Expression monitoring for gene function identification

• US 6,733,969 B2
• Filed: 04/18/2001
• Issued: 05/11/2004
• Est. Priority Date: 01/13/1997
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Mack, David H.
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US09/836,278
• Publication Number:
  US 20020028454A1
• Time in Patent Office: 1,119 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• 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 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

This invention provides methods, compositions and apparatus for mapping the regulatory relationship among genes by massive parallel monitoring gene expression. In some embodiments, mutations in the up-stream regulatory genes are detected by monitoring the change in down-stream gene expression. Similarly, the function of a specific mutation in a up-stream gene is determined by monitoring the down-stream gene expression. In addition, regulatory function of a target gene can be determined by monitoring the expression of a large number of down-stream genes. The invention also provides specific embodiments for detecting p53 functional homozygous and heterozygous mutations and for determining the function of p53 mutations.

Claims

1. A method for detecting a functional mutation in a target up-stream regulatory gene comprising:

preparing a reference sample from reference cells having a wild-type up-stream regulatory gene corresponding to said target up-stream regulatory gene;

preparing a target sample from target cells suspected of having a mutation in said target up-stream regulatory gene, said target cells being otherwise substantially similar to said reference cells;

detecting the expression of a plurality of down-stream genes in said reference sample to obtain a reference expression pattern, said down-stream genes being up or down regulated by said wild-type up-stream regulatory gene;

detecting the expression of said plurality of down-stream genes in said target sample to obtain a target expression pattern; and

comparing said reference expression pattern with said target expression pattern to detect functional mutation in or inactivation of said target gene.

2. The method of claim 1, wherein said down-stream genes are transcriptionally regulated by said wild-type up-stream regulatory gene and said reference and target expression patterns are detected by measuring the amount of transcripts of said down-stream genes in said reference and target samples.

3. The method of claim 2, wherein said amount of transcripts is detected with a high density nucleic acid array.

4. The method of claim 1, wherein said reference and target expression patterns are detected by measuring the amount of protein products of said down-stream genes in said reference and target samples.

5. The method of claim 1 further comprising the steps of:

detecting expression of a plurality of control genes in said reference and target samples, said control genes being unresponsive to the function of said up-stream regulatory gene; and

comparing reference and target expression patterns of control genes to provide a baseline for detecting a significant difference in expression patterns.

6. The method of claim 1 further comprising the step of:

indicating a loss of wild-type function in said target gene if a significant number of said down regulated genes are expressed relatively higher in said target sample than in said reference sample or if a significant number of said up-regulated genes are expressed lower in said target sample than in said reference sample.

7. The method of claim 1 further comprising the step of:

indicating a gain of function mutation in said target gene if a significant portion of said down regulated genes are expressed lower in said target sample than in said reference sample or if a significant portion of said up-regulated genes are expressed higher in said target sample than in said reference sample.

8. A method for detecting a p53 gene functional mutation in target cells comprising the steps of:

preparing a reference sample from reference cells having a wild-type p53 gene, said reference cells being otherwise substantially similar to said target cells;

detecting the expression of a plurality of more than 100 down-stream genes in said reference cells and said target cells to obtain a target expression pattern and a reference expression pattern, said down-stream genes being up- or down-regulated by said wild-type p53 gene; and

comparing said reference expression pattern with said target expression pattern to detect said p53 functional mutation.

9. The method of claim 8, wherein said down-stream genes are transcriptionally regulated by said wild-type p53 gene and the expression of said down-stream genes is detected by measuring the amount of transcripts of said down-stream genes in said reference and target cells.

10. The method of claim 9, wherein said amount of transcripts is measured with high density nucleic acid array.

11. The method of claim 9, wherein said down-stream genes comprise p53 up-regulated gadd45, cyclin G, p21waf1, Bax, IGF-BP3 and Thrombospondin genes and p53 down-regulated c-myc and PCNA genes.

12. An in-cell functional assay for a p53 sequence alteration comprising the steps of:

preparing a target sample from target cells having said p53 sequence alteration;

preparing a reference sample from reference cells having a wild-type p53 gene, said reference cells being otherwise substantially similar to said target cells;

detecting the expression of a plurality of more than 100 down-stream genes in said reference cells to obtain a reference expression pattern and in said target cells to obtain a target expression pattern, said down-stream genes being selected from the group consisting of p53 up-regulated genes and p53 down-regulated genes; and

comparing said reference expression pattern with said target expression pattern to determine the function of said p53 sequence alteration.

13. The method of claim 12, wherein said down-stream genes are transcriptionally regulated by said wild-type p53 gene and the expression of said down-stream genes is detected by measuring the amount of transcripts of said down-stream genes in said reference and target cells.

14. The method of claim 13, wherein said amount of transcripts is measured with a high density nucleic acid array.

15. The method of claim 13, wherein said down-stream genes comprise p53 up regulated gadd45, cyclin G, p21waf1, Bax, IGF-BP3 and Thrombospondin genes and p53 down regulated c-myc and PCNA genes.