DNA diagnostics based on mass spectrometry

• US 6,258,538 B1
• Filed: 04/06/1999
• Issued: 07/10/2001
• 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: Braun, Andreas, Köster, Hubert, Little, Daniel P.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/287,679
• Time in Patent Office: 826 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 436/94
• 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 sequences in the molecules are provided. Depending upon the sequence to be detected, the processes, for example, can be used to diagnose a genetic disease or a chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogen, or for determining identity or heredity.

Claims

1. A process for identifying a target nucleotide present in a nucleic acid molecule, comprising the steps of:

a) obtaining a nucleic acid molecule that contains a target nucleotide;

b) optionally immobilizing the nucleic acid molecule onto a solid support, to produce an immobilized nucleic acid molecule;

c) hybridizing the nucleic acid molecule with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide;

d) contacting the product of step c) with a complete set of dideoxynucleoside triphosphates or 3′

-deoxynucleoside triphosphates and a polymerase, so that only the dideoxynucleoside or 3′

-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer;

e) ionizing and volatilizing the product of step d); and

f) determining the molecular mass of the extended primer by mass spectrometry, thereby identifying the target nucleotide.

2. A process of claim 1, wherein the primer oligonucleotide is an oligonucleotide mimetic.

3. A process of claim 2, wherein the oligonucleotide mimetic is a peptide nucleic acid.

4. A process of claim 1, wherein the nucleic acid molecule is immobilized on a solid support.

5. A process of claim 4, wherein the nucleic acid molecule is immobilized by hybridizing a portion of the nucleic acid molecule, which is distinct from the site immediately adjacent to the target nucleotide, to a complementary capture nucleic acid sequence, which is immobilized to the solid support.

6. A process of claim 4, wherein the nucleic acid molecule is immobilized by direct bonding to the solid support.

7. A process of claim 1, wherein, prior to mass spectrometry, a portion of the nucleic acid molecule comprising the target nucleotide is amplified.

8. A process of claim 1, wherein the nucleic acid molecule is DNA and the polymerase is a DNA dependent DNA polymerase.

9. A process of claim 1, wherein the nucleic acid molecule is DNA and the polymerase is a DNA dependent RNA polymerase.

10. A process of claim 1, wherein the nucleic acid molecule is RNA and the DNA polymerase is a RNA dependent DNA polymerase.

11. A process of claim 4, wherein the solid support is selected from the group consisting of beads, flat surfaces, pins, combs and wafers.

12. A process of claim 4, wherein the nucleic acid molecule is one of a plurality of nucleic acid molecules, which are immobilized by hybridizing a portion of the nucleic acid molecules, which is distinct from the site immediately adjacent to the target nucleotide, to an array of complementary capture nucleic acid sequences, which are immobilized to the solid support.

13. A process of claim 12, wherein the complementary capture nucleic acid sequences are oligonucleotide mimetics.

14. A process of claim 13, wherein the oligonucleotide mimetics are peptide nucleic acids.

15. A process of claim 4, wherein the immobilization is reversible.

16. A process of claim 4, wherein the immobilization is via a thiol linkage.

17. A process of claim 15, wherein the immobilization is via a photocleavable linkage.

18. A process of claim 1, wherein the mass spectrometry is selected from the group consisting of matrix-assisted laser desorption/ionization time-of-flight, electrospray, ion cyclotron resonance, Fourier transform, and combinations thereof.

19. A process of claim 1, wherein, prior to mass spectrometry, the product of step c) is conditioned.

20. A process of claim 1, wherein the nucleic acid molecule comprises one of a plurality of nucleic acid molecules, and said hybridizing is performed using at least two primer oligonucleotides, each of which is complementary to a site immediately adjacent to the target nucleotide in a nucleic acid molecule in the plurality, wherein the products of step d) are mass differentiated to detect and distinguish the target nucleotides in the plurality of nucleic acid molecules simultaneously.

21. A process of claim 20, wherein mass differentiation is achieved by differences in the length or sequence of the products.

22. A process of claim 20, wherein mass differentiation is achieved by the introduction of mass modifying functionalities in a base, sugar or phosphate moiety of the products.

23. A process of claim 20, wherein mass differentiation is achieved by exchange of cations at the phosphodiester bonds of the products.

24. A process of claim 1, wherein the nucleic acid molecule is obtained from an individual, and the target nucleotide provides a DNA fingerprint or is indicative of a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection.

57. The method of claim 1, wherein the presence of a target nucleotide is indicative of the presence of a mutation.

25. A process for identifying target nucleotides present in a plurality of nucleic acid molecules, comprising the steps of:

a) hybridizing nucleic acid molecules in the plurality with primer oligonucleotides that are complementary to a site immediately adjacent to a target nucleotide, wherein a primer that hybridizes to one nucleic acid molecule in the plurality, or an extension product of the primer, is distinguishable from each primer, or extension product thereof, that hybridizes to a different nucleic acid molecule in the plurality, thereby producing one or more hybridized primers;

b) contacting the hybridized primers with a complete set of dideoxynucleoside triphosphates or 3′

-deoxynucleoside triphosphates and a DNA dependent DNA polymerase, so that only a dideoxynucleoside triphosphate or 3′

-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto a hybridized primer; and

c) determining the molecular masses of the products of step b) by mass spectrometry, thereby identifying the target nucleotides present in the plurality of nucleic acid molecules.

26. A process of claim 25, wherein the products of step b) are mass air differentiated and, thereby, distinguishable from each other.

27. A process of claim 26, wherein the products are mass differentiated due to differences in the length or sequence of the products.

28. A process of claim 26, wherein the products are mass differentiated due to the introduction of mass modifying functionalities in a base, sugar or phosphate moiety of the products.

29. A process of claim 26, wherein the products are mass differentiated due to an exchange of cations at the phosphodiester bonds of the products.

30. A process of claim 25, wherein each nucleic acid molecule in the plurality is immobilized to a solid support.

31. A process of claim 30, wherein the solid support is selected from the group consisting of beads, flat surfaces, pins, combs and wafers.

32. A process of claim 30, wherein the nucleic acid molecules in the plurality are immobilized in an array.

33. A process of claim 30, wherein the nucleic acid molecules in the plurality are reversibly immobilized.

34. A process of claim 33, wherein the nucleic acid molecules in the plurality are immobilized via a bond that can be cleaved by a process selected from the group consisting of a chemical process, an enzymatic process and a physical process.

35. A process of claim 34, wherein the bond is a photocleavable bond.

36. A process of claim 25, wherein, prior to mass spectrometry, a portion of the nucleic acid molecules comprising the target nucleotide is amplified.

37. A process of claim 25, wherein the nucleic acid molecules in the plurality are alleles of a gene.

38. A process of claim 25, wherein the nucleic acid molecules in the plurality are obtained from one or more individuals, and the target nucleotide provides a DNA fingerprint or is indicative of a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection.

58. The method of claim 25, wherein the presence of a target nucleotide is indicative of the presence of a mutation.

39. A process for detecting a target nucleotide present in a biological sample, comprising the steps of:

a) obtaining a nucleic acid molecule that contains a target nucleotide;

b) hybridizing the nucleic acid molecule with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide;

c) contacting the product of step b) with a dideoxynucleoside or a 3′

-deoxynucleoside triphosphate and a DNA polymerase, whereby if the dideoxynucleoside or 3′

-deoxynucleoside triphosphate is complementary to the target nucleotide it is extended onto the primer;

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

e) detecting the primer by mass spectrometry, to determine the identity of the target nucleotide.

40. The method of claim 39, further comprising:

before hybridizing, immobilizing the nucleic acid molecule onto a solid support, to produce an immobilized nucleic acid molecule.

41. A process for detecting a target nucleotide present in a biological sample, comprising the steps of:

a) obtaining a nucleic acid molecule that contains a target nucleotide;

b) hybridizing the nucleic acid molecule with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide;

c) contacting the product of step b) with dideoxynucleosides or 3′

-deoxynucleoside triphosphates and a DNA polymerase, whereby if a dideoxynucleoside or 3′

-deoxynucleoside triphosphate is complementary to the target nucleotide it is extended onto the primer;

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

e) detecting the primer by mass spectrometry, to determine the identity of the target nucleotide.

42. A process for determining whether a target nucleotide is present in a nucleic acid molecule, comprising:

a) hybridizing the nucleic acid molecule to a primer oligonucleotide, which is complementary to a sequence of the target nucleic acid molecule, wherein the sequence is adjacent to the region suspected of containing the target nucleotide, thereby producing a hybridized primer;

b) contacting the hybridized primer with

i) three deoxyribonucleoside triphosphates,

ii) a chain terminating nucleotide selected from the group consisting of a dideoxyribonucleoside triphosphate and a 3′

-deoxynucleoside triphosphate, wherein the chain terminating nucleotide corresponds to the missing deoxyribonucleoside triphosphate, and

iii) a DNA polymerase,

whereby the hybridized primer is extended until a chain terminating nucleotide is incorporated to produce an extended primer; and

c) determining the molecular mass of the extended primer by mass spectrometry, thereby determining whether the target nucleotide is present in the nucleic acid molecule.

43. The process of claim 42, wherein the primer oligonucleotide is an oligonucleotide mimetic.

44. The process of claim 43, wherein the oligonucleotide mimetic is a peptide nucleic acid.

45. The process of claim 42, wherein the presence of a target nucleotide is indicative of the presence of a mutation, and the mutation is selected from the group consisting of a point mutation, a deletion and an insertion.

46. The process of claim 42, wherein the mass spectrometry format is selected from the group consisting of matrix-assisted laser desorption/ionization time-of-flight, electrospray, ion cyclotron resonance, Fourier transform and combinations thereof.

47. The process of claim 42, wherein the nucleic acid molecule is immobilized on a solid support.

48. The process of claim 42, wherein the nucleic acid molecule is obtained from an individual, and the extended primer provides a DNA fingerprint or is indicative of a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection.

59. The method of claim 42, wherein the presence of a target nucleotide is indicative of the presence of a mutation.

49. A process for determining whether a target nucleotide is present in a plurality of nucleic acid molecules, comprising:

a) hybridizing each nucleic acid molecule in a plurality of nucleic acid molecules with a primer oligonucleotide, which is complementary to a sequence of a nucleic acid molecule that is adjacent to a region suspected of containing the target nucleotide, wherein a primer that hybridizes to one nucleic acid molecule in the plurality is distinguishable from each primer that hybridizes to a different nucleic acid molecule in the plurality, thereby producing hybridized primers;

b) contacting the hybridized primers with

i) three deoxynucleoside triphosphates, and

ii) a chain terminating nucleotiuj selected from the group consisting of a dideoxynucleoside triphosphate and a 3′

-deoxynucleoside triphosphate, wherein the chain terminating nucleotide corresponds to the missing deoxynucleoside triphosphate, and

iii) a DNA polymerase,

whereby the hybridized primers are extended until a chain terminating nucleotide is incorporated, thereby producing extended primers; and

c) determining the molecular masses of the extended primers by mass spectrometry, thereby determining whether a target nucleotide is present in a nucleic acid molecule among the plurality.

50. The process of claim 49, wherein:

the extended primers are mass differentiated and, thereby, distinguishable from each other.

51. A process of claim 50, wherein length or sequence differences among the extended primers render them distinguishable.

52. The process of claim 50, wherein the mass differentiation of the extended primers results from the presence of mass modifying functionalities in a base, sugar or phosphate moiety of the extended primers.

53. The process of claim 50, wherein the mass differentiation of the extended primers results from an exchange of cations at the phosphodiester bonds of the extended primers.

54. The process of claim 49, wherein each nucleic acid molecule in the plurality is immobilized to a solid support.

55. The process of claim 49, wherein, prior to determining the molecular masses of the extended primers, a portion of the nucleic acid molecules suspected of a target nucleotide is amplified.

56. The process of claim 49, wherein the nucleic acid molecules in the plurality are obtained from one or more individuals, and an extended primer provides a DNA fingerprint or is indicative of a disease or condition selected from the group consisting of a genetic disease, a chromosomal abnormality, a genetic predisposition, a viral infection, a fungal infection, a bacterial infection and a protist infection.