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

• US 7,964,343 B2
• Filed: 05/12/2004
• Issued: 06/21/2011
• Est. Priority Date: 05/13/2003
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Hofstadler, Steven A., Cummins, Lendell L.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US10/844,938
• Publication Number:
  US 20050164215A1
• Time in Patent Office: 2,596 Days
• Field of Search: 435/6, 536/23.1
• US Class Current: N/A
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• 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

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 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 nucleic acids for mass spectrometry analysis comprising:

mixing said solution comprising nucleic acids with one or more magnetic beads linked to at least one anion exchange functional group having a pKa of 9 or greater to yield a suspension of said one or more magnetic beads in said solution wherein said nucleic acids bind to said at least one anion exchange functional group linked to said one or more magnetic beads;

isolating said one or more magnetic beads from said solution by application of a magnetic field;

removing said solution from said one or more magnetic beads;

washing said one or more magnetic beads to remove one or more contaminants with one or more wash buffers while said nucleic acids are bound to said at least one anion exchange functional group linked to said one or more magnetic beads; and

eluting said nucleic acids from said at least one anion exchange functional group linked to said one or more magnetic beads with an elution buffer compatible with electrospray ionization mass spectrometry wherein said elution buffer does not comprise a metal cation salt.

2. The method of claim 1 further comprising analyzing said nucleic acids 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 mixing is accomplished by pipetting, vortexing, sonication, or shaking.

6. The method of claim 1 wherein said one or more wash buffers comprises ammonium acetate.

7. The method of claim 1 wherein said one or more wash buffers comprises ammonium bicarbonate.

8. The method of claim 1 wherein said one or more wash buffers comprises methanol.

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

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

11. The method of claim 1 wherein said nucleic acids are the products of a nucleic acid amplification reaction.

12. The method of claim 11 wherein said nucleic acid amplification reaction is selected from the group consisting of polymerase chain reaction, ligase chain reaction or strand displacement amplification reaction.

13. The method of claim 1 wherein said one or more contaminants is selected from the group consisting of buffer additives, salts, detergents, polymers, stabilizers, metal cations, proteins and deoxynucleotide triphosphates.

14. The method of claim 6 wherein said one or more wash buffers comprise 50 mM to 200 mM ammonium acetate.

15. The method of claim 7 wherein said one or more wash buffer comprise 50 mM to 200 mM ammonium bicarbonate.

16. The method of claim 8 wherein said one or more wash buffer comprise 20% to 50% methanol.

17. The method of claim 9 wherein said elution buffer comprises 2% to 6% ammonium hydroxide.

18. The method of claim 10 wherein said elution buffer comprises 10 mM to 50 mM piperidine, 10 mM to 50 mM imidazole, and methanol.

19. The method of claim 18 wherein said methanol is selected from the group consisting of 30% methanol and 35% methanol.

20. The method of claim 18 wherein said elution buffer comprises 25 mM piperidine, 25 mM imidazole, and methanol.

21. The method of claim 20 wherein said methanol is selected from the group consisting of 30% methanol and 35% methanol.

22. The method of claim 1, wherein said anion exchange functional group is polyethyleneimine.

23. The method of claim 1, wherein said anion exchange functional group is a diethylaminomethyl.

24. The method of claim 1, wherein said anion exchange functional group is a diethylaminoethyl.