a first to a second state at a different applied stimulus from the stimulus which causes the second, engrafted polymer to transition from the first to the second state.
  • the first polymer which is flowable at a first elevated temperature and forms a solid, porous matrix at a second temperature.
  • the second, engrafted polymer could change position, thereby filling the pores of the first polymer and changing the sieving properties of the media.
  • the engrafted polymer may then change position so as to substantially fill the pores of the first polymer.
  • the change in position of the engrafted polymer may be proportional to the change in temperature, providing for control over the degree of filling of the pore size of the media and the possibility of dynamically changing the pore size of the media, as will be discussed further below.
  • Illustrative graft co- polymers having temperature reversible viscosity changes include N-isopropyl acrylamide grafted with acrylic acid, poly(oxyethylene), poly (hydroxyethylmethacrylate), and the like (see Rempt and Franta, Advances in Polymer Science 58, Springer-Berleg. 1984, Macromonomersr Synthesis, Characterization, and Applications; Chen and Hoffman (1995) Nature 373:49-52; de Vos and Moller, Makromol. Chem. , Macromol. Symp. 75:223-229 (1993)). Combinations of the various un-crosslinked thermoreversible polymers may be employed where 2 or more, usually not more than 4, may be used in a single composition.
  • Each of the component polymers may vary from about 5 to 95 weight % of the total polymer composition. Where 2 polymers are used, the amount of each polymer will be in the range of about 5 to 95, usually from about 15 to 85, more usually from about 20 to 80, and often from about 25 to 75 weight % of the total polymer composition.
  • Non-temperature sensitive polymers of interest include homopolymers and copolymers of both synthetic and natural origin.
  • the un- crosslinked non-temperature sensitive polymers may be derivatized, branched, grafted and the like.
  • Specific un-crosslinked non-temperature sensitive polymers of interest include polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethylcellulose, and the like. Each of the component polymers may vary from about 5 to 95 weight percent of the total polymer composition.
  • the amount of each polymer will be in the range of about 5 to 95, usually from about 15 to 85, more usually from about 20 to 80, and often from about 25 to 75 weight % of the total polymer composition.
  • the media containing only the un-crosslinked temperature sensitive polymers may not form a gel.
  • other additives may be provided which provide for gelation.
  • Gelling or binding agents which may be present in the media in conjunction with the thermoreversible un-crosslinked polymer may be any natural, synthetic or derivatized natural substance which, in combination with the un-crosslinked polymer, does not interfere with the temperature reversible viscosity change, nor the sieving properties of the gel.
  • the total concentration of the un-crosslinked polymer and gelling agent will generally be at least about 0.1 and not more than about 25 weight percent, preferably not more than about 10 weight percent of the hydrated gel.
  • the total concentration of the gelling agent will generally be in the range of about 0.1 to 25 weight percent, preferably not more than about 10 weight percent, usually not more than about 5 weight percent of the hydrated gel.
  • Binding or gelling agents may include agar, agarose, carrageenan, curdlan, gelatin, polyacrylylglycinamide, polymethacrylylglycinamide, tamarind seed polysaccharide, or the like, where the gelling agent may have a viscosity profile which is temperature sensitive or non-temperature sensitive, as temperature sensitive is defined above.
  • the binding or gelling agent may be introduced by mixing with the viscosity temperature responsive un-crosslinked polymer during preparation of the electrophoresis medium or by soaking the medium contained in a permeable mold in a solution of the binding agent.
  • the subject media may also comprise the un-crosslinked temperature sensitive polymers in conjunction with a crosslinked polymer.
  • the crosslinked polymer will form large pores in which the temperature sensitive un-crosslinked polymers are entrapped, i.e. the crosslinked polymer serves as a framework for the un-crosslinked temperature sensitive polymers.
  • Media comprising un-crosslinked temperature sensitive polymers entrapped in a crosslinked polymer framework provide for the possibility of dynamic porosity during electrophoresis, as described in greater detail below.
  • Specific crosslinked polymers that may be included in the media to provide a framework for the un-crosslinked temperature sensitive polymers include: crosslinked polyacrylamide, and the like.
  • the crosslinked polymeric framework can be synthesized in a solution of the temperature sensitive, un-crosslinked polymer(s).
  • the media according to the subject invention may include surfactants, stabilizers, denaturants, and the like. Included among the electrophoretic devices in which the subject media find use are slab devices, column devices, microchannel devices, capillary devices, and the like.
  • gels may be pre-prepared or safely prepared by the end user. Therefore, for the most part, where the gels are to be pre- cast, the gels will have the sieving high viscosity characteristic at room temperature.
  • the first step is the introduction of the medium into the gel holder of the device.
  • the medium will be introduced into the gel holder in the first state of low viscosity. Since the medium is readily pourable and flowable in the first state, it will fill the gel holder without voids, bubbles, or other i ⁇ egularities.
  • the medium Following introduction of the medium into the gel holder, by providing for a temperature change which results in the transition of the media from the first to the second state, the medium will then be in a state for electrophoresis.
  • the stimulus may be applied using any convenient heating or cooling means.
  • electrophoresis of the sample in the device may then be carried out by introducing the sample into the gel medium and applying a sufficient voltage gradient across the gel.
  • the buffers employed and the subject gels may be used in accordance with conventional techniques used for other electrophoretic gels, such as crosslinked polyacrylamide and agarose. Any sample amenable to electrophoresis may be employed, where the sample may comprise a single or plurality of components.
  • Samples employed for separation or identification may include nucleic acids, proteins, carbohydrates, combinations thereof, under naturing or denaturing conditions. If desired, following electrophoresis, the medium may be returned to the first state of low viscosity through application of the appropriate stimulus, for removal from the electrophoretic device, and other processing.
  • the subject media are suitable for use in any electrophoretic device comprising a gel holder, the media are particularly applicable for use in slab, capillary or microchannel gel electrophoresis devices.
  • the medium is first introduced into the separation chamber or interior space of a capillary using any convenient means, such as injection or suction by vacuum where the medium is in the first state of low viscosity. Once the medium is introduced into the capillary, the medium is converted into the second state of high viscosity to provide a sievable composition. Electrophoresis of the sample may then be performed.
  • the medium may be maintained in a constant state, so as to provide for constant sieving charactenstics du ⁇ ng electrophoresis of the sample.
  • the conditions of the media may be modulated du ⁇ ng electrophoresis, thereby changing the sieving properties of the media.
  • dynamic sieving conditions du ⁇ ng electrophoresis where the sieving conditions may be optimized for a particular sample to achieve more efficient resolution of the separated sample compounds over the course of the electrophoretic run. For example, a first set of conditions may be employed and the sample partially separated. After partial separation, the conditions may be changed, whereby more defined separation may be obtained
  • One may use stepwise changing of the conditions or gradual dynamic changing of the conditions.
  • the magnitude of temperature change used to achieve dynamic porosity in the medium will generally range from about 1 to 70 °C, usually from about 5 to 50 °C, more usually from about 5 to 20 °C, where the temperature modulation will usually take place over a penod of minutes to hours depending on the time scale of the preparation.
  • the gel may be reused, depending upon the nature of the sample which had been employed. Where the gel is to be discarded, the capillary can be easily flushed free of medium by changing the medium conditions back to the low viscosity first condition under which the capillary was filled.
  • the subject gels provide for good separation of both large and small sample components. Separations can be achieved which are the equivalent of gels compnsing approximately 3.5 to 10% w/v polyacrylamide and 3.33 % crosslinking. For slab gel electrophoresis, good resolution is obtained with differences of ten nucleotides with nucleotides with dsDNA having from 10 to 300 bp.
  • HEC Hydroxyethylcellulose
  • EHEC Ethylhydroxyethylcellulose
  • N,N'-d ⁇ methylacrylam ⁇ de and N,N'-d ⁇ ethylacrylam ⁇ de were used to synthesize linear poly-DMA/DEA (50/50).
  • the viscous solution was transferred to dialysis tubing (12,000 MW cutoff) and dialyzed in a five-gallon tank containing deionized distilled water. The water was changed daily for three days to ensure removal of unreacted monomer and low molecular weight oligomers.
  • the solution was transferred to freeze-dry in a flask and frozen at -20° C. The polymer was freeze dried for four days under a vacuum of 0.04 mbar. The resulting polymer was a low density foam-like material which could be readily dissolved in an aqueous medium to provide the subject compositions.
  • HEC 0.75 g of HEC (MW 90,000-105,000) and 0.75 g of HPC (MW 300,000) were dissolved in 98.5 g of 0.5 X TBE by vigorous manual shaking followed by turning the solution overnight. 0.75 g of HEC and 0.75 g of HPC were dissolved in
  • the viscosity behavior for solutions containing un-crosslinked temperature sensitive polymers and un-crosslinked non-temperature sensitive polymers in combination with a gelling agent is similar to that for solutions containing only un-crosslinked polymers which do not gel, as described in Example 2.
  • the viscosity of media containing a gelling agent or binding agent such as agarose increases gradually until the vicinity of the transition temperature (42 °C in this example), whereupon the viscosity increases sharply and dramatically with further cooling. If the gelling or binding agent is present in sufficient quantity, such as in this example, the solution will gel, and the viscosity will become effectively infinite.
  • Example 4 Slab Gel Electrophoresis of dsDNA fragments in a Composite Matnx Containing agarose, a derivatized Natural Polymer with Temperature Sensitivity, and a Non-Temperature Sensitive Polymer
  • HEC MW 90,000-105,000
  • HPC MW 300,000
  • DNA samples (from left to nght in the gel) were- 10 bp ladder, pBR322/ SP /, 100 bp ladder, ⁇ X174/H ⁇ e ///, 10 bp ladder, pBR322/Msp /, ⁇ XHA/Hae III, and 100 bp ladder.
  • the resultant gel gave good resolution of the DNA fragments.
  • the 10 bp ladder was resolved up to 150 bp, and the 100 bp ladder was resolved completely (100-1500 bp). All of the ⁇ Xl74/HaeIII fragments were resolved (72, 118, 194, 234, 271 , 281 , 310, 603, 872, 1078, and 1353 bp), See Fig. 1 , and all of the pER322/Msp I fragments were resolved (67, 76, 90, 1 10, 123, 147, 160, 180, 190, 201 , 217, 242/238, 307, 404, 527, 622 bp) except that the 238 and 242- bp fragments appeared as one band.
  • 2.5 grams of a blend contaimng equal parts of ⁇ EC
  • Example 5 Slab Gel Electrophoresis of dsDNA fragments in a Composite Matrix Containing agarose, a Synthetic, Temperature-Sensitive Polymer, and a Non- Temperature Sensitive Polymer
  • the solution was sti ⁇ ed to dissolve the ⁇ EC (MW 90,000-105,000) and dilute the un- crosslinked polymer. After the addition of 0.4 g agarose, the solution was heated with stirring to dissolve the agarose. Ethidium bromide was added to the solution to a final concentration of 0.5 ⁇ g/mL. The solution was allowed to cool to approximately 60°C before pouring into a 15 by 20 cm casting tray ( ⁇ oefer). Electrophoresis was run for 2 hours at 5.8 V/cm in 0.5 X TBE buffer.
  • DNA samples (from left to right) are: pBR322/ s/> /, 100 bp ladder, ⁇ XllA/Hae UI fragment, 10 bp ladder, 10 bp ladder, 100 bp ladder, ⁇ X174/H ⁇ e ///, and pBR322/Ms/> /.
  • the resultant gel gave good resolution.
  • the 10 bp ladder was resolved up to 150 bp, and the 100 bp ladder was resolved completely (100-1500 bp).
  • All of the ⁇ Xll IHaelll fragments were resolved (72, 118, 194, 234, 271 , 281 , 310, 603, 872, 1078, and 1353 bp), See Fig. 2, and all of the pBR322/M ⁇ / fragments were resolved (67, 76, 90, 110, 123, 147, 160, 180, 190, 201, 217, 242/238, 307, 404, 527, 622 bp) except that the 238 and 242- bp fragments appeared as one band.
  • EHEC electrokinetically for 20 seconds at 10 kV.
  • Electrophoresis was performed using a Beckman P/ACE System 2100 at 14 kV. DNA was detected via absorbance at 260 nm. All fragments were resolved except for the 271 and 281 bp-fragments, which appeared as one peak on the electropherogra .
  • HPC MW 300,000
  • HPC solution MW 300,000
  • a 57.5-cm capillary was pressure- loaded with the HPC solution at 350 psi.
  • the 75-micron-ID capillary was coated with polyacrylamide following the procedure of Hjerten (Hjerten 1985).
  • DNA fragments of ⁇ XMAIHaelll were loaded electrokinetically for four seconds at 10 kV. Electrophoresis was performed using a Beckman P/ACE System 2100 at 15 kV.
  • Example 7 Capillary Electrophoresis ofdsDNA Fragments in a Solution Containing Temperature-Sensitive and Non-Temperature-Sensitive Polymer
  • HEC HEC
  • HPC HPC
  • 0.4 g of HEC (MW 90,000-105,000) and 1.5 g of HPC (MW 300,000) were dissolved in 98.1 g of 0.5 X TBE by vigorous manual shaking followed by turning overnight.
  • a 57-cm capillary was pressure-loaded with the HEC/HPC solution at 350 psi.
  • the 75-micron-ID capillary was coated with polyacrylamide following the procedure of Hjerten (Hjerten, 1985).
  • DNA fragments of pBR322/ ⁇ / were loaded electrokinetically for seven seconds at 10 kV.
  • Electrophoresis was performed using a Beckman P/ACE System 2100 at 14 kV. Excellent resolution of all fragments was obtained, including the two 147-bp fragments which differ only in sequence. See Fig. 3.
  • Example 8 Capillary Electrophoresis ofdsDNA Fragments in a Solution Containing Temperature-Sensitive and Non-Temperature-Sensitive Polymer at two different temperatures
  • HEC 1.25 g HEC (MW 90-105,000) and 0.25 g HPC (MW 300,000) were dissolved in 98.5 g lxTBE by vigorous manual shaking followed by turning overnight. A 57 cm capillary was pressure loaded with the HEC/HPC solution at 350 psi. The 75 micron ID capillary was coated with polyacrylamide following the procedure of Hjerten (Hjerten, 1985).
  • Figure 4a is an electropherogram for a run conducted at 25 °C.
  • Figure 4b is an electropherogram for a run conducted at 38 °C.
  • DNA fragments of ⁇ X174/HaeIII were loaded electrokinetically for 10 seconds (Fig. 4a) or 7 seconds (Fig. 4b) at 10 kV. Electrophoresis was performed using a Beckman P/ACE System 2100 at 11 kV.
  • Example 9 Capillary Electrophoresis in a Separation Medium Comprising a
  • N,N-diethylacrylamide and N,N-dimethylacrylamide were used to synthesize linear poly-DMA/DEA (30/70).
  • the solution was prepared at 6% T.
  • a solution was prepared containing 7.2 g DMA, 16.8 g DEA and 376 mL deionized distilled water. Helium was bubbled for 1 hour through the solution while stirring.
  • To the solution was added rapidly 320 ⁇ l of 20 weight percent ammonium persulfate and 320 ⁇ l of TEMED. The bottle was sealed and stirring continued slowly at room temperature for 30 minutes. The bottle was then transfe ⁇ ed to a refrigerator at 4°C and the reaction was allowed to proceed for 24 hours.
  • the viscous solution was transferred to dialysis tubing (6-8,000 MW cutoff) and dialyzed in a five gallon tank for three days to ensure removal of unreacted monomer and low molecular weight oligomers.
  • the polymer solution was transferred to a freeze-drying flask and placed in a freezer for 48 hours. The frozen polymer solution was then lyophilized for three days. The resulting dried polymer was redissolved in lxTBE, 7M urea at a concentration of 6 weight percent.
  • the viscosity of the 6 weight % solution of DEA/DMA copolymer was measured as a function of temperature using a Brookfield DVII+ viscometer as before. Viscosity data is shown in Fig. 5a. The viscosity transition occurs in the vicinity of 55 °C for this preparation.
  • a 70 cm, 75 micron inner diameter, polyacrylamide coated capillary was pressure loaded at 50 psi.
  • DNA from an M13mpl8 sequencing reaction for T- terminated fragments was loaded electrokinetically for 15 second at 12 kV. Fragments were prepared via primer extension using the Sequanase Sequencing Kit (Amersham Life Science). Electrophoresis was performed at 12 kV using a Beckman P/ACE System 2100, and the results are shown in Fig. 5b. Single-base resolution was obtained up to 150 bases, demonstrating the feasibility of this type of temperature sensitive matrix for DNA sequencing.
  • the subject invention provides for greatly improved applications for gel electrophoresis, by providing for gels which can be readily pre-cast, easily loaded, even into microchannels and capillaries, and provide for excellent separation, even of closely related sample components.
  • the gel may then be removed by changing the temperature to the pourable state and the gel removed. Being able to change the viscosity also allows for ease of separation of the components being separated.
  • the subject gels provide for excellent clarity, handling properties and, as needed, mechanical strength.

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Abstract

Cette invention se rapporte à des supports d'électrophorèse, comprenant au moins des polymères déréticulés qui, selon la température, peuvent passer de façon réversible d'un état faiblement visqueux à un état hautement visqueux, de façon à être coulants à une première température et à offrir des propriétés de crible à une seconde température. Dans ces polymères déréticulés qui ont un pouvoir de changement de viscosité réversible en fonction de la température peuvent être inclus des agents gélifiants et des polymères qui n'ont pas ce pouvoir de changement de viscosité réversible selon la température. De telles compositions possèdent d'excellentes propriétés en ce qui concerne leur transparence, leur aptitude à subir des séparations et des résolutions, leur capacité à être manipulées et leur résistance mécanique. Ces compositions sont compatibles avec le transfert des constituants séparés du gel vers une membrane accepteur.
PCT/US1996/018113 1995-11-13 1996-11-12 Supports polymeres dereticules pour electrophorese Ceased WO1997018463A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77280/96A AU7728096A (en) 1995-11-13 1996-11-12 Un-crosslinked polymeric media for electrophoresis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US664695P 1995-11-13 1995-11-13
US60/006,646 1995-11-13
US08/589,150 US5885432A (en) 1992-11-05 1996-01-22 Un-crosslinked polymeric media for electrophoresis
US08/589,150 1996-01-22

Publications (1)

Publication Number Publication Date
WO1997018463A1 true WO1997018463A1 (fr) 1997-05-22

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/018113 Ceased WO1997018463A1 (fr) 1995-11-13 1996-11-12 Supports polymeres dereticules pour electrophorese

Country Status (2)

Country Link
AU (1) AU7728096A (fr)
WO (1) WO1997018463A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5104954A (en) * 1987-10-05 1992-04-14 Ciba-Geigy Corporation Thermostropic biphilic hydrogels and hydroplastics

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5104954A (en) * 1987-10-05 1992-04-14 Ciba-Geigy Corporation Thermostropic biphilic hydrogels and hydroplastics

Also Published As

Publication number Publication date
AU7728096A (en) 1997-06-05

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