Single label comparative hybridization

• US 20050260665A1
• Filed: 05/19/2005
• Published: 11/24/2005
• Est. Priority Date: 05/20/2004
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Mohammed, Mansoor S., Kim, Jaeweon, McCaskill, Christopher, Dzidic, Natasa
• Granted Patent: US 8,911,942 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2539/101 Subtraction analysis
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention provides methods of detecting and mapping chromosomal or genetic abnormalities associated with various diseases or with predisposition to various diseases, or to detecting the phenomena of large scale copy number variants. In particular, the present invention provides advanced methods of performing array-based comparative hybridization that allow reproducibility between samples and enhanced sensitivity by using the same detectable label for both test sample and reference sample nucleic acids. Invention methods are useful for the detection or diagnosis of particular disease conditions such as cancer, and detecting predisposition to cancer based on detection of chromosomal or genetic abnormalities and gene expression level. Invention methods are also useful for the detection or diagnosis of hereditary genetic disorders or predisposition thereto, especially in prenatal samples. Moreover, invention methods are also useful for the detection or diagnosis of de novo genetic aberrations associated with post-natal developmental abnormalities.

Claims

1. A method of performing comparative hybridization, said method comprising, comparing the amount of test and reference nucleic acids hybridized to a nucleic acid array, said amount of hybridized test and reference nucleic acids determined by detecting a signal from the hybridized nucleic acids which are labeled with the same detectable label.

2. The method of claim 1 wherein said detectable label is attached to the test and reference nucleic acids before hybridization.

3. The method of claim 1 wherein said detectable label is attached to the test and reference nucleic acids after hybridization.

4. The method of claim 1 wherein said detectable label is attached to the test and reference nucleic acids via a ligand-receptor interaction or by hybridization between complementary nucleotide sequences.

5. The method of claim 1 wherein one of said test or reference nucleic acids is associated with the detectable label during hybridization and the detectable label is attached to the other of said test or reference nucleic acids after hybridization.

6. The method of claim 1 wherein the amount of hybridized test and reference nucleic acids are determined by:

a) determining a signal for the detectable label hybridized to the array representing the total of hybridized test and reference nucleic acid;

b) treating the hybridized nucleic acids to selectively remove said detectable label from one of said test or reference nucleic acids, wherein said detectable label is linked to the test and reference nucleic acids through a first and second chemical linkage, respectively, and wherein one of said first linkage or said second linkage is susceptible to the treatment step;

c) determining a signal for the detectable label hybridized to the array following step b), which represents one of said hybridized test or reference nucleic acid; and

d) determining a signal for the other of the hybridized test or reference by using the signal from c) and b).

7. The method of claim 6 wherein said detectable label is attached to the test and reference nucleic acids before hybridization.

8. The method of claim 6 wherein said detectable label is attached to the test and reference nucleic acids after hybridization.

9. The method of claim 6 wherein said detectable label is attached to the test and reference nucleic acids via a ligand-receptor interaction or by hybridization between complementary nucleotide sequences.

10. The method of claim 6 wherein one of said test or reference nucleic acids is associated with the detectable label during hybridization and the detectable label is attached to the other of said test or reference nucleic acids after hybridization.

11. The method of claim 1 wherein the amount of hybridized test and reference nucleic acids are determined by:

a) determining a signal representing one of the test or reference nucleic acid hybridized to the array by contacting the hybridized array with a first complex comprising a detectable label and a first entity, wherein said first complex selectively reacts with one of said test or reference nucleic acid via a first tag attached to said test or reference nucleic acid, said first entity and tag being members of a specific binding pair;

b) determining a signal representing the total of the hybridized test and reference nucleic acid by contacting the hybridized array of step a) with a second complex comprising the detectable label and a second entity, wherein said second complex selectively reacts with the other of said test or reference nucleic acid via a second tag attached to the other of said test or reference nucleic acid detected in step b), said second entity and second tag being members of a specific binding pair; and

c) using the signal from a) and b) to obtain the signal for the other of said hybridized test or reference nucleic acid not detected in step a).

12. The method of claim 1 wherein the amount of hybridized test and reference nucleic acids are determined by:

a) determining a signal representing one of the test or reference nucleic acid hybridized to the array by contacting the hybridized array with a first complex comprising a detectable label and a first entity, wherein said first complex selectively reacts with one of said test or reference nucleic acid via a first tag attached to said test or reference nucleic acid;

b) determining a signal representing the other of said test or reference nucleic acids not detected in a) by contacting a duplicate of the hybridized array used in a) with a second complex comprising a detectable label and a second entity, wherein said second complex selectively reacts with the other of said test or reference nucleic acid via a second tag attached to said other of said test or reference nucleic acid.

13. The method of claim 11 wherein the amount of hybridized test and reference nucleic acids are determined by:

a) determining a signal representing one of the test or reference nucleic acid hybridized to the array, wherein said detectable label has been attached before hybridization;

b) determining a signal representing the total of the hybridized test and reference nucleic acid by contacting the hybridized array with a first complex comprising a detectable label and an entity, wherein said first complex selectively reacts with the other of said test or reference nucleic acid via a tag attached to said other of the test or reference nucleic acid;

c) using the signal from a) and b) to obtain the signal for the other of said hybridized test or reference nucleic acid not detected in step a).

14. The method of claim 1 wherein the amount of hybridized test and reference nucleic acids are determined by:

a) determining a signal representing one of the test or reference nucleic acid hybridized to the array reading the array, wherein said detectable label is attached to one of said test and reference nucleic acids before hybridization;

b) determining a signal representing the other of said test or reference nucleic acids not detected in a) by contacting a duplicate of the hybridized array used in a) with a complex comprising a detectable label and an entity, wherein said complex selectively reacts with the other of said test or reference nucleic acid via a tag attached to said other of said test or reference nucleic acid, said duplicate array differs from the first array in that no detectable label is attached to the hybridizing nucleic acid.

15. The method of any of claims 11-12, wherein said first tag and said second tag each comprises a unique oligonucleotide capture sequence.

16. The method of claim 15, wherein said first complex and said second complex each comprises an oligonucleotide complementary to one of said unique oligonucleotide capture sequences.

17. The method of claim 15, wherein said first complex and said second complex each comprises an oligonucleotide complementary to one of said unique oligonucleotide capture sequences in a dendrimeric construct.

18. The method of claim any of claims 11-12 wherein said first entity and said first tag are each attached to a member of a first specific binding pair and said second entity and second tag are each attached to a member of a second specific binding pair wherein said first and second binding pair members do not cross-react.

19. The method of any of claims 1-14 wherein said test or reference nucleic acids represent a subset of the nucleic acid of a cell.

20. The method of any of claims 1-14 wherein said nucleic acid array comprises a plurality of nucleic acid segments each contained within an artificial chromosome.

21. The method of claim 20, wherein said artificial chromosome is a bacterial artificial chromosome (BAC).

22. The method of claim 20, wherein said artificial chromosome is P-1 derived artificial chromosome (PAC).

23. The method of claim 1-14 wherein said nucleic acid array comprises a plurality of nucleic acid segments, each nucleic acid segment of said plurality being between about 1,000 and about 1,000,000 nucleotides in length.

24. The method of claim 1-14 wherein said nucleic acid array comprises a plurality of nucleic acid segments each nucleic acid segment of said plurality being between about 100,000 and about 300,000 nucleotides in length.

25. The method of claim 1-14, wherein said nucleic acid array comprises a plurality of nucleic acid segments that collectively comprise the nucleic acid sequence of at least one chromosome.

26. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 3-4 megabases in length.

27. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 2-3 megabases in length.

28. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 1-2 megabases in length.

29. The method of claim 25, wherein said plurality of nucleic acid segments collectively comprise the nucleic acid sequence of all chromosomes of a cell.

30. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 3-4 megabases in length.

31. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 2-3 megabases in length.

32. The method of claim 25, wherein said plurality of nucleic acid segments comprise segments of chromosomal sequence of about 1-2 megabases in length.

33. The method of any of claims 1-14, wherein said nucleic acid array comprises a plurality of nucleic acid segments that collectively comprise at least about 50 genomic regions of interest.

34. The method of any of claims 1-14, wherein said nucleic acid array comprises at least about 300 discrete locations where a nucleic acid of the plurality of nucleic acid segments is present.

35. The method of any of claims 1-14, wherein said nucleic acid array comprises at least about 500 discrete locations where a nucleic acid of the plurality of nucleic acid segments is present.

36. The method of any of claims 1-14, wherein said detectable label is detected using optical means.

37. The method of any of claims 1-14, wherein said detectable label is a fluorophore.

38. The method of any of claims 6-10, wherein said first linkage and said second linkage differ in susceptibility to temperature.

39. The method of claim 38, wherein said first linkage and said second linkage both comprise oligonucleotides with different melting temperatures (T_m).

40. The method of any of claims 6-10, wherein said first linkage and said second linkage differ in susceptibility to pH hydrolysis.

41. The method of claim 40, wherein said first linkage and said second linkage both comprise peptides.

42. The method of any of claims 6-10, wherein said first linkage and said second linkage differ in susceptibility to radiation.

43. The method of claim 42, wherein said first linkage and said second linkage both comprise oligonucleotides comprising nucleotide stretches having different susceptibility to ultraviolet radiation.

44. The method of any of claims 6-10, wherein said first linkage and said second linkage differ in susceptibility to a change in oxidative conditions.

45. The method of any of claim 6-10, wherein said first linkage and said second linkage differ in susceptibility to a change in atmospheric conditions.

46. The method according to claim 45, wherein said change in atmospheric conditions comprises exposing said surface to ozone.

47. The method of any of claim 6-10, wherein either said first linkage and said second linkage is hydrolyzed upon contact with an agent causing hydrolysis.

48. The method of any of claims 6-10, wherein either said first linkage and said second linkage is hydrolyzed upon contact with an enzyme.

49. The method of claim 48, wherein either said first linkage and said second linkage comprises an oligonucleotide containing a recognition site for a restriction endonuclease or a homing endonuclease.

50. The method of claim 49, wherein either said first linkage and said second linkage is cleaved by a rare-cutting endonuclease to selectively remove the detectable label from either nucleic acids of said test sample or nucleic acids of said reference sample upon cleavage.

51. The method of claim 49, wherein said oligonucleotide containing a recognition site for a restriction endonuclease or a homing endonuclease is in a dendrimeric construct.

52. The method of any of claims 6-10, wherein said first linkage and said second linkage differ in susceptibility to a change in buffer conditions.

53. The method of any of claims 1-14, wherein said test sample is obtained from a patient.

54. The method of claim 53, wherein said test sample obtained from a patient is suspected of comprising cancerous cells.

55. The method of any of claims 1-14, wherein said test sample is obtained from a prenatal specimen.

56. The method of any of claims 1-14, wherein said test sample is obtained from an embryo or fetus.

57. The method of any of claims 1-14, wherein said test sample is from an individual and the method is used to detect a genetic abnormality.

58. The method of any of claims 1-14, wherein said test sample is from an individual and the method is used to diagnose a genetic abnormality.

59. The method of any of claims 1-14, wherein said test nucleic acids and reference nucleic acids are genomic nucleic acids.

60. A method of comparing the expression of genes in a test sample versus that of a reference sample, said method comprising

comparing the amount of cDNA prepared from mRNA of a test sample and cDNA prepared from mRNA of a reference sample hybridized to a nucleic acid array, said amount of hybridized test and reference cDNA determined by detecting a signal from the hybridized cDNA which is labeled with the same detectable label.

61. The method of claim 60 wherein said detectable label is attached to the test and reference cDNA before hybridization.

62. The method of claim 60 wherein said detectable label is attached to the test and cDNA after hybridization.

63. The method of claim 60 wherein said detectable label is attached to the test and cDNA via a ligand-receptor interaction or by hybridization between complementary nucleotide sequences.

64. The method of claim 60 wherein the amount of hybridized test and reference cDNAs are determined by:

a) determining a signal for the detectable label hybridized to the array representing the total of hybridized test and reference cDNA;

b) treating the hybridized nucleic acids to selectively remove said detectable label from one of said test or reference cDNA, wherein said detectable label is linked to the test and reference cDNA through a first and second chemical linkage, respectively, and wherein one of said first linkage or said second linkage is susceptible to the treatment step;

c) determining a signal for the detectable label hybridized to the array following step b), which represents one of said hybridized test or reference cDNA; and

d) determining a signal for the detectable label selectively removed in b) by subtracting c) from b), wherein said signal from c) and d) represents the signal for one of said test and reference cDNA hybridized to the nucleic acid array.

65. The method of claim 64 wherein said detectable label is attached to the test and reference cDNAs before hybridization.

66. The method of claim 64 wherein said detectable label is attached to the test and reference cDNAs after hybridization.

67. The method of claim 64 wherein said detectable label is attached to the test and reference cDNAs via a ligand-receptor interaction or by hybridization between complementary nucleotide sequences.

68. The method of claim 60 wherein the amount of hybridized test and reference cDNAs are determined by:

a) determining a signal representing one of the test or reference cDNA hybridized to the array by contacting the hybridized array with a first complex comprising a detectable label and a first entity, wherein said first complex selectively reacts with one of said test or reference cDNA via a first tag attached to said test or reference cDNA, said first entity and tag being members of a specific binding pair;

b) determining a signal representing the total of the hybridized test and reference cDNA by contacting the hybridized array of step a) with a second complex comprising the detectable label and a second entity, wherein said second complex selectively reacts with the other of said test or reference nucleic acid via a second tag attached to the other of said test or reference cDNA detected in step b), said second entity and second tag being members of a specific binding pair; and

c) subtracting a) from b) to obtain the signal for the other of said hybridized test or reference cDNA not detected in step a).

69. The method of claim 60 wherein the amount of hybridized test and reference cDNAs are determined by:

a) determining a signal representing one of the test or reference cDNA hybridized to the array by contacting the hybridized array with a first complex comprising a detectable label and a first entity, wherein said first complex selectively reacts with one of said test or reference cDNA via a first tag attached to said test or reference cDNA;

b) determining a signal representing the other of said test or reference cDNA not detected in a) by contacting a duplicate of the hybridized array used in a) with a second complex comprising a detectable label and a second entity, wherein said second complex selectively reacts with the other of said test or reference cDNA via a second tag attached to said other of said test or reference cDNA.