DETECTION AND QUANTIFICATION OF BIOMOLECULES USING MASS SPECTROMETRY

• US 20080305479A1
• Filed: 12/04/2007
• Published: 12/11/2008
• Est. Priority Date: 12/05/2006
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: VAN DEN BOOM, Dirk Johannes
• Granted Patent: US 8,133,701 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 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 uses the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed oligonucleotide probes from hybridized duplexes and release labels for detection by mass spectrometry. This process is easily incorporated into a PCR amplification assay. The method also includes embodiments directed to quantitative analysis of target nucleic acids.

Claims

1. A method of detecting a target nucleic acid sequence in a sample, comprising the steps of:

(a) contacting a sample comprising a target nucleic acid with an (i) oligonucleotide primer, comprising a 3′

end and a 5′

end, and containing a sequence complementary to a region of the target nucleic acid and a (ii) detector oligonucleotide, comprising a 3′

end and a 5′

end, and containing a sequence complementary to a second region of the target nucleic acid sequence, thereby forming a (iii) mixture of duplexes under hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the oligonucleotide primer and to the detector oligonucleotide such that the 3′

end of the oligonucleotide primer is upstream of the 5′

end of the detector oligonucleotide;

(b) exposing the sample of step (a) to a cleavage agent under conditions sufficient to cleave and release the annealed detector oligonucleotide or at least one fragment thereof, thereby producing one or more mass-distinguishable products; and

(c) detecting the one or more mass-distinguishable products by mass spectrometry, thereby detecting the presence or absence of the target nucleic acid sequence in the sample.

4. The method of claim 1 or 3, wherein steps a) and b) are performed simultaneously in a single, closed reaction vessel.

7. The method of claim 1, 2 or 3, wherein two or more target nucleic acids are detected in a single, multiplexed reaction.

13. The method of claim 1, wherein the cleavage agent has 5′

to 3′

nuclease activity.

16. The method of claim 1, 2 or 3, wherein more than one mass-distinguishable product from the same detector oligonucleotide is detected by mass spectrometry, and further wherein said more than one mass-distinguishable product produces a mass-specific detection signature that corresponds to a target nucleic acid.

17. The method of claim 1, 2 or 3, wherein the detector oligonucleotide comprises a sequence of nucleotides which is non-complementary to the target nucleic acid.

18. The method of claim 1, 2 or 3, wherein the detector oligonucleotide comprises one or more nucleoside modifications.

19. The method of claim 18, wherein the nucleoside modification is the incorporation of one or more locked nucleic acids (LNAs).

20. The method of claim 1, 2 or 3, wherein the detector oligonucleotide comprises a detection moiety.

21. The method of claim 20, wherein the detection moiety is selected from the group consisting of a sugar, protein, antibody, chemical compound, mass tag, fluorescent tag, charge tag and hydrophobic tag.

22. The method of claim 1, 2 or 3, wherein the one or more mass-distinguishable products are capable of binding to a solid support upon release.

23. The method of claim 1, 2 or 3, wherein the one or more mass-distinguishable products are amplified after their release.

24. The method of claim 1, 2, or 3, wherein the detector oligonucleotide selectively binds to a methylation-specific sequence based on the methylation status of the target nucleic acid.

25. The method of claim 1, 2 or 3, wherein the detection is done by a mass spectrometer selected from the group consisting of MALDI-TOF MS, Tandem MS, ESI-TOF, ESI-iontrap, LC-MS, GC-MS and LOI-MS.

26. The method of claim 1, 2 or 3, wherein the detection is done in real-time.

27. The method of claim 1, 2 or 3, wherein a competitor template nucleic acid is introduced, and further wherein the competitor template nucleic acid serves as an internal control.

28. The method of claim 1, 2 or 3, wherein the quantity of the target nucleic acid sequence in the sample is determined.

29. The method of claim 1, 2 or 3, wherein the detector oligonucleotide comprises a minor groove binding moiety.

2. An amplification method of detecting a target nucleic acid sequence in a sample, comprising the steps of:

(a) providing to an amplification reaction containing the sample, a set of oligonucleotide primers, wherein a first primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence;

(b) providing at least one detector oligonucleotide containing a sequence complementary to a region of the target nucleic acid, wherein said detector oligonucleotide anneals within the target nucleic acid sequence bounded by the oligonucleotide primers of step (a) thereby creating an annealed duplex, and further wherein each oligonucleotide primer is selected to anneal to its complementary template upstream of any detector oligonucleotide annealed to the same nucleic acid strand;

(c) amplifying the target nucleic acid sequence employing an enzyme having 5′

to 3′

nuclease activity as a template-dependent polymerizing agent under conditions which are permissive for amplification cycling steps of (i) annealing of oligonucleotide primers and detector oligonucleotide to a template nucleic acid sequence contained within the target sequence, and (ii) extending the primer oligonucleotide wherein said nucleic acid amplification enzyme synthesizes a primer extension product while the 5′

to 3′

nuclease activity of the nucleic acid amplification enzyme simultaneously releases mass-distinguishable products from the annealed duplexes comprising detector oligonucleotides and its complementary template nucleic acid sequences, thereby creating one or more mass-distinguishable products; and

(d) detecting the one or more mass-distinguishable products by mass spectrometry, thereby determining the presence or absence of the target sequence in the sample.

5. The method of claim 2, wherein steps a), b) and c) are performed simultaneously in a single, closed reaction vessel.

6. The method of claim 2 or 3 wherein the detector oligonucleotide is non-extendable by an enzyme.

3. A method of detecting a target nucleic acid, comprising the steps of:

(a) annealing an oligonucleotide primer to the target nucleic acid, annealing a detector oligonucleotide to the same target nucleic acid;

(b) introducing an enzyme to extend the oligonucleotide primer in the direction of the detector oligonucleotide, wherein the enzyme cleaves and thereby releases at least a portion of the detector oligonucleotide, thereby producing one or more mass-distinguishable products; and

(c) detecting the one or more mass-distinguishable products by mass spectrometry.

11. The method of claim 3, wherein a second oligonucleotide is introduced that binds to the synthesis product of the first oligonucleotide, whereby exponential amplification can subsequently occur.

12. The method of claim 11, wherein the target nucleic acid is initially a single-stranded nucleic acid molecule.

14. The method of claim 3, wherein the enzyme has 5′

to 3′

nuclease activity.

15. The method of claim 3, wherein the enzyme has polymerase activity capable of amplifying target nucleic acid.

8. The method of 1, 2 or 3, wherein more than one detector oligonucleotide is used to detect more than one target nucleic acid in a multiplexed reaction.

9. The method of 1, 2 or 3, wherein the target nucleic acid comprises a mixture of nucleic acid.

10. The method of claim 9, wherein the mixture of nucleic acid comprises maternal and fetal nucleic acid, further wherein said mixture of nucleic acid has been obtained from a sample from a pregnant female.

30. A method of detecting a target biomolecule, comprising the steps of:

(a) introducing a detectable probe to a sample containing a target biomolecule, wherein the detectable probe binds to or otherwise comes in contact with a target biomolecule, further wherein the detectable probe comprises an oligonucleotide that serves as a template nucleic acid;

(b) annealing an oligonucleotide primer to the template nucleic acid, annealing a detector oligonucleotide to the same template nucleic acid;

(c) introducing an enzyme to extend the oligonucleotide primer in the direction of the detector oligonucleotide, wherein the enzyme cleaves and thereby releases at least a portion of the detector oligonucleotide, thereby producing one or more mass-distinguishable products; and

(d) detecting the one or more mass-distinguishable products by mass spectrometry.

31. The method of claim 30, wherein more than one detectable probe is introduced in step (a).

32. The method of claim 30, wherein the detectable probe is specifically bound to the target biomolecule.

33. The method of claim 31, wherein the more than one detectable probes comprise oligonucleotides that ligate to form a template nucleic acid when the detectable probes are in contact with the target biomolecule.

34. A method for detecting a target nucleic acid sequence, which comprises analyzing a nucleic acid sample containing mass-distinguishable products by mass spectrometry, wherein the mass-distinguishable products result from (a) annealing an oligonucleotide primer to a target nucleic acid;

(b) annealing a detector oligonucleotide to the same target nucleic acid; and

(c) contacting the target nucleic acid with an enzyme that extends the oligonucleotide primer in the direction of the detector oligonucleotide, wherein;

the detector oligonucleotide or portion thereof is complementary to the target nucleic acid sequence, and

the enzyme cleaves and thereby releases at least a portion of the detector oligonucleotide, thereby producing one or more mass-distinguishable products;

whereby the target nucleic acid sequence is detected by identifying the mass-distinguishable products by mass spectrometry.

36. The method of claim 34 or 35, wherein a second oligonucleotide is introduced that binds to the synthesis product of the first oligonucleotide, whereby exponential amplification can subsequently occur.

35. A method of detecting a target nucleic acid sequence, which comprises analyzing a nucleic acid sample containing mass-distinguishable products by mass spectrometry, wherein the mass-distinguishable products result from (a) contacting a target biomolecule with a detectable probe containing an oligonucleotide that serves as a template nucleic acid under conditions in which the detectable probe specifically binds to the target biomolecule;

(b) annealing an oligonucleotide primer to the template nucleic acid;

(c) annealing a detector oligonucleotide to the same template nucleic acid; and

(d) contacting the template nucleic acid with an enzyme that extends the oligonucleotide primer in the direction of the detector oligonucleotide, wherein;

the detector oligonucleotide or portion thereof is complementary to the target nucleic acid sequence, and

the enzyme cleaves and thereby releases at least a portion of the detector oligonucleotide, thereby producing one or more mass-distinguishable products;

whereby the target nucleic acid sequence is detected by identifying the mass-distinguishable products by mass spectrometry.