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 eluting is carried out under conditions effective to denature the secondary structure of said RNA molecules.
- 3. A method of claim 1 including collecting mobile phase fractions containing said RNA molecules.
- 4. A method of claim 1, wherein said mobile phase comprises a counterion agent and an organic solvent, wherein said organic solvent is water soluble.
- 5. A method of claim 4, wherein said solvent is selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof.
- 6. A method of claim 4, wherein said solvent comprises acetonitrile.
- 7. 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.
- 8. A method of claim 7 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.
- 9. A method of claim 2 wherein said eluting is carried out at a temperature greater than about 60° C.
- 10. A method of claim 9 wherein said eluting is carried out at a temperature within the range of about 40° C. to about 80° C.
- 11. A method of claim 1 wherein the pH of said mobile phase is within the range of about pH 5 to about pH 9.
- 12. A method of claim 11 wherein the pH of said mobile phase is about pH 7.
- 13. A method of claim 1 wherein said mixture comprises RNA molecules exceeding about 1,000 nucleotides.
- 14. A method of claim 1 wherein said mixture comprises RNA molecules having up to about 20,000 nucleotides.
- 15. 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.
- 16. A method of claim 15 wherein said eluting is carried out under conditions effective to minimize the secondary structure of said RNA molecules.
- 17. A method of claim 15 wherein said eluting is carried out at a temperature within the range of about 40° C. to about 80° C.
- 18. A method of claim 15 wherein said ID is greater than about 7 mm.
- 19. A method of claim 15 wherein said ID is greater than about 10 mm.
- 20. A method of claim 15 wherein said ID is greater than about 50 mm.
- 21. A method of claim 15 wherein said ID is in the range of about 5 mm to about 1 m.
- 22. 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.
- 23. A column of claim 22 wherein said ID is greater than about 7 mm.
- 24. A system for segregating a mixture or RNA molecules by Matched ion Polynucleotide Chromatography, comprising:
the column of claim 22.
CROSS REFERENCE TO RELATED CO-PENDING APPLICATIONS
[0001] This application is a continuation in part of U.S. patent application Ser. No. 09/183,123 filed Oct. 30, 1998, which is a continuation in part of U.S. patent application Ser. No. 09/058,580 filed Apr. 10, 1998, which is a continuation in part of 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] 60/130,824 filed Apr. 23, 1999;
[0003] 60/135,314 filed May 20, 1999;
[0004] 60/155,685 filed Sep. 23, 1999;
[0005] 60/163,197 filed Nov. 3, 1999;
[0006] 60/187,979 filed Mar. 9, 2000.
Divisions (1)
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Number |
Date |
Country |
Parent |
09557424 |
Apr 2000 |
US |
Child |
10126055 |
Apr 2002 |
US |
Continuation in Parts (3)
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Number |
Date |
Country |
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 |