Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture

• US 20050130196A1
• Filed: 09/17/2004
• Published: 06/16/2005
• Est. Priority Date: 05/13/2003
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hofstadler, Steven A., Cummins, Lendell L.
• Granted Patent: US 8,158,354 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: H01J 49/165 Electrospray ionisation

Abstract

The present invention provides a method for rapid solution capture purification of nucleic acids for subsequent analysis by electrospray mass spectrometry which is efficient and cost-effective relative to existing methods. The present invention also provides for kits useful for practicing rapid solution capture of nucleic acids so that purified samples are in condition for analysis by electrospray mass spectrometry.

Claims

1. A method of purifying a solution comprising a nucleic acid comprising:

mixing said solution with an anion exchange resin to yield a suspension of said anion exchange resin in said solution wherein said nucleic acids bind to said anion exchange resin;

isolating said anion exchange resin from said solution;

washing said anion exchange resin to remove one or more contaminants with one or more wash buffers while retaining bound nucleic acids;

eluting said nucleic acids from said ion exchange resin with an elution buffer.

2. The method of claim 1 further comprising analyzing said nucleic acid by mass spectrometry.

3. The method of claim 1 wherein said mixing, washing, and eluting steps are performed by a liquid handler.

4. The method of claim 1 wherein said mixing, washing, isolating, and eluting steps are performed by a robot.

5. The method of claim 1 wherein said anion exchange resin is a strong anion exchange resin.

6. The method of claim 5 wherein the functional group of said strong anion exchange resin is a quaternary amine.

7. The method of claim 1 wherein said anion exchange resin is a weak anion exchange resin.

8. The method of claim 7 wherein the functional group of said weak anion exchange resin comprises polyethyleneimine, a charged aromatic amine, diethylaminomethyl, or diethylaminoethyl.

9. The method of claim 7 wherein said weak anion exchange resin comprises one of the following commercial products:

Baker polyethyleneimine resin, Baker diethylaminomethyl resin, Dionex ProPac™

WAX, Millipore polyethyleneimine, and Applied Biosystems POROS™

PI.

10. The method of claim 1 wherein said mixing is accomplished by pipetting, vortexing, sonication, or shaking.

11. The method of claim 1 wherein said isolating step is accomplished by filtration.

12. The method of claim 11 wherein said filtration is carried out in a 96-well plate, a 384-well plate, or a micro filter tube.

13. The method of claim 11 wherein said filtration is accomplished employing one of the following membranes:

polytetrafluoroethylene, polyvinyldifluoroethylene, polypropylene, polyethylene, glass fiber, polycarbonate or polysulfone.

14. The method of claim 11 wherein said filtration is accomplished by vacuum, centrifugation or positive pressure displacement with fluids or gases.

15. The method of claim 1 wherein said anion exchange resin comprises an anion exchange functional group bound to a magnetic bead.

16. The method of claim 15 wherein said isolating step is accomplished by application of a magnetic field to compress said magnetic beads and aspirating said solution.

17. The method of claim 1 wherein said wash buffer comprises ammonium acetate.

18. The method of claim 1 wherein said wash buffer comprises ammonium bicarbonate.

19. The method of claim 1 wherein said wash buffer comprises methanol.

20. The method of claim 1 wherein said elution buffer comprises ammonium hydroxide.

21. The method of claim 1 wherein said elution buffer is compatible with electrospray ionization (ESI).

22. The method of claim 1 wherein said elution buffer comprises a combination of piperidine, imidazole and methanol.

23. The method of claim 1 wherein said solution of nucleic acids is the product of a nucleic acid amplification reaction.

24. The method of claim 23 wherein said amplification reaction is effected by the polymerase chain reaction, the ligase chain reaction or strand displacement amplification.

25. The method of claim 1 wherein said contaminants comprise any of the following:

buffer salts, stabilizers, metal cations, proteins and deoxynucleotide triphosphates.

26. A method of purifying a solution comprising a nucleic acid for subsequent analysis by electrospray mass spectrometry comprising:

adding a weak anion exchange resin to said solution and mixing to yield a suspension of said weak anion exchange resin in said solution wherein said nucleic acids bind to said weak anion exchange resin;

isolating said weak anion exchange resin from said solution;

washing said weak anion exchange resin to remove contaminants with one or more wash buffers while retaining bound nucleic acids; and

eluting said nucleic acids from said weak ion exchange resin with an elution buffer.

27. The method of claim 26 further comprising analyzing said nucleic acid by mass spectrometry.

28. The method of claim 26 wherein the functional group of said weak anion exchange resin comprises a polyethyleneimine, charged aromatic amine, diethylaminomethyl, or diethylaminoethyl.

29. The method of claim 26 wherein said weak anion exchange resin comprises one of the following commercial products:

Baker polyethyleneimine resin, Baker diethylaminomethyl resin, Dionex ProPac WAX, Millipore polyethyleneimine, and Applied Biosystems POROS PI.

30. A method of purifying a solution of nucleic acids for subsequent analysis by electrospray mass spectrometry comprising:

adding ProPac™

WAX weak anion exchange resin to said solution and mixing to yield a suspension of said weak anion exchange resin in said solution wherein said nucleic acids bind to said weak anion exchange resin;

isolating said weak anion exchange resin from said solution;

washing said weak anion exchange resin to remove contaminants with one or more wash buffers while retaining bound nucleic acids; and

eluting said nucleic acids from said weak ion exchange resin with an elution buffer comprising piperidine, imidazole and methanol.

31. The method of claim 30 further comprising analyzing said nucleic acid by mass spectrometry.

32. A kit for purification of nucleic acids comprising:

a resin source plate containing anion exchange resin;

a sample mixing plate;

a filter plate or a microtube rack comprising a plurality of micro filter tubes;

one or more resin wash solutions; and

an ESI-MS-compatible elution buffer.

33. The kit of claim 32 wherein said filter plate is a 96-well plate or a 384-well plate.

34. The kit of claim 32 wherein said anion exchange resin comprises a weak anion exchange functional group.

35. The kit of claim 32 wherein said anion exchange resin comprises ProPac™

WAX.

36. The kit of claim 32 wherein said anion exchange resin comprises a weak anion exchange functional group bound to a magnetic bead.

37. The kit of claim 32 wherein said weak anion exchange resin is ProPac™

WAX.

38. The kit of claim 32 wherein said one or more wash solutions comprises about 50 mM to about 200 mM ammonium acetate or about 50 mM to about 200 mM ammonium bicarbonate.

39. The kit of claim 32 wherein said at least one anion exchange wash buffer comprises about 20% to about 50% methanol.

40. The kit of claim 32 wherein said ESI-MS compatible elution buffer comprises about 2% to about 6% ammonium hydroxide.

41. The kit of claim 32 wherein said ESI-MS compatible elution buffer comprises about 10 mM to about 50 mM imidazole, about 10 mM to about 50 mM piperidine and about 20% to about 50% methanol.

42. The kit of claim 32 wherein said ESI-MS compatible elution buffer comprises about 10 mM to about 50 mM imidazole, about 10 mM to about 50 mM piperidine and about 20% to about 50% methanol.