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WO1997007395A1 - Milieu de separation pour electrophorese capillaire - Google Patents

Milieu de separation pour electrophorese capillaire Download PDF

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Publication number
WO1997007395A1
WO1997007395A1 PCT/US1996/012765 US9612765W WO9707395A1 WO 1997007395 A1 WO1997007395 A1 WO 1997007395A1 US 9612765 W US9612765 W US 9612765W WO 9707395 A1 WO9707395 A1 WO 9707395A1
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WO
WIPO (PCT)
Prior art keywords
separation
matrix
buffer
capillary
separation medium
Prior art date
Application number
PCT/US1996/012765
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English (en)
Inventor
Barbara A. Siles
Dennis J. Reeder
Bruce G. Collier
Original Assignee
Trevigen, Inc.
UNITED STATES OF AMERICA, as represented by THE SECRETARY OF COMMERCE, NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Trevigen, Inc., UNITED STATES OF AMERICA, as represented by THE SECRETARY OF COMMERCE, NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY filed Critical Trevigen, Inc.
Publication of WO1997007395A1 publication Critical patent/WO1997007395A1/fr

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Classifications

    • 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

  • the present invention relates to an improved polymer medium for the separation of DNA fragments and other biomolecules via capillary electrophoresis.
  • the present invention also relates to the separation of similar molecular species, including plasmid conformers and oiigonucleotides.
  • matrices have been used previously for the separation of DNA fragments in capillary electrophoresis (CE) . These matrices fall into two categories, chemically crosslinked and physically entangled. Chemically crosslinked matrices, most often composed of polyacrylamide, present a variety of problems, including heat sensitivity leading to bubble formation and loss of electrical conductivity during separation; sample-to-sample carryover contamination limiting the lifetime of the gels. Dubrow, CAPILLARY ELECTROPHORESIS: THEORY AND PRACTICE, Academic Press, Inc. (1992) , pages 133-157. But chemically crosslinked gels do provide single-base or base-pair resolution and extremely high separation efficiencies.
  • the so-called "physically entangled" polymeric matrices that have been employed as separation media in capillary electrophoresis include but are not limited to linear (uncrosslinked) polyacrylamide (Guttman, HANDBOOK OF CAPILLARY ELECTROPHORESIS, CRC Press (1994) , page 129); hydroxyethyl cellulose (Grossman et al. , J. Chroma togr. 559: 257 (1991)); hydroxypropylmethyl cellulose (Baba et al . , loc. ci t . 653: 329 (1993)) ; methyl cellulose (MacCrehan et al . , J. Liq. Chromatogr .
  • a low viscosity separation medium for capillary electrophoresis comprising an aqueous, buffered solution that contains (i) a derivatized agarose and (ii) an adjuvant polymer that interacts non- covalently with said agarose to neutralize charges thereon in solution such that electroendosmotic flow is decreased, wherein said solution has a total polymer concentration in the range of about 0.02% and about 1.0% by weight and is non-gelling at ambient temperatures.
  • the adjuvant polymer is dextran, polyvinyl alcohol, or a galactomannan, such as clarified locust bean gum.
  • the derivatized agarose and the adjuvant polymer are present in a weight ratio of between about 1:2 and 5:1.
  • the buffered solution is a TPE buffer of pH 7.
  • Figure 1 shows the effect of a lower matrix fraction range -- 0.050 (A), 0.10 (B) and 0.20 % (C) -- of the new polymer matrix on the separation of the 1 Kbp DNA ladder at 30°C, -3 kV, 47 cm X 50 ⁇ m i.d. DB-17 capillary (-64 V/cm) in TPE buffer at pH 8.3.
  • Figure 2 shows the effect of a higher mass fraction range (up to 0.50 %) of the new polymer matrix on the separation of the X174/Hae III DNA standard at 30°C, -5 kV, 36 cm X 50 ⁇ m i.d. BioRad BioFocus linear polyacrylamide coated capillary (-139 V/cm) in TPE buffer at pH 7.
  • Figure 3 shows the effect of buffer pH and buffer composition on the separation of the ⁇ X174/Hae III DNA standard at 30°C, -5 kV, 35.5 cm X 75 ⁇ m i.d. Supelco H-75 methyl coated capillary (-140 V/cm) using 0.20 % of the new matrix in buffer.
  • Figure 4 shows the effect of applied field strength on the separation of the ⁇ X174/Hae III DNA standard at 30°C, 35.5 cm X 75 ⁇ m i.d. Supelco H-75 methyl coated capillary, using 0.20 % of the new matrix in TPE buffer at pH 7.
  • Figure 5 shows the comparison of the new polymer matrix, (0.02 % in TPE at pH 7; (C) and (D) ) with two commercially available separation matrix solutions ( (A) and (B))for the separation of the ⁇ X174/Hae III DNA standard at the indicated recommended capillary temperatures and field strengths, using a 36 cm X 50 ⁇ m BioRad BioCAP linear polyacrylamide-coated capillary.
  • Figure 6 shows a comparison of the background UV absorbances at 260 and 280 nm of the new polymer matrix (0.02 % in TPE at pH 7) with two commercially available separation matrix solutions.
  • Figure 7 shows the electropherograms of a DNA standard (A) and the pTZ18R plasmid DNA (B) and two restriction enzyme cleavage products ( (C) and (D) ) at 30°C, 35.5 cm X 75 ⁇ m i.d. Supelco H-75 methyl coated capillary, using 0.20 % of the new matrix in TPE buffer at pH 7.
  • Figure 8 shows the separation of two oiigonucleotides differing by three nucleotides at 30°C, 35.5 cm X 75 ⁇ m i.d. Supelco H-75 methyl coated capillary, using 0.20 % of the new matrix in TPE buffer at pH 7.
  • a polymer matrix comprised of derivatized agarose and an adjuvant polymer adopts a low-viscosity form that is especially suitable for capillary electrophoresis, in part because it does not form a gel at ambient temperatures and, hence, is readily loaded into capillaries and used in a CE regimen without special temperature constraints.
  • the low viscosity and non-gelling character of the inventive matrix composition is surprising in light of known separation compositions in which agarose interacts with an adjuvant polymer to yield an "entangled" structure associated with higher viscosity gels. See U.S. patent No. 4,290,911 and H. Schwartz & A.
  • the separation medium of the present invention provides a level of molecular-weight resolution in biomolecule separations that is unexpected in view of conventional separation compositions.
  • agarose denotes a polysaccharide generally comprised of galactose and altered anhydrogalactose residues, with sulfate residues.
  • a "derivatized agarose” is one that has been reacted with an alcohol so that hydroxymethyl or hydroxyethyl groups are added to the polysaccharide molecule.
  • Agaroses thus derivatized typically have a gelling temperature below about 18°C, which is advantageous in the context of the present invention.
  • the production of derivatized agaroses are described, for example, in U.S. patents No. 5,143,646 and No. 3,956,273, the respective contents of which are hereby incorporated by reference.
  • a matrix composition according to the present invention comprises an aqueous, buffered solution of derivatized agarose and another polymer, denoted the “adjuvant polymer,” which interacts non-covalently with the agarose to neutralize charges on the outside of the agarose molecule in solution, thereby decreasing electroendosmotic (EEO) flow.
  • the suitable adjuvant polymers for this purpose are galactomannans, particularly clarified locust bean gum (cLBG) , and dextran, both of which adopt helical conformations in solution such that charges on the molecule are lined up in a manner that complements charges on the hydrated agarose molecule.
  • polyvinyl alcohol also could fulfill the role of charge-neutralizing adjuvant polymer.
  • the derivatized agarose and adjuvant polymer are present in a ratio adjusted to meet requirements of viscosity and pore size associated with CE.
  • an agarose:polymer weight ratio of between about 1:2 and about 5:1 is suitable, and more preferably is between 1:2 and 3:1.
  • Other ratios may be determined empirically to meet the requirements of a given CE regimen.
  • the total polymer concentration in a separation medium of to the present invention generally is between about 0.02 % and about 1.0% by weight, and more preferably is between about 0.05 % and 0.5 % by weight.
  • the present invention thus contemplates the use of the above-described matrix composition for the separation via CE of biopolymers, including but not limited to polynucleotides and polypeptides.
  • DNA's typically less than 600 base pairs in size, while higher field strengths are recommended for larger DNA's.
  • the inventive matrix offers high run-to-run migration time reproducibility and more rapid and enhanced separation relative to existing polymer solutions for this purpose.
  • DNA fragments in the 100 to 10,000 base pair size range can be separated according to the present invention.
  • separations were performed using either a Hewlett-Packard 3D CE system or a BioRad BioFocus 3000 CE system, each in the reversed polarity mode, with the cathode (negatively-charged electrode) at the capillary inlet and the anode (positively-charged electrode) at the capillary outlet.
  • Detection was by UV absorbance at 260 nm with a bandwidth of 30 nm.
  • the response time of each detector was set at 1.0 second.
  • the reference wavelength was set at 370 nm with a bandwidth of 100 nm.
  • Electrophoresis units equipped with a UV detector, with optics optimized for path lengths in the range of 25 to 100 ⁇ m, and a high voltage power supply up to 7500 V.
  • Capillaries are recommended that are of 50 to 75 ⁇ m i.d. and 24 to 50 cm in length. The use of a shorter capillary is limited by the capillary housing of the capillary electrophoresis unit. Longer capillary lengths proportionally increase migration times of the fragments, in some instances with loss of separation efficiency due to extended residence time of the fragment in the capillary leading to diffusion.
  • the capillaries preferably are coated internally with moderately hydrophobic functionalities, thereby masking or "deactivating" hydrophilic functionalities otherwise presented by the glass or plastic surface, so as nearly to eliminate EEO flow.
  • the temperature of the capillary contained within the Hewlett Packard capillary cartridge was equilibrated and maintained at 30.0°C with a Peltier cooling/heating device.
  • the capillary contained within the BioRad capillary cartridge was equilibrated and maintained at 30.0°C by liquid cooling, circulating a 50/50 mixture of methanol/water.
  • the capillary temperature in the range of 20°C and 40°C was not found to have a significant impact on the separation ability of — o —
  • the capillary was conditioned (flushed) with matrix solution for three minutes, each at the maximum pressure of each unit.
  • the Hewlett Packard CE system with a maximum pressure of 14 p.s.i. was not capable of loading matrix solutions with mass fractions of greater than 0.20 %.
  • the BioRad BioFocus CE system with a maximum pressure of 100 p.s.i.
  • the capillary was capable of loading up to 0.50 % mass fraction solutions of the matrix.
  • the capillary was rinsed with an aqueous solution of HCl (pH 2.5) or NaOH (pH 11) for 1 minute and then with HPLC-grade water for two minutes.
  • the acidic pH rinse preserves the integrity of the capillary coating better than the basic pH rinse.
  • Covalent capillary coatings are most susceptible to aqueous solution pHs that are outside the range of 2.5 to 7.
  • the concentration of the matrix in buffer solution can be varied for optimization of the size range of DNA to be separated.
  • Lower matrix mass fractions for example, in the range of about 0.05 % to about 0.20 % by weight, provide high separation ability for larger DNA fragments, i.e., greater than 500 bp in length.
  • Higher matrix mass fractions (0.30 to 0.50 %) in buffer solution provide high separation ability for smaller DNA fragments less than 500 base pairs in length.
  • the intermediate concentration of 0.20 % (w/w) of the new polymer matrix provides high separation efficiency for oiigonucleotides and plasmid conformers as well.
  • the buffer composition in which the new separation matrix is dissolved affects the separation of the DNA fragments.
  • the inventors have investigated four different buffer compositions: Tris-phosphate-EDTA (1 X TPE) at pH 7 and pH 8; Tris-acetate-EDTA (1 X TAE) at pH 8; Tris-borate-EDTA (1 X TBE) at pH 8.
  • the buffers were prepared with HPLC-grade water, as 0.100 mol/L Tris base and 0.100 mol/L phosphoric acid, acetic acid or boric acid (Fisher Scientific) and 0.001 mol/L EDTA. Each buffer was filtered through a 0.45 ⁇ m porosity nylon Acrodisc syringe filter (Gelman Sciences) before use.
  • Matrix solutions were prepared by dissolving a weighed quantity of matrix into 10 mL of the appropriate buffer in a 25 mL screw-cap volumetric test tube at 95°C in a water bath. Heating the solutions in a microwave oven was not required since the matrix dissolved readily into each buffer at a mass fraction of less than or equal to 0.50 %.
  • TBE buffer was not able to separate DNA fragments that were shorter than 600 base pairs in length.
  • TAE buffer was less effective in the separation of smaller fragments versus TPE buffer, especially the 271/281 pair.
  • the separation efficiency of the 100 bp DNA ladder increased as the counterion in the buffer was varied according to the following series; borate ⁇ acetate ⁇ phosphate. Separation efficiency (N) is defined by the equation:
  • N 5.54 (t/w 1/2 ) 2 , where t is the migration time and w 1/2 is the peak width at half height.
  • the widespread use of the other, more commonly used buffers in CE is due to their lower background currents during electrophoresis and from extensive previous use in slab gel electrophoresis.
  • the absolute value of the background current increased in the order, borate ⁇ acetate ⁇ phosphate.
  • the absolute values of the background, current for a 35.5 cm X 75 ⁇ m for the three buffers (pH 8) at the applied field strength of 141 V/cm were 7.8, 54 and 86 ⁇ amps, respectively.
  • the absolute value of the background current is dependent on the capillary dimensions including internal diameter and length, the applied run voltage and the buffer composition.
  • the matrix of the present invention in TPE buffer allows more efficient separations than that observed in TAE or TBE buffers, even though the background current obtained with TPE buffer was higher.
  • the interactions between the matrix and the acidic buffer component appear to override joule heating effects which often contribute to loss of efficiency and overall loss of resolution in CE. See Knox, Chromatographia 26: 329-37 (1988) . It is believed that complexation between the components of the three different buffers and the new polysaccharide matrix alters the characteristics of the polymer network formed by the matrix in this mass fraction range and, hence, its separation ability.
  • TPE buffer In addition, the lowest UV absorbance background is observed with the use of TPE buffer. The detectability of DNA fragments is severely limited with the use of TBE buffer.
  • Other commercially available polymer solutions for the separation of DNA fragments by CE are dissolved TBE buffer at pH 8.3.
  • TPE buffer of pH 7.0 with this new matrix provides higher separation efficiency relative to the use of pH 8.0 of the same buffer components.
  • the lower buffer pH also extends the lifetime of the capillary coating.
  • the applied field strength may be varied from 70 to 210 V/cm for applied voltages of 2500 to 7500 V for a 35.5 cm X 75 ⁇ m i.d. capillary.
  • the use of the highest applied field strength is recommended in order to minimize the time required for separation and to optimize the efficiency of separation for larger DNA fragments, which may migrate in a biased manner, i . e . , may be subject to a biased reptation mode of migration, through the polymeric matrix during electrophoresis.
  • An intermediate applied field strength of 140 V/cm was optimal for smaller DNA fragments, less than 600 bp in length.

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  • Chemical & Material Sciences (AREA)
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Abstract

Une nouvelle matrice de polysaccharides dans un EDTA Tris-phosphate ou dans un autre tampon approprié apporte des avantages surprenants dans la séparation des oligonucléotides, y compris l'ADN double brin, parmi d'autres biomolécules. La matrice de séparation confère une aptitude améliorée à la séparation, une viscosité moins importante, et une absorbance UV de fond inférieure à 260 ou 280 nm pour une détectabilité perfectionnée, par rapport aux autres solutions polymères disponibles sur le marché et que l'on utilise dans ce contexte. La figure 5 fournit une illustration de la séparation améliorée en comparant les résultats de séparation des solutions polymères actuelles (A) et (B) à la solution polymère des inventions (C) et (D).
PCT/US1996/012765 1995-08-21 1996-08-12 Milieu de separation pour electrophorese capillaire WO1997007395A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US257595P 1995-08-21 1995-08-21
US60/002,575 1995-08-21

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WO1997007395A1 true WO1997007395A1 (fr) 1997-02-27

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2180229C1 (ru) * 2001-01-19 2002-03-10 ООО Научно-производственное фармацевтическое предприятие "Полидан" Способ получения растворов натрия нуклеоспермата

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4321121A (en) * 1981-01-19 1982-03-23 Beckman Instruments, Inc. Electrophoretic technique for separating lactate dehydrogenase isoenzymes and improved electrophoretic gel for use therein
JPH03113357A (ja) * 1989-09-28 1991-05-14 Shimadzu Corp 電気泳動用緩衝液及びキャピラリー電気泳動法
JPH04248460A (ja) * 1991-02-04 1992-09-03 Kurita Water Ind Ltd ゾーン電気泳動用ゲル
JPH07113785A (ja) * 1993-10-15 1995-05-02 Kurita Water Ind Ltd キャピラリー電気泳動用緩衝液
JPH07128287A (ja) * 1993-06-29 1995-05-19 Ina Food Ind Co Ltd 等電点電気泳動用アガロース
US5455344A (en) * 1993-09-03 1995-10-03 Fmc Corporation Agarose compositions for nucleic acid sequencing

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4321121A (en) * 1981-01-19 1982-03-23 Beckman Instruments, Inc. Electrophoretic technique for separating lactate dehydrogenase isoenzymes and improved electrophoretic gel for use therein
JPH03113357A (ja) * 1989-09-28 1991-05-14 Shimadzu Corp 電気泳動用緩衝液及びキャピラリー電気泳動法
JPH04248460A (ja) * 1991-02-04 1992-09-03 Kurita Water Ind Ltd ゾーン電気泳動用ゲル
JPH07128287A (ja) * 1993-06-29 1995-05-19 Ina Food Ind Co Ltd 等電点電気泳動用アガロース
US5455344A (en) * 1993-09-03 1995-10-03 Fmc Corporation Agarose compositions for nucleic acid sequencing
JPH07113785A (ja) * 1993-10-15 1995-05-02 Kurita Water Ind Ltd キャピラリー電気泳動用緩衝液

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2180229C1 (ru) * 2001-01-19 2002-03-10 ООО Научно-производственное фармацевтическое предприятие "Полидан" Способ получения растворов натрия нуклеоспермата

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