Claims
- 1. A chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides, said method comprising:
a) applying a solution of said fragments and counterion reagent to a column containing polymeric beads having non-polar surfaces, wherein said beads have an average diameter of about 1 to about 100 microns; b) eluting said RNA molecules with a mobile phase which includes said counterion reagent and an organic component.
- 2. A method of claim 1 wherein said surfaces are characterized by being substantially free from multivalent cations which are free to bind with said RNA.
- 3. A method of claim 1 wherein said eluting is carried out at a minimum denaturing temperature.
- 4. A method of claim 1 including collecting mobile phase fractions containing said RNA molecules.
- 5. A method of claim 1 further including detecting RNA molecules during step (b).
- 6. A method of claim 1 wherein said segregating is performed by Matched Ion Polynucleotide Chromatography.
- 7. A method of claim 1 wherein said surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation.
- 8. A method of claim 1 wherein said multivalent metal cations are selected from the group consisting for Fe(III), Cu(II), Cr(III), and colloidal metal.
- 9. A method of claim 1 wherein said mobile phase includes a multivalent cation binding agent.
- 10. A method of claim 9 wherein said cation binding agent comprises EDTA.
- 11. A method of claim 1 wherein said medium comprises polymer beads having an average diameter of 0.5 to 100 microns, the non-polar surfaces being unsubstituted or having bound thereto a hydrocarbon group having from 1 to 1,000,000 carbons.
- 12. A method of claim 1, wherein said mobile phase comprises a counterion agent and an organic solvent, wherein said organic solvent is water soluble.
- 13. A method of claim 12, wherein said solvent is selected from the group consisting of alcohol, acetonitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof.
- 14. A method of claim 12, wherein said solvent comprises acetonitrile.
- 15. A method of claim 1 wherein said counterion agent is selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkyammonium salt, quaternary ammonium salt, and mixtures of one or more thereof.
- 16. A method of claim 15 wherein said counterion agent is selected from the group consisting of octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, triethylammonium hexafluoroisopropyl alcohol, and mixtures of one or more thereof.
- 17. A method of claim 1 wherein said medium has been subjected to acid wash treatment to remove any residual metal contaminants.
- 18. A method of claim 1 where said medium has been subjected to treatment with multivalent cation binding agent.
- 19. A method of claim 2 wherein said eluting is carried out at a temperature greater than about 60° C.
- 20. A method of claim 19 wherein said eluting is carried out at a temperature within the range of about 60° C. to about 90° C.
- 21. A method of claim 1 wherein the pH of said mobile phase is within the range of about pH 5 to about pH 9.
- 22. A method of claim 21 wherein the pH of said mobile phase is about pH 7.
- 23. A method of claim 1 wherein said mixture comprises RNA molecules exceeding about 1,000 nucleotides.
- 24. A method of claim 1 wherein said mixture comprises RNA molecules having up to about 20,000 nucleotides.
- 25. A chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides, said method comprising:
a) applying a solution of said fragments and counterion reagent to a column containing polymeric beads having non-polar surfaces, wherein said beads have an average diameter of about 1 to about 100 microns; b) eluting said RNA molecules at a minimum denaturing temperature.
- 26. A method of claim 25 wherein step (b) includes eluting said RNA molecules with a mobile phase which includes said counterion reagent and an organic solvent.
- 27. A method of claim 25 wherein said temperature is in the range of about 60° C. to about 100° C.
- 28. A method of claim 25 wherein said surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation.
- 29. A chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides, said method comprising:
a) flowing the mixture through a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, said beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from 1 to 1,000,000 carbons, and b) segregating said mixture of RNA molecules at a minimum denaturing temperature.
- 30. A chromatographic method for segregating a mixture of RNA molecules, said method comprising:
a) flowing the mixture through a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, said beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from 1 to 1,000,000 carbons, wherein said beads are characterized by being substantially free from multivalent cations which are free to bind with said RNA molecules, and b) segregating said mixture of RNA molecules at a minimum denaturing temperature.
- 31. An improved method for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, said mixture comprising molecules having lengths exceeding about 100 nucleotides, the method comprising:
a) applying a solution of said molecules and counterion reagent to a column containing polymeric separation beads having non-polar surfaces, wherein said separation beads have an average diameter of 1 to 100 microns, said column having an ID greater than about 5 mm; b) eluting said RNA molecules with a mobile phase which includes said counterion reagent and an organic component.
- 32. A method of claim 31 wherein said eluting is carried out at a temperature within the range of about 60° C. to about 90° C.
- 33. A method of claim 31 wherein said ID is greater than about 7 mm.
- 34. A method of claim 31 wherein said ID is greater than about 10 mm.
- 35. A method of claim 31 wherein said ID is greater than about 50 mm.
- 36. A method of claim 31 wherein said ID is in the range of about 5 mm to about 1 m.
- 37. An improved reverse phase chromatography column for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, the mixture comprising molecules having lengths exceeding about 100 nucleotides, the column comprising:
a cylinder containing polymer beads, said beads having an average diameter of 1 to 100 microns, said beads being unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons, said column having an ID greater than about 5 mm.
- 38. A column of claim 37 wherein said ID is greater than about 7 mm.
- 39. A chromatography system for segregating a mixture of RNA molecules by Matched Ion Polynucleotide Chromatography, comprising:
the column of claim 37.
- 40. A chromatographic method for segregating a mixture of RNA molecules having lengths exceeding about 100 nucleotides, said method comprising:
a) applying a solution of said fragments and counterion reagent to a column containing polymeric beads having non-polar surfaces, wherein said beads have an average diameter of about 1 to about 100 microns, wherein said surfaces are characterized by being substantially free from multivalent cations which are free to bind with said RNA; b) eluting said RNA molecules with a mobile phase which includes said counterion reagent and an organic component.
- 41. A method of claim 40 wherein said eluting is carried out at a minimum denaturing temperature.
- 42. A method for segregating a mixture comprising RNA molecules, the method comprising:
a) applying said mixture to a polymeric separation medium having non-polar surfaces, wherein said surfaces are characterized by being substantially free from multivalent cations which are free to interfere with RNA segregation, and b) eluting said RNA molecules with a mobile phase which includes counterion reagent and an organic component.
- 43. A method of claim 42 wherein said multivalent metal cations are selected from the group consisting of Fe(III), Cu(II), Cr(III), and colloidal metal.
- 44. A method of claim 42 wherein said medium has been subjected to acid wash treatment to remove any residual metal contaminants.
- 45. A method of claim 42 where said medium has been subjected to treatment with multivalent cation binding agent.
- 46. A method of claim 42 wherein step (b) includes, with a mobile phase containing a counterion agent, eluting said mixture at a minimum denaturing temperature.
- 47. A method of claim 46 wherein said eluting is carried out at a temperature greater than about 55° C.
- 48. A method of claim 47 wherein said eluting is carried out at a temperature within the range of about 60° C. to about 90° C.
- 49. A method of claim 42 wherein said medium comprises polymeric beads having non-polar surfaces, wherein said beads have an average diameter of about 1 to about 100 microns.
- 50. A method of claim 42 wherein the non-polar surfaces comprise the surfaces of interstitial spaces of a polymeric monolith.
- 51. A method of claim 50 wherein said surfaces are unsubstituted or substituted with a hydrocarbon group having from 1 to 1,000,000 carbons.
- 52. A method of claim 50 wherein said monolith comprises a member selected from the group consisting of monovinyl substituted aromatic compound, divinyl substituted aromatic compound, acrylate, methacrylate, polyolefin, polyester, polyurethane, polyamide, polycarbonate, fluoro-substituted ethylene, and combinations of one or more thereof.
CROSS REFERENCE TO RELATED CO-PENDING APPLICATIONS
[0001] This application is a continuation in part of U.S. patent application Ser. no. 09/557,424 filed Apr. 10, 2000, which is a continuation in part of U.S. patent application Ser. No. 09/183,123 filed Oct. 30, 1998 (now U.S. Pat. No. 6,066,258), which is a continuation in part of U.S. patent application Ser. No. 09/058,580 filed Apr. 10, 1998 (abandoned), which is a continuation in part of U.S. patent application Ser. No. 08/748,376 filed Nov. 13, 1996 (now U.S. Pat. No. 5,772,889). This application is a regular U.S. patent application under 35 U.S.C. §111(a) and 35 U.S.C. §1.53(b) and claims priority from the following co-pending, commonly assigned provisional applications, each filed under 35 U.S.C. §111(b), each of which is incorporated herein by reference:
[0002] 60/187,979 filed Mar. 9, 2000
[0003] 60/224,918 filed Aug. 11, 2000;
[0004] 60/215,208 filed Jun. 29, 2000;
[0005] 60/220,119 filed Jul. 21, 2000.
Provisional Applications (4)
|
Number |
Date |
Country |
|
60187979 |
Mar 2000 |
US |
|
60224918 |
Aug 2000 |
US |
|
60215208 |
Jun 2000 |
US |
|
60220119 |
Jul 2000 |
US |
Continuation in Parts (4)
|
Number |
Date |
Country |
Parent |
09557424 |
Apr 2000 |
US |
Child |
09753856 |
Jan 2001 |
US |
Parent |
09183123 |
Oct 1998 |
US |
Child |
09557424 |
Apr 2000 |
US |
Parent |
09058580 |
Apr 1998 |
US |
Child |
09183123 |
Oct 1998 |
US |
Parent |
08748376 |
Nov 1996 |
US |
Child |
09058580 |
Apr 1998 |
US |