High-viscosity polymer matrix and methods

• US 5,164,055 A
• Filed: 01/29/1990
• Issued: 11/17/1992
• Est. Priority Date: 01/29/1990
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Dubrow, Robert S.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Starsiak, Jr., John S.
• Application Number:
  US07/472,045
• Time in Patent Office: 1,023 Days
• Field of Search: 204/299 R, 204/180.1, 204/183.2, 204/182.8, 204/182.9
• US Class Current: 204/455
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44795 Isoelectric focusing

Abstract

A supported, substantially uniform matrix for use in electric field-induced separation of molecular components in a sample. The matrix is prepared by forming a viscoelastic polymer matrix and pumping the matrix into a capillary or preparative-scale tube. The polymer type, concentration, and molecular weight are selected to optimize separation of protein or nucleic acid components. The matrix can be expelled from the tube, after fractionation, for analysis and/or recovery of fractionated components.

Claims

1. A supported matrix for use in electric field-induced separation of nucleic acids in a sample comprising:

a support containing an elongate separation chamber, and

contained within said chamber, and filling the chamber substantially uniformly, an aqueous viscoelastic matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer having a molecular weight of at least about 200,000 daltons and having a concentration in the matrix of between about 0.1 to 5 percent by weight, said polymer selected from the group consisting of polyvinyl alcohol and a water-soluble, hydroxylated cellulose compound, and

(ii) a viscosity of at least about 100,000 centipoise.

2. The matrix of claim 1 for use in separating nucleic acids wherein the polymer is a mixture of a first polymer selected from the group consisting of polyethylene oxide, polyacrylamide, and polymethacrylamide, and a second polymer selected from the group consisting of a water-soluble, hydroxylated cellulose compound and polyvinyl alcohol.

3. The matrix of claim 1, for use in isoelectric focusing, wherein the polymer matrix contains ampholyte species effective to establish a substantially continuous pH gradient over a selected pH range.

4. A method of electrophoretic separation of molecular component sin a sample comprising:

adding a sample to one end region of a support defining an elongate separation chamber which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer having a molecular weight of at least 5-10 kilodaltons, and

(ii) a viscosity of at least 5,000 centipoise,

separating said components by applying an electric field across opposite end regions of the chamber, until a desired degree of electrophoretic separation of the components is achieved,

removing a selected region of the polymer matrix containing a separated sample component,

liquefying the removed region to form a low-viscosity liquid, and

isolating the sample component from the polymer in the liquid.

5. The method according to claim 4, wherein said liquefying is carried out by diluting the solution in an aqueous medium.

6. A method of fractionating a sample of nucleic acid components in the size range less than about 100 bases, comprising

adding said sample to one end region of a support defining an elongate separation chamber which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by:

(i) a mixture of polymers, one of which is selected from selected from the group consisting of polyethylene oxide, polyacrylamide, and polymethacrylamide, and another of which is selected from the group consisting of a water-soluble hydroxylated cellulose compound,

(ii) polymer molecular weights of molecular weight of at least about 100,000 daltons, and

(iii) a viscosity of at least about 100,000 centipoise, and

separating nucleic acid components by applying an electric field across opposite end regions of the chamber, until a desired degree of electrophoretic separation of the components is achieved.

7. The method of claim 6, wherein the polymer mixture contains at least about 5 weight percent of each of the polymers in the mixture.

8. The method of claim 6, for separation of oligonucleotides having sizes less than about 50 bases, wherein the polymer mixture has a viscosity, as measured by as measured by the pressure needed t pump the matrix through a 30 cm length of 75 μ

capillary tube, at a flow rate of 100 μ

l/min, at room temperature, is at least about 3,000 psi.

9. A method of separating molecular components in a sample by isoelectric focusing comprising:

adding the sample to a matrix supported in an elongate separation chamber which is filled, substantially uniformly, with an aqueous ampholyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer having a molecular weight of at least about 5-10 kilodaltons, and

(ii) a viscosity of at least 5,000 centipoise, and

separating said components by applying an electric field across opposite end regions of the chamber, until the ampholyte has equilibrated to produce a selected pH gradient across the end regions of the chamber, and the molecular components have migrated to their isoelectric points within the chamber,

removing a selected region of the polymer matrix containing a separated sample component,

liquefying the removed region to form a low-viscosity liquid, and

isolating the sample component from the polymer in the liquid.

10. The method of claim 9, wherein said liquefying is carried out by diluting the solution in an aqueous medium.

11. A method of electrophoretic separation of molecular components in a sample comprising:

adding a sample to one end region of an electrophoresis tube which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer having a molecular weight of at least 5-10 kilodaltons, and

(ii) a viscosity of at least 5,000 centipoise,

separating said components by applying an electric field across opposite end regions of the tube, until a desired degree of electrophoretic separation of the components is achieved,

forming the matrix out of the tube, and

monitoring the matrix for the presence of discrete sample component bands as the matrix is forced from the tube.

12. A method of electrophoretic separation of oligonucleotides in a sample comprising:

adding a sample of to one end region of an electrophoresis tube which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked hydroxylated cellulose compound having a molecular weight of at least 5-10 kilodaltons, and

(ii) a viscosity of at least 5,000 centipoise, and

separating said components by applying an electric field across opposite end regions of the chamber, until a desired degree of electrophoretic separation of the components is achieved.

13. A method of electrophoretic separation of oligonucleotides in a sample comprising:

adding a sample to one end region of an electrophoresis tube which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer mixture having a molecular weight of at least 5-10 kilodaltons, said polymer matrix comprising a first polymer selected from the group consisting of polyethylene oxide, polyacrylamide and polymethacrylamide and a second polymer selected from the group consisting of a water-soluble, hydroxylated cellulose compound and polyvinyl alcohol,

(ii) a viscosity of at least 5,000 centipoise, and

separating said components by applying an electric field across opposite end regions of the chamber, until a desired degree of electrophoretic separation of the components is achieved.

14. A method of electrophoretic separation of a phosphorylated low molecular weight nucleic acid component from its non-phosphorylated analog in a sample comprising:

adding a sample to one end region of an electrophoresis tube which is filled, substantially uniformly, with an aqueous electrolyte-containing polymer matrix characterized by;

(i) a viscosity of at least 5,000 centipoise, and

separating said components by applying an electric field across opposite end regions of the chamber, until a desired degree of electrophoretic separation of the components is achieved.

15. A method of separating molecular components in a sample by isoelectric focusing comprising:

adding the sample to a matrix supported in a tube which is filled, substantially uniformly, with an aqueous ampholyte-containing polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer having a molecular weight of at least about 5-10 kilodaltons, and

(ii) a viscosity of at least 5,000 centipoise,

separating said components by applying an electric field across opposite end regions of the tube, until the ampholyte has equilibrated to produce a selected pH gradient across the end regions of the tube, and the molecular components have migrated to their isoelectric points within the tube,

forcing the matrix out of the tube, and

monitoring the matrix for the presence of discrete sample component bands as the matrix is forced from the tube.

16. A method of preparing a supported, substantially homogeneous matrix for use in electric field-induced separation of nucleic acids in a sample comprising:

forming a viscoelastic, flowable aqueous polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer, having a molecular weight of at least about 5-10 kilodaltons,

(ii) a viscosity of at least about 5,000 centipoise, and

(iii) containing an electrolyte; and

pumping the polymer matrix, in its high-viscosity state, into an elongate separation chamber, to fill the chamber uniformly with the matrix, wherein the polymer is a hydroxylated polymer selected from the group consisting of a water-soluble hydroxylated cellulose compound and polyvinyl alcohol.

17. A method of preparing a supported, substantially homogeneous matrix for use in electric field-induced separation of nucleic acids in a sample comprising:

forming a viscoelastic, flowable aqueous polymer matrix characterized by;

(i) a water-soluble, substantially non-crosslinked polymer, having a molecular weight of at least about 5-10 kilodaltons,

(ii) a viscosity of at least about 5,000 centipoise, and

(iii) containing an electrolyte; and

pumping the polymer matrix, in its high-viscosity state, in to an elongate separation chamber, to fill the chamber uniformly with the matrix, wherein the polymer is a mixture of a first polymer selected from the group consisting of polyethylene oxide, polyacrylamide, and polymethacrylamide, and a second polymer selected from the group consisting of a water-soluble, hydroxylated cellulose compound and polyvinyl alcohol.