Mass spectrometric methods for detecting mutations in a target nucleic acid

• US 7,074,563 B2
• Filed: 02/24/2003
• Issued: 07/11/2006
• Est. Priority Date: 03/17/1995
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Köster, Hubert
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/375,714
• Publication Number:
  US 20030228594A1
• Time in Patent Office: 1,233 Days
• Field of Search: 435/6, 436/94
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 35/1067 for transfer to or from containers having different spacing
• CPC Class Codes: H01J 49/00 : Particle spectrometers or separator tubes
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 : Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Provided herein are mass spectrometric methods for detecting a mutation in a target nucleic acid. In an embodiment, provided is a method which comprises digesting a target nucleic acid molecule; capturing digested fragments on a solid support that comprises oligonucleotides complementary thereto; and detecting hybrids and the molecular weights of captured fragments by mass spectrometry, thereby identifying mutations in the target nucleic acid molecule.

Claims

1. A method for detecting mutations in a target nucleic acid, comprising:

digesting a target nucleic acid molecule;

capturing digested fragments on a solid support that comprises oligonucleotides complementary thereto;

detecting hybrids and the molecular weights of captured fragments by mass spectrometry, thereby identifying mutations in the target nucleic acid molecule.

2. The method of claim 1, wherein the target molecule is RNA.

3. The method of claim 2, wherein the RNA is produced by transcription of a target DNA molecule.

4. The method of claim 1, wherein the solid support comprises a flat surface.

5. The method of claim 1, wherein the solid support comprises a matrix for performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.

6. The method of claim 1, wherein the solid support is selected from the group consisting of glass fiber filters, glass surfaces and metal surfaces.

7. The method of claim 1, wherein the solid support is selected from the group consisting of steel, gold, sliver, aluminum, copper and silicon.

8. The method of claim 1, wherein the solid support is silicon.

9. The method of claim 1, wherein the solid support is a silicon wafer.

10. The method of claim 1, wherein the oligonucleotides on the solid support are linked to the support via a linker or a bond cleavable under the conditions of mass spectrometric analysis.

11. The method of claim 10, wherein the oligonucleotides are linked to the support via a linker or bond that is photocleavable.

12. The method of claim 1, wherein the oligonucleotides comprise oligoribonucleotides, oligodeoxyribonucleotides, nucleotide analogs, or protein nucleic acid (PNA).

13. The method of claim 12, wherein the nucleotide analogs comprise a thio-modified phosphodiester or phosphotriester backbone.

14. The method of claim 1, wherein the oligonucleotides are linked to the support by an irreversible bond.

15. The method of claim 1, wherein the oligonucleotides are linked to the support by a disulfide bond, a biotin/streptavidin linkage, a heterobifunctional derivative of a trityl ether group, a levulinyl group, an arginine-arginine bond, a lysine-lysine bond, a pyrophosphate bond or a charge transfer complex.