DNA diagnostics based on mass spectrometry

• US 6,043,031 A
• Filed: 03/18/1996
• Issued: 03/28/2000
• Est. Priority Date: 03/17/1995
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Higgens, G. Scott, Little, Daniel P., Koster, Hubert
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/617,256
• Time in Patent Office: 1,471 Days
• Field of Search: 435/6, 435/91.2, 435/91.52, 536/25.3, 536/25.4, 935/77, 935/78, 436/54, 436/173, 250/282, 250/423 P
• 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

The invention provides fast and highly accurate mass spectrometer based processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

Claims

1. A process for identifying a target nucleic acid sequence present in a biological sample as being normal or mutant, comprising the steps of:

a) obtaining the target nucleic acid sequence from a biological sample;

b) hybridizing the target nucleic acid sequence with

a mutant primer (M) having sufficient 3'"'"' terminal base complementarity to hybridize to a mutation containing portion of the target nucleic acid sequence, or

a normal primer (N), which is distinguishable from M, having sufficient 3'"'"' terminal base complementarity to hybridize to a wildtype sequence in the same portion of the target nucleic acid as M;

c) contacting the product of step b) with a polymerase enzyme and a nucleoside triphosphate, whereby

extension from N occurs, if N has hybridized to the target nucleic acid sequence and the nucleoside triphosphate is complementary to the next base in the template target nucleic acid sequence, or

extension from M occurs, if M has hybridized to the target acid sequence and the nucleoside triphosphate is complementary to the next base in the template target nucleic acid sequence;

d) ionizing and volatizing the product of step c); and

e) detecting the product of step d) by mass spectrometry, wherein the molecular weight of the product indicates whether the target nucleic acid sequence is normal or mutant.

3. A process of claim 1, wherein prior to step b), the target nucleic acid is immobilized onto a solid support to produce an immobilized target nucleic acid sequence.

4. A process of claim 3, wherein immobilization is accomplished by hybridization between a complementary capture nucleic acid molecule, which has been previously immobilized to a solid support, and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

5. A process of claim 3, wherein immobilization is accomplished via direct bonding between the solid support and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

6. A process of claim 3, wherein the target nucleic acid is reversibly immobilized.

7. A process of claim 6, wherein the target nucleic acid can be cleaved from the solid support by a chemical, enzymatic or physical process.

8. A process of claim 6, wherein immobilization is accomplished via a photocleavable bond.

9. A process of claim 6, wherein the target nucleic acid is cleaved from the support during the ionizing and volatizing step.

10. A process of claim 3, wherein the solid support is selected from the group consisting of:

beads, flat surfaces, chips, capillaries, pins, combs and wafers.

11. A process of claim 3, wherein immobilization is accomplished by hybridization between an array of complementary capture nucleic acid molecules, which have been previously immobilized to a solid support, and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

12. A process of claim 1, wherein prior to step b), an amplification step is performed on the target nucleic acid.

13. A process of claim 12, wherein the amplification step is selected from the group consisting of:

cloning, transcription, the polymerase chain reaction (PCR), the ligase chain reaction (LCR) and strand displacement amplification (SDA).

14. A process of claim 1, wherein the mass spectrometer is selected from the group consisting of:

Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), Electrospray (ES), Ion Cyclotron Resonance (ICR) and Fourier Transform.

15. A process of claim 1, wherein prior to step b), the target nucleic acid is purified.

16. A process of claim 1, wherein the primer or extended primer is conditioned.

17. A process of claim 14, wherein the primer or extended primer is conditioned by phosphodiester backbone modification.

18. A process of claim 17, wherein the phosphodiester backbone modification is a cation exchange.

19. A process of claim 16, wherein the primer or extended primer is conditioned by contact with an alkylating agent or trialkylsilyl chloride.

20. A process of claim 16, wherein the primer or extended primer is conditioned by containing at least one nucleotide which reduces sensitivity for depurination.

21. A process of claim 20, wherein the nucleotide is an N7- or N9-deazapurine nucleotide or 2'"'"' fluoro 2'"'"' deoxy nucleotide.

22. A process of claim 1, wherein the target nucleic acid sequence is indicative of a disease or a condition selected from the group consisting of:

a genetic disease, a chromosomal abnormality, a genetic predisposition, a cancer and an infection.

27. A process of claim 1, wherein the mass spectrometer format is Matrix Assisted Laser Desorption (MALDI).

2. A process for determining at least one base in a target nucleic acid sequence present in a biological sample, comprising the steps of:

a) obtaining the target nucleic acid sequence from a biological sample;

b) performing at least one hybridization of the target nucleic acid sequence with a set of ligation educts for each DNA strand and a DNA ligase, thereby forming a ligation product;

c) ionizing and volatizing the product of step b); and

d) detecting the ligation product by mass spectrometry and comparing the value obtained with a known value to determine at least one base in the target nucleic acid sequence.

23. A process of claim 2, wherein either the first or second ligation educt for each DNA strand contains a 5'"'"' phosphate.

24. A process of claim 2, which is performed in a cyclic manner to obtain an amplified ligation product on which to perform detection.

25. A process of claim 2, wherein the ligase is a thermostable DNA ligase.

26. A process of claim 2, wherein the target nucleic acid is indicative of a disease or condition selected from the group consisting of a genetic disease;

a chromosomal abnormality, a cancer, a genetic predisposition to a disease and an infection.

28. A process of claim 2, wherein prior to step b), the target nucleic acid is immobilized onto a solid support to produce an immobilized target nucleic acid sequence.

29. A process of claim 28, wherein immobilization is accomplished by hybridization between a complementary capture nucleic acid molecule, which has been previously immobilized to a solid support, and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

30. A process of claim 28, wherein immobilization is accomplished via direct bonding between the solid support and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

31. A process of claim 28, wherein the target nucleic acid is reversibly immobilized.

32. A process of claim 31, wherein the target nucleic acid can be cleaved from the solid support by a chemical, enzymatic or physical process.

33. A process of claim 31, wherein immobilization is accomplished via a photocleavable bond.

34. A process of claim 31, wherein the target nucleic acid is cleaved from the support during the ionizing and volatizing step.

35. A process of claim 28, wherein the solid support is selected from the group consisting of:

beads, flat surfaces, chips, capillaries, pins, combs and wafers.

36. A process of claim 28, wherein immobilization is accomplished by hybridization between an array of complementary capture nucleic acid molecules, which have been previously immobilized to a solid support, and a portion of the nucleic acid molecule, which is distinct from the target nucleic acid sequence.

37. A process of claim 2, wherein the mass spectrometer is selected from the group consisting of:

Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), Electrospray (ES), Ion Cyclotron Resonance (ICR) and Fourier Transform.

38. A process of claim 2, wherein prior to step b), the target nucleic acid is purified.

39. A process of claim 2, wherein the ligation product is conditioned.

40. A process of claim 37, wherein the ligation product is conditioned by phosphodiester backbone modification.

41. A process of claim 40, wherein the phosphodiester backbone modification is a cation exchange.

42. A process of claim 39, wherein the ligation product is conditioned by contact with an alkylating agent or trialkylsilyl chloride.

43. A process of claim 39, wherein the ligation product is conditioned by containing at least one nucleotide which reduces sensitivity for depurination.

44. A process of claim 43, wherein the nucleotide is an N7- or N9-deazapurine nucleotide or 2'"'"' fluoro 2'"'"' deoxy nucleotide.

45. A process of claim 2, wherein the target nucleic acid sequence is indicative of a disease or a condition selected from the group consisting of:

a genetic disease, a chromosomal abnormality, a genetic predisposition, a cancer and an infection.

46. A process of claim 2, wherein the mass spectrometer format is Matrix Assisted Laser Desorption (MALDI).