Chip-based speciation and phenotypic characterization of microorganisms

• US 7,252,948 B2
• Filed: 02/04/2005
• Issued: 08/07/2007
• Est. Priority Date: 02/08/1996
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Mack, David, Chee, Mark S., Berno, Anthony J., Gingeras, Thomas R., Ghandour, Ghassan, Stryer, Lubert, Wang, Ching
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US11/050,647
• Publication Number:
  US 20060204970A1
• Time in Patent Office: 914 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.15
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

This invention provides oligonucleotide based arrays and methods for speciating and phenotyping organisms, for example, using oligonucleotide sequences based on the Mycobacterium tuberculosis rpoB gene. The groups or species to which an organism belongs may be determined by comparing hybridization patterns of target nucleic acid from the organism to hybridization patterns in a database.

Claims

1. A method for assigning an organism to a group or species by detecting at least one polymorphism at a position of interrogation in a target nucleic acid sequence, comprising:

(a) providing a plurality of different probes complementary to one or more reference nucleic acid sequences, said probes immobilized in an array on a solid surface, wherein said probes differ from each other at the position of interrogation;

(b) hybridizing the target nucleic acid sequence to said probes in the array;

(c) hybridizing the reference nucleic acid sequence to said probes in a separate, identical array;

(d) comparing the hybridization pattern of step (b) to the hybridization pattern of step (c); and

(e) detecting at least one polymorphism at the position of interrogation, wherein said detecting allows assignment of an organism to a group or species.

2. The method of claim 1, wherein said probes are arranged in a plurality of probe sets.

3. The method of claim 2, wherein each probe differs from its predecessor in the same probe set by the acquisition of a 5′

nucleotide and the loss of a 3′

nucleotide.

4. The method of claim 2, wherein said probes are arranged in four probe sets.

5. The method of claim 3, wherein said array is a tiled array.

6. The method of claim 4, wherein the probes of said four probe sets differ from each other by a single nucleotide at the interrogation position.

7. The method of claim 6, wherein the probes of said four probe sets contain a complete set of permutations of nucleotides A, C, G and T/U at the position of interrogation.

8. The method of claim 1, wherein said probes comprise trailing or flanking sequences.

9. The method of claim 1, wherein said probes comprise at least 3,4,7,9, 15, 21 or 25 nucleotides.

10. The method of claim 1, wherein said probes are oligodeoxyribonucleotides (DNA) or oligoribonucleotides (RNA).

11. The method of claim 1, wherein said reference sequence comprises about 2,5, 10,20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000 or 100,000 nucleotides.

12. The method of claim 1, wherein said one or more reference sequences are oligodeoxyribonucleotides (DNA) or oligoribonucleotides (RNA).

13. The method of claim 12, wherein said one or more reference DNA or RNA sequences are selected from the group consisting of 16SrRNA, the rpoB gene, the katG gene, the inhA gene, the gyrA gene, the 23SnRNA gene, the rrs gene, the pncA gene, and the rpsL gene.

14. The method of claim 12, wherein said one or more reference DNA or RNA sequences are from a highly conserved gene.

15. The method of claim 1, wherein said at least one polymorphism is in a coding segment of the gene.

16. The method of claim 15, wherein said gene is a cytochrome b gene.

17. The method of claim 15, wherein said gene is a rpoB gene of a microorganism.

18. The method of claim 17, wherein said microorganism is M. tuberculosis.

19. The method of claim 1, wherein said at least one polymorphism is in a noncoding segment of a gene.

20. The method of claim 1, wherein at least two polymorphisms are detected.

21. The method of claim 1, wherein at least ten polymorphisms are detected.

22. The method of claim 1, wherein said organism is resistant to an antibiotic drug.

23. The method of claim 22, wherein the drug is selected from the group consisting of rifampacin, rifabutin, isoniazid, streptomycin, pyrazinamide, ethambutol.

24. The method of claim 1, wherein the comparing is used to derive one or more sets of differences between the target nucleic acid and the reference sequence, each set being associated with a probability that the target possesses a particular phenotype.

25. The method of claim 24, wherein the probability associated with each set of differences is used to derive a combined probability greater than a desired confidence level that the target possesses a particular phenotype.

26. The method of claim 1, wherein the comparing identifies one or more species-specific polymorphisms and these species-specific polymorphisms are used to confirm the assignment of species.

27. The method of claim 1, wherein the comparing identifies one or more shared polymorphisms and these shared polymorphisms are used to confirm the assignment of species.

28. The method of claim 1, wherein the target nucleic acid is amplified from a biological sample.

29. The method of claim 28, wherein the target nucleic acid is fluorescently labeled.

30. The method of claim 1, wherein the hybridizing is performed in a fluid volume of 250 p1 or less.

31. The method of claim 1, wherein the array has between 100 and 1,000,000 probes.

32. The method of claim 31, wherein the array has approximately 2,800 probes.

33. The method of claim 1, wherein the probes are linked to the support via a spacer.

34. A method for assigning an organism to a group or species by detecting at least one polymorphism at a position of interrogation in a target nucleic acid sequence, comprising:

(a) providing a plurality of different probes complementary to one or more reference nucleic acid sequences, said probes immobilized in an array on a solid surface;

wherein said probes differ from each other at the position of interrogation;

(b) labeling the target nucleic acid sequence fluorescently with one color; and

labeling the reference nucleic acid sequence fluorescently with another color;

(c) hybridizing the labeled reference and target nucleic acid sequences simultaneously to said probes in the array;

(d) analyzing the hybridization pattern of step (c) to identify said at least one polymorphism.

35. The method of claim 34, wherein the reference nucleic acid sequence is labeled with phycoerythrin (red).

36. The method of claim 34, wherein the target nucleic acid sequence is labeled with fluorescein (green).

37. The method of claim 34, wherein said probes are arranged in a plurality of probe sets.

38. The method of claim 37, wherein each probe differs from its predecessor in the same probe set by the acquisition of a 5′

nucleotide and the loss of a 3′

nucleotide.

39. The method of claim 37, wherein said probes are arranged in four probe sets.

40. The method of claim 39, wherein said array is a tiled array.

41. The method of claim 39, wherein the probes of said four probe sets differ from each other by a single nucleotide at the interrogation position.

42. The method of claim 41, wherein the probes of said four probe sets contain a complete set of permutations of nucleotides A, C, G and T/U at the position of interrogation.

43. The method of claim 34, wherein said probes comprise trailing or flanking sequences.

44. The method of claim 34, wherein said probes comprise at least 3, 4, 7, 9, 15, 21 or 25 nucleotides.

45. The method of claim 34, wherein said probes are oligodeoxyribonucleotides (DNA) or oligoribonucleotides (RNA).

46. The method of claim 34, wherein said reference sequence comprises about 2, 5, 10, 20, 50, 100, 500, 1,000, 5,000, 10,000, 20,000 or 100,000 nucleotides.

47. The method of claim 34, wherein said one or more reference sequences are oligodeoxyribonucleotides (DNA) or oligoribonucleotides (RNA).

48. The method of claim 47, wherein said one or more reference DNA or RNA sequences are selected from the group consisting of 16SrRNA, the rpoB gene, the katG gene, the inhA gene, the gyrA gene, the 23SnRNA gene, the rrs gene, the pncA gene, and the rpsL gene.

49. The method of claim 47, wherein said one or more reference DNA or RNA sequences are from a highly conserved gene.

50. The method of claim 34, wherein said at least one polymorphism is in a coding segment of the gene.

51. The method of claim 50, wherein said gene is a cytochrome b gene.

52. The method of claim 50, wherein said gene is a rpoB gene of a microorganism.

53. The method of claim 52, wherein said microorganism is M. tuberculosis.

54. The method of claim 34, wherein said at least one polymorphism is in a noncoding segment of a gene.

55. The method of claim 34, wherein at least two polymorphisms are detected.

56. The method of claim 34, wherein at least ten polymorphisms are detected.

57. The method of claim 34, wherein said organism is resistant to an antibiotic drug.

58. The method of claim 57, wherein the drug is selected from the group consisting of rifampacin, rifabutin, isoniazid, streptomycin, pyrazinamide, ethambutol.

59. The method of claim 34, wherein the analyzing is used to derive one or more sets of differences between the target nucleic acid and the reference sequence, each set being associated with a probability that the target possesses a particular phenotype.

60. The method of claim 59, wherein the probability associated with each set of differences is used to derive a combined probability greater than a desired confidence level that the target possesses a particular phenotype.

61. The method of claim 34, wherein the analyzing identifies one or more species-specific polymorphisms and these species-specific polymorphisms are used to confirm the assignment of species.

62. The method of claim 34, wherein the analyzing identifies one or more shared polymorphisms and these shared polymorphisms are used to confirm the assignment of species.

63. The method of claim 34, wherein the target nucleic acid is amplified from a biological sample.

64. The method of claim 34, wherein the hybridizing is performed in a fluid volume of 250 μ

l or less.

65. The method of claim 34, wherein the array has between 100 and 1,000,000 probes.

66. The method of claim 65, wherein the array has approximately 2,800 probes.

67. The method of claim 34, wherein the probes are linked to the support via a spacer.