Mass spectrometric methods for detecting mutations in a target nucleic acid

• US 7,759,065 B2
• Filed: 05/22/2008
• Issued: 07/20/2010
• 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:
  US12/125,857
• Publication Number:
  US 20090092977A1
• Time in Patent Office: 789 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/5
• 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

Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and mutations in the molecules are provided. In some embodiments, a process comprises: amplifying one or more target nucleic acid molecules from a biological sample; ionizing and volatilizing the amplified product; and analyzing a product by mass spectrometry to determine an observed molecular mass of the product.

Claims

1. A process for characterizing a target nucleic acid from a biological sample, comprising:

a) amplifying a target nucleic acid from a biological sample by polymerase chain reaction (PCR);

b) ionizing and volatilizing the product of step (a); and

c) analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, or (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product;

whereby the target nucleic acid is characterized based on the analysis of (c).

2. The process of claim 1, wherein step (c) comprises analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, and (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product.

3. The process of claim 1, wherein the target nucleic acid comprises at least one mutation.

4. The process of claim 1, wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.

5. The process of claim 4, wherein characterizing the target nucleic acid identifies different individuals or strains within the same species.

6. The process of claim 1, wherein the biological sample comprises material obtained from a human.

7. The process of claim 1, wherein the biological sample comprises material obtained from an infectious organism.

8. The process of claim 7, wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.

9. The process of claim 1, wherein the product of step (a) is conditioned.

10. The process of claim 9, wherein said conditioning comprises cation exchange.

11. The process of claim 10, wherein the target nucleic acid is subjected to cation exchange directly after step (a), and step (b) is performed directly after the cation exchange.

12. The process of claim 1, wherein steps b) and c) are performed directly after step a).

13. The process of claim 1, wherein step c) comprises the use of electrospray (ES) mass spectrometry.

14. The process of claim 1, wherein step c) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.

15. The process of claim 1, wherein the target nucleic acid is not specifically digested.

16. The process of claim 15, wherein the target nucleic acid is not specifically digested by a nuclease.

17. The process of claim 1, wherein the target nucleic acid is double stranded.

18. The process of claim 1, wherein the target nucleic acid is DNA.

19. The process of claim 1, wherein, in solution, (i) the target nucleic acid is isolated from the biological sample, and (ii) the isolated target nucleic acid directly is subjected to step (a).

20. The process of claim 1, wherein the biological sample comprises cells, and the target nucleic acid is isolated from the biological sample by lysing the cells.

21. The process of claim 1, which comprises collecting the biological sample.

22. The process of claim 1, which comprises isolating the target nucleic acid from the biological sample.

44. The process of claim 1, wherein the product of step (a) is from about 7 to about 50 bases.

45. The process of claim 1, wherein the product of step (a) is about 40 bases, about 50 bases, about 100 bases, or about 200 bases.

23. A process for characterizing a target nucleic acid from a biological sample, comprising:

a) providing a biological sample;

b) isolating a target nucleic acid from the biological sample;

c) amplifying the target nucleic acid of (b) by polymerase chain reaction (PCR);

d) ionizing and volatilizing the product of step (c); and

e) analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, or (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product;

whereby the target nucleic acid is characterized based on the analysis of (e).

24. The process of claim 23, wherein step (e) comprises analyzing (i) a forward strand PCR product by mass spectrometry to determine an observed molecular mass of the forward strand PCR product, and (ii) analyzing a reverse strand PCR product by mass spectrometry to determine an observed molecular mass of the reverse strand PCR product.

25. The process of claim 23, wherein two or more biological samples are provided and the nucleic acid is isolated from the two or more biological samples.

26. The process of claim 23, wherein the target nucleic acid comprises at least one mutation.

27. The process of claim 23, wherein the biological sample comprises material obtained from a human, animal, plant, fungus, parasite or virus.

28. The process of claim 27, wherein characterizing the target nucleic acid identifies different individuals or strains within the same species.

29. The process of claim 23, wherein the biological sample comprises material obtained from a human.

30. The process of claim 23, wherein the biological sample comprises material obtained from an infectious organism.

31. The process of claim 29, wherein the target nucleic acid is a sequence present in an infectious organism and distinct from host cell sequences.

32. The process of claim 23, wherein the product of step (c) is conditioned.

33. The process of claim 32, wherein said conditioning comprises cation exchange.

34. The process of claim 33, wherein the target nucleic acid is subjected to cation exchange directly after step (c), and step (d) is performed directly after the cation exchange.

35. The process of claim 23, wherein steps d) and e) are performed directly after step c).

36. The process of claim 23, wherein step e) comprises the use of electrospray (ES) mass spectrometry.

37. The process of claim 23, wherein step e) comprises the use of matrix assisted laser desorption ionization (MALDI) mass spectrometry.

38. The process of claim 23, wherein the target nucleic acid is not specifically digested.

39. The process of claim 38, wherein the target nucleic acid is not specifically digested by a nuclease.

40. The process of claim 23, wherein the target nucleic acid is double stranded.

41. The process of claim 23, wherein the target nucleic acid is DNA.

42. The process of claim 23, wherein step (c) is performed directly after step (b) and each step is performed in solution.

43. The process of claim 23, wherein the biological sample comprises cells, and the target nucleic acid molecule is isolated from the biological sample by lysing the cells.

46. The process of claim 23, wherein the product of step (a) is from about 7 to about 50 bases.

47. The process of claim 23, wherein the product of step (a) is about 40 bases, about 50 bases, about 100 bases, or about 200 bases.