Detection and quantification of biomolecules using mass spectrometry

• US 8,383,795 B2
• Filed: 03/03/2011
• Issued: 02/26/2013
• Est. Priority Date: 12/05/2006
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Van Den Boom, Dirk Johannes, Oeth, Paul Andrew, Mahboubi, Payam
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/040,056
• Publication Number:
  US 20110160093A1
• Time in Patent Office: 726 Days
• Field of Search: 435/6.1, 435/91.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/26.6
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention is directed in part to a method for detecting a target nucleic acid using detector oligonucleotides detectable by mass spectrometry. This method takes advantage of the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and releases labels for detection by mass spectrometry. This process is easily incorporated into a polymerase chain reaction (PCR) amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.

Claims

1. A detector oligonucleotide comprising:

a complementary portion that is complementary to a target nucleic acid, which complementary portion comprises a non-cleavable nucleotide incorporated at its 5′

end, and

a contiguous non-complementary portion linked to the 5′

end of the complementary portion, that is susceptible to cleavage by a cleavage agent, which cleavage can produce a mass distinguishable product (MDP).

2. The detector oligonucleotide of claim 1, wherein the non-cleavable nucleotide is a locked nucleic acids (LNA) or amadite.

3. The detector oligonucleotide of claim 1, further comprising a modification that increases the melting temperature of the detector oligonucleotide.

4. The detector oligonucleotide of claim 3, wherein the modification is the incorporation of one or more minor groove binders to the detector oligonucleotide.

5. The detector oligonucleotide of claim 1, wherein the non-complementary portion further comprises a capture mechanism that allows for the mass-distinguishable product (MDP) to bind to a solid support upon its cleavage from the detector oligonucleotide by a cleavage agent.

6. The detector oligonucleotide of claim 5, wherein the capture mechanism is a biotin molecule.

7. The detector oligonucleotide of claim 1, wherein the mass-distinguishable product is amplified after its release from the detector oligonucleotide.

8. The detector oligonucleotide of claim 7, wherein the detector oligonucleotide comprises a universal primer hybridization site and is amplified using a universal primer.

9. The detector oligonucleotide of claim 5 that further comprises one or more releasing mechanisms for subsequent release of the MDP or fragment thereof after it binds to the solid support.

10. The detector oligonucleotide of claim 9, wherein the one or more releasing mechanisms are selected from the group consisting of enzymatic cleavage, photocleavage, exchange reactions and temperature-sensitive release.

11. A library comprising two or more detector oligonucleotides of claim 1, wherein each mass distinguishable product corresponds to a unique target nucleic acid.

12. The library of claim 11 which comprises 10 or more detector oligonucleotide species.

13. The library of claim 11 which comprises 50 or more detector oligonucleotide species.

14. The library of claim 11 which comprises 100 or more detector oligonucleotide species.

15. The library of claim 11 which comprises 250 or more detector oligonucleotide species.