Methods for mapping the chromosomal loci of genes expressed by a cell

• US 20030124542A1
• Filed: 02/08/2002
• Published: 07/03/2003
• Est. Priority Date: 12/28/2001
• Status: Abandoned Application

• Current Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Original Assignee: Spectral Genomics, Inc. (Revvity, Inc.)
• Inventors: Shah, Shishir, Mohammed, Mansoor
• Application Number:
  US10/071,837
• Publication Number:
  US 20030124542A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The invention provides novel methods to map the location and measure the expression of some or all the genes expressed by a cell using array-based nucleic acid hybridizations. The use and applicability of the invention includes identification of tissue specific genes and identifying different tissue types during growth and development. The methods also can be used to identify genes expressing abnormal types of transcripts or abnormal levels or transcripts. Thus, the invention can be used to identify genes encoding transcripts associated with disease processes, such as cancer.

Claims

1. An array-based method for identifying a chromosomal loci of a gene expressed by a cell, the method comprising:

(a) providing an array comprising a plurality of cloned genomic nucleic acid segments, wherein each genomic nucleic acid segment is immobilized to a discrete and known spot on a substrate surface to form an array and the cloned genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome;

(b) providing a sample comprising a nucleic acid sequence comprising a detectable label, wherein the nucleic acid sequence comprises the same sequence or a sequence complementary to a sequence of a transcript expressed by a cell;

(c) contacting the sample of step (b) with the array of step (a) under conditions wherein the labeled nucleic acid can specifically hybridize to the genomic nucleic acid segments immobilized on the array; and

,

(d) identifying to which discrete and known spots on the substrate surface are specifically hybridized to a labeled nucleic acid segment, wherein the identification is made by detecting a specifically hybridized nucleic acid sequence, thereby identifying a chromosomal loci of a gene expressed by a cell.

2. The method of claim 1, wherein the labeled nucleic acid sequence comprises the same sequence or a sequence complementary to a subset of transcripts expressed by the cell.

3. The method of claim 2, wherein the labeled nucleic acid sequence comprises the same sequence or a sequence complementary to all of the transcripts expressed by the cell.

4. The method of claim 1, wherein the transcript comprises a HER2/neu or a Neu/ErbB2 transcript.

5. The method of claim 1, wherein the transcript comprises a c-fos, a c-fas or a c-jun transcript.

6. The method of claim 1, wherein the transcript comprises a DAX-1 or a DAX-2 transcript.

21. The method of claim 1 or claim 7, wherein the nucleic acid sequence comprises an RNA.

22. The method of claim 1 or claim 7, wherein the nucleic acid sequence comprises a DNA.

23. The method of claim 1 or claim 7, wherein the nucleic acid sequence comprises a cDNA.

24. The method of claim 1 or claim 7, wherein the nucleic acid sequence comprises an expressed sequence tag (EST).

25. The method of claim 1 or claim 7, wherein the nucleic acid sequence complementary to a transcript comprises a sequence representative of the full length of the transcript.

26. The method of claim 25, wherein the nucleic acid sequence complementary to a transcript is between about 12 to about 500 bases in length.

27. The method of claim 26, wherein the nucleic acid sequence complementary to a transcript is between about 25 to about 250 bases in length.

28. The method of claim 27, wherein the nucleic acid sequence complementary to a transcript is between about 50 to about 150 bases in length.

29. The method of claim 1, wherein the cloned genomic nucleic acid segment is cloned in a construct comprising an artificial chromosome.

30. The method of claim 29, wherein the artificial chromosome comprises a bacterial artificial chromosome (BAC).

31. The method of claim 29, wherein the artificial chromosome is selected from the group consisting of a human artificial chromosome (HAC) a yeast artificial chromosome (YAC), a transformation-competent artificial chromosome (TAC) and a bacteriophage P1-derived artificial chromosome (PAC).

32. The method of claim 1, wherein a cloned nucleic acid segment is cloned in a construct comprising a vector selected from the group consisting of a cosmid vector, a plasmid vector and a viral vector.

33. The method of claim 1, wherein the cloned nucleic acid segment is between about 50 kilobases (0.5 megabase) to about 500 kilobases (5 megabases) in length, between about 100 kilobases (1 megabase) to about 400 kilobases (4 megabases) in length, or, is about 300 kilobases (3 megabases) in length.

34. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a body fluid sample, a cell sample or a tissue sample.

35. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a cancer cell or a tumor cell sample.

36. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a biopsy sample.

37. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a blood sample.

38. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a urine sample.

39. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a cerebral spinal fluid (CSF) sample.

40. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises an amniotic fluid sample.

41. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a chorionic villus sample.

42. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises an embryonic cell or embryo tissue sample.

43. The method of claim 1, wherein the cell expressing the transcript of step (b) comprises a mammalian cell.

44. The method of claim 43, wherein the mammalian cell comprises a human cell.

45. The method of claim 1, further comprising a washing step, wherein nucleic acid in the sample not specifically hybridized to the genomic nucleic acid segments immobilized on the array are removed.

46. The method of claim 45, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.02 molar at pH 7 at a temperature of at least about 50°

C.

47. The method of claim 45, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.15 M at a temperature of at least about 72°

C. for about 15 minutes.

48. The method of claim 45, wherein the washing step comprises use of a solution comprising a salt concentration of about 0.2X SSC at a temperature of at least about 50°

C. for at least about 15 minutes.

49. A kit comprising the following components:

(a) an array comprising a plurality of cloned genomic nucleic acid segments, wherein each genomic nucleic acid segment is immobilized to a discrete and known spot on a substrate surface to form an array and the cloned genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome; and

,

(b) instructions for using the array for identifying a chromosomal loci of a gene expressed by a cell as set forth in claim 1 or claim 7.

50. The kit of claim 49, further comprising materials to prepare a sample comprising a genomic nucleic acid for application to the array.

51. The kit of claim 49, further comprising materials to isolate, clone or amplify the transcripts of a cell.

52. The kit of claim 49, further comprising materials to prepare a cDNA library.

53. The kit of claim 49, further comprising materials to label a sample.

54. The kit of claim 49, further comprising an array-immobilized nucleic acid.

55. The kit of claim 49, further comprising a sample of wild type, or normal, nucleic acid.

56. The kit of claim 55, wherein the wild type, or normal, nucleic acid is labeled.

57. The kit of claim 56, wherein the wild type, or normal, nucleic acid comprises a human wild type genomic nucleic acid.

58. The kit of claim 54, wherein the array comprises a G-CHIP™

, a SPECTRALCHIP™

Mouse BAC Array or a SPECTRALCHIP™

Human BAC Array.

7. An array-based method for identifying a chromosomal loci of a gene differentially expressed by different cells or by a cell under different conditions by performing an array-based comparative genomic hybridization (CGH), comprising the following steps:

(a) providing an array comprising a plurality of cloned genomic nucleic acid segments, wherein each genomic nucleic acid segment is immobilized to a discrete and known spot on a substrate surface to form an array and the cloned genomic nucleic acid segments comprise a substantially complete genome or a known subset of a genome;

(b) providing a first sample, wherein the sample comprises a plurality of nucleic acid sequences comprising a first detectable label, wherein the nucleic acid sequence comprises the same sequence or a sequence complementary to all of the transcripts expressed by a first cell or a subset of transcripts expressed by the first cell;

(c) providing a second sample, wherein the sample comprises a plurality of nucleic acid sequences comprising a second detectable label, wherein the nucleic acid sequence comprises the same sequence or a sequence complementary to all of the transcripts expressed by a second cell or a subset of transcripts expressed by the second cell;

(d) contacting the samples of step (b) and step (c) with the array of step (a) under conditions wherein the nucleic acid in the samples can specifically hybridize to the immobilized nucleic acid of step (a); and

(e) identifying to which discrete and known spots on the substrate surface are specifically hybridized to a nucleic acid sequence of step (b) and identifying to which discrete and known spots on the substrate surface are specifically hybridized to a nucleic acid sequence of step (c), wherein the identification is made by detecting a specifically hybridized first and a specifically hybridized second sample nucleic acid sequence, thereby identifying a chromosomal loci of a gene differentially expressed by a first cell compared to a second cell.

8. The method of claim 7, in step (e) further comprising measuring the amount of specifically hybridized first and second label on each discrete and known spot and comparing the amount of first and second sample nucleic acid sequence on each discrete and known spot, thereby determining the level of expression of the differentially expressed gene in the first cell as compared to the second cell.

9. The method of claim 7, wherein the first cell is a normal cell and the second cell has an abnormal phenotype.

10. The method of claim 9, wherein the abnormal phenotype comprises a disease phenotype.

11. The method of claim 9, wherein the abnormal phenotype comprises a neoplastic or hyperplastic phenotype.

12. The method of claim 11, wherein neoplastic phenotype is selected from the group consisting of breast cancer and bone cancer.

13. The method of claim 7, wherein the first cell is an unstimulated cell and the second cell is the unstimulated cell after stimulation.

14. The method of claim 7, wherein the first cell is an undifferentiated cell and the second cell is the undifferentiated cell after stimulation.

15. The method of claim 7, wherein the first cell is a normal cell and the second cell has phenotype of an injured cell.

16. The method of claim 7, wherein the first cell is a normal cell and the second cell has an altered phenotype because it has been exposed to an environmental stress.

17. The method of claim 16, wherein the environmental stress comprises a high or a low or a change in temperature.

18. The method of claim 16, wherein the environmental stress comprises an exposure to a chemical.

19. The method of claim 18, wherein the chemical is a carcinogen.

20. The method of claim 18, wherein the carcinogen is a drug or a medicine.