Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping

• US 6,811,977 B2
• Filed: 07/27/2001
• Issued: 11/02/2004
• Est. Priority Date: 07/27/2000
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Davis, Mark E., Wold, Barbara J., Tirrell, David A., Kirshenbaum, Kent, Murphy, John F.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/918,687
• Publication Number:
  US 20020137057A1
• Time in Patent Office: 1,194 Days
• Field of Search: 435/6, 435/91.2, 436/94, 436/173, 250/282
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• 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 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• 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

The invention provides methods of identifying one or more nucleic acids in a sample. The nucleic acids, for example, expressed genes in a cell, can be identified by contacting the nucleic acids with oligonucleotides having detector tags, and selector tags to form tagged oligonucleotides. Each nucleic acid can be uniquely identified by mass-spectrophotometric analysis of the detector tag.

Claims

1. A method of detecting a specific nucleic acid in a sample comprising:

(a) contacting the nucleic acid with a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a peptoid detector tag, in a reaction mixture under conditions that allow the first and second oligonucleotides to specifically hybridize with the nucleic acid such that the first oligonucleotide is located immediately adjacent to the second oligonucleotide, thereby forming adjacently hybridized first and second oligonucleotides;

(b) ligating the adjacently hybridized first and second oligonucleotides to form a ligated oligonucleotide;

(c) de-linking the detector tag from the ligated oligonucleotide; and

(d) identifying the de-linked detector tag, thereby detecting a specific nucleic acid in a sample.

2. The method of claim 1, wherein identifying the de-linked detector tag further comprises separating, using the selector tag, the ligated oligonucleotide from the reaction mixture.

3. The method of claim 2, wherein using the selector tag comprises contacting the selector tag with an agent that specifically binds to the selector tag.

4. The method of claim 2, wherein de-linking the detector tag is performed by subjecting the ligated oligonucleotide to a de-linking agent selected from the group consisting of an acid condition, an alkaline condition, a visible light radiation, a UV radiation, heat, a reducing condition and an oxidizing condition.

5. The method of claim 1, wherein identifying the de-linked detector tag comprises using mass spectrometry.

6. The method of claim 5, further comprising using chromatography.

7. The method of claim 1, wherein the selector tag is a fluorescent moiety.

30. The method of claim 1, wherein the method further comprises identifying an isotopic internal standard along with the de-linked detector tag.

31. The method of claim 30, wherein the method further comprises quantitating the relative amount of the de-linked detector tag relative to the isotopic internal standard.

8. A method of detecting a plurality of specific nucleic acids in a sample comprising:

(a) contacting each specific nucleic acid with an oligonucleotide pair in a reaction mixture under conditions that allow the oligonucleotide pair to specifically hybridize to the nucleic acid such that the oligonucleotide pair members are located immediately adjacent to each other thereby forming an adjacently hybridized oligonucleotides pair, wherein each oligonucleotide pair comprises a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a peptoid detector tag;

(b) ligating each adjacently hybridized oligonucleotide pair to form one or more ligated oligonucleotides;

(c) de-linking the detector tag from the one or more ligated oligonucleotides; and

(d) identifying one or more de-linked detector tags.

9. The method of claim 8, wherein identifying the linked detector tags further comprises separating, using the selector tag, the ligated oligonucleotide from the reaction mixture.

10. The method of claim 9 wherein using the selector tag comprises contacting the selector tag with an agent that specifically binds to the selector tag.

11. The method of claim 9 wherein de-linking the detector tags is performed by subjecting the ligated oligonucleotide to a de-linking agent selected from the group consisting of an acid condition, an alkaline condition, a visible light radiation, a UV radiation, heat, a reducing condition and an oxidizing condition.

12. The method of claim 8, wherein identifying the linked detector tag comprises using mass spectrometry.

13. The method of claim 12 further comprising using chromatography.

14. The method of claim 8, wherein the selector tag is a fluorescent moiety.

15. The method of claim 8, wherein each first oligonucleotide linked to selector tag has an identical selector tag.

16. The method of claim 8, wherein each first oligonucleotide linked to selector tag has a different tag.

17. The method of claim 8, wherein each second oligonucleotide linked to detector tag has a different tag.

32. The method of claim 17, wherein the method further comprises identifying a corresponding isotopic internal standard for each dc-linked detector tag.

33. The method of claim 32, wherein the method further comprises quantitating the relative amount of each type of de-linked detector tag relative to its corresponding isotopic internal standard.

18. A method of detecting a nucleic acid in a sample comprising:

(a) amplifying the nucleic acid with a primer pair to form a dual-tagged amplification product in a reaction mixture, wherein the primer pair is a first oligonucleotide linked to a selector tag and a second oligonucleotide linked to a peptoid detector tag;

(b) de-linking the detector tag from the ligated oligonucleotide; and

(c) identifying the de-linked detector tag, thereby detecting the nucleic acid in a sample.

19. The method of claim 18, wherein identifying the detector tag further comprises separating, using the selector tag, the amplification product from the reaction mixture prior to identifying the detector tag associated with the amplification product.

20. The method of claim 19, wherein using the selector tag comprises contacting the selector tag with an agent that specifically binds to the selector tag.

21. The method of claim 18, further comprising removing the detector tag from the amplification product prior to step (b).

22. The method of claim 18, wherein de-linking the detector tag is performed by subjecting the ligated oligonucleotide to a de-linking agent selected from the group consisting of an acid condition, an alkaline condition, a visible light radiation, a UV radiation, heat, a reducing condition and an oxidizing condition.

23. The method of claim 18, wherein the selector tag is a fluorescent moiety.

24. The method of claim 18, wherein identifying the detector tag comprises using mass spectrometry.

25. A method of detecting a nucleic acid in a sample comprising:

(a) contacting the nucleic acid with an oligonucleotide linked to a peptoid detector tag under conditions that allow the oligonucleotide to specifically hybridize to the nucleic acid to form a mixture of hybridized oligonucleotide and unhybridized oligonucleotide;

(b) separating the hybridized oligonucleotide from the unhybridized oligonucleotide;

(c) de-linking the detector tag from the hybridized oligonucleotide; and

(d) identifying the de-linked detector tag, thereby detecting the nucleic acid.

26. The method of claim 25, wherein separating comprises contacting the mixture with an agent that binds to a polyA tail.

27. The method of claim 25, wherein separating comprises contacting the mixture with an agent that binds to a 5′

-capped nucleic acid.

28. A method of generating a double-tagged oligonucleotide duplex comprising:

(a) contacting a single-stranded nucleic acid with a first oligonucleotide linked to a selector tag and a second oligonucleotide removably linked to a peptoid detector tag, under conditions that allow the first and second oligonucleotide to specifically hybridize to the nucleic acid such that the first oligonucleotide is located immediately adjacent to the second oligonucleotide thereby forming an adjacently hybridized oligonucleotide;

(b) ligating the first and second oligonucleotides thereby forming a duplex having detector and selector tags.

29. The method of claim 28, wherein the selector tag and the detector tag are a peptoid and wherein the selector tag and detector tag are different.