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

• US 8,158,354 B2
• Filed: 09/17/2004
• Issued: 04/17/2012
• 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.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US10/943,344
• Publication Number:
  US 20050130196A1
• Time in Patent Office: 2,769 Days
• Field of Search: 435/6, 435/91.2, 536/23.1
• US Class Current: 435/6.12
• 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 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 plurality of nucleic acids for subsequent analysis by electrospray mass spectrometry comprising:

providing amine-terminated magnetic beads, wherein each of said amine-terminated magnetic beads comprises a magnetic bead linked to at least one amine molecule;

combining said amine-terminated magnetic beads with said solution and mixing to yield a suspension of said amine-terminated magnetic beads in said solution, wherein said plurality of nucleic acids bind to said at least one amine molecule on said amine-terminated magnetic beads;

isolating said amine-terminated magnetic beads from said solution;

washing said amine-terminated magnetic beads to remove contaminants with one or more wash buffers while retaining said bound plurality of nucleic acids; and

eluting said one or more plurality of nucleic acids from said amine-terminated magnetic beads with an elution buffer wherein said elution buffer does not comprise a metal cation salt.

2. The method of claim 1, further comprising analyzing said plurality of nucleic acids by mass spectrometry.

3. The method of claim 1 wherein said isolating step is accomplished by application of a magnetic field to separate said amine terminated magnetic beads and aspirating said solution.

4. The method of claim 1 wherein said amine terminated magnetic beads are amine-terminated supraparamagnetic beads.

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

6. The method of claim 5 wherein said elution buffer comprises from about 10 mM to about 50 mM imidazole, from about 10 mM to about 50 mM piperidine, and from about 20% to about 50% methanol.

7. The method of claim 6 wherein said elution buffer comprises 25 mM piperidine, 25 mM imidazole, and 35% methanol.

8. The method of claim 1 wherein at least one of said one or more wash buffers comprises from about 20% to about 50% methanol.

9. The method of claim 8 wherein at least one of said one or more wash buffers comprises 50% methanol.

10. The method of claim 1 wherein at least one of said one or more wash buffers comprises from about 50 mM to about 200 mM ammonium acetate or from about 50 mM to about 200 mM ammonium bicarbonate.

11. The method of claim 10 wherein said at least one of said one or more wash buffers comprises 50 mM ammonium bicarbonate and 50% methanol.

12. The method of claim 10 wherein said at least one of said one or more wash buffers comprises 100 mM ammonium bicarbonate and 50% methanol.

13. The method of claim 1 wherein said plurality of nucleic acids comprise the product of an amplification reaction.

14. The method of claim 1 wherein the amine-terminated magnetic bead comprises an amine having a pKa of greater than 9.

15. The method of claim 14 wherein the amine is a primary, secondary or tertiary alkyl amine.

16. The method of claim 1 wherein the amine-terminated magnetic bead comprises an iron particle.