Methods of analysis of alternative splicing in mouse

• US 20050214823A1
• Filed: 01/13/2005
• Published: 09/29/2005
• Est. Priority Date: 01/13/2004
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Williams, Alan J., Blume, John E.
• Granted Patent: US 7,341,835 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention provides nucleic acid sequences which are complementary, in one embodiment, to a wide variety of mouse genes. The invention provides the sequences in such a way as to make them available for a variety of analyses. In one embodiment the nucleic acid sequences provided are present as an array of probes that may be used to measure gene expression of different mature RNA isoforms from at least 5,000 alternatively spliced mouse genes. As such, the invention relates to diverse fields impacted by the nature of molecular interaction, including chemistry, biology, medicine, pharmacology and medical diagnostics.

Claims

1. A probe array comprising a plurality of nucleic acid probes, wherein each probe in the plurality of nucleic acid probes comprises one of the sequences listed in SEQ ID Nos. 1-991,174 and wherein the plurality of nucleic acid probes of the array comprises at least 100,000 of the sequences listed in SEQ ID Nos. 1-991,174, each sequence present as a different feature of the array.

2. The array of claim 1 wherein said plurality of nucleic acid probes is attached to a solid support.

3. The array of claim 1 wherein the array comprises a plurality of beads wherein the probes are attached to the beads and the probes on a bead consist essentially of one of the sequences listed in SEQ ID Nos. 1-991,174.

4. The probe array of claim 1 wherein the array consists of a single contiguous solid support.

5. The probe array of claim 1 wherein the array consists of a plurality of solid supports.

6. A comprising at least 10,000 different exon probe sets wherein the array comprises a first, second and third probe set for each gene in a plurality of genes, wherein the plurality of genes comprises at least 1,000 mouse genes, wherein each gene has at least three exons, and wherein the first, second and third probe sets are complementary to a first, second and third exon, respectively, in each gene in the plurality.

7. The array of claim 6 wherein the plurality of genes comprises at least 5,000 mouse genes.

8. The array of claim 6 wherein the array comprises an exon probe set for each of at least four exons in each of at least 2,000 mouse genes.

9. The array of claim 6 wherein the array comprises an exon probe set for each of at least five exons in each of at least 1,000 mouse genes.

10. A nucleic acid array comprising a plurality of at least 100,000 probe sets wherein each probe set comprises:

a plurality of different perfect match probes, wherein the probes of each probe set are complementary to a single probe selection region, and wherein each probe selection region is a single exon or a subsequence of an exon.

11. A probe array comprising a plurality of exon probe sets, wherein each exon probe set comprises at least one probe that is complementary to an exon of a multi-exon gene;

wherein each probe in each exon probe set is complementary to the same exon and wherein the array comprises probe sets that are complementary to at least 1,000 different exons in the mouse genome.

12. The probe array of claim 11 wherein the plurality of exon probe sets comprises at least 5,000 different probe sets.

13. The probe array of claim 11 wherein the plurality of exon probe sets comprises at least 10,000 different probe sets.

14. The probe array of claim 11 wherein the plurality of exon probe sets comprises at least 250,000 different probe sets.

15. The probe array of claim 11 wherein the plurality of exon probe sets comprises at least 500,000 different probe sets.

16. A probe array comprising a plurality of probe sets, wherein each probe set is complementary to a single exon in a transcript, the plurality of probe sets comprising probe sets complementary to a plurality of multi-exon mouse gene and wherein the plurality of probe sets includes at least one probe set complementary to each exon in each gene in the plurality of multi-exon mouse genes.

17. The probe array of claim 16 wherein there are at least 1000 different exons in the plurality of multi-exon genes and the array comprises a probe set that is specifically complementary to each of the at least 1000 different exons.

18. The probe array of claim 16, wherein the plurality of multi-exon mouse genes comprises at least 1000 genes, and wherein each multi-exon mouse gene in the plurality comprises at least 3 exons.

19. The probe array of claim 16, wherein the plurality of multi-exon mouse genes comprises at least 1000 genes, and wherein each multi-exon mouse gene in the plurality comprises at least 4 exons and the array comprises a probe set that is specifically complementary to each exon in each multi-exon mouse gene in the plurality.

20. The probe array of claim 16 further comprising a plurality of control probes wherein said control probes are antigenomic background probes or genomic background probes.

21. A kit comprising a probe array according to claim 16, a T7-N6 primer, random primers, a T7 RNA polymerase, dUTP, UDG and optionally an AP endonuclease.

22. A method of detecting a plurality of mature RNA isoforms from each of a plurality of mouse genes in a biological sample from a mouse comprising:

obtaining a nucleic acid derived from the biological sample;

labeling the nucleic acid;

hybridizing the labeled nucleic acid to an array comprising a plurality of exon probe sets comprising probes that are complementary to a plurality of exons in a plurality of at least 1,000 mouse multi exon genes, wherein for each multi exon gene there is a probe set on the array for each of at least two exons from the gene;

detecting the hybridization pattern; and

analyzing the hybridization pattern to detect a plurality of mature RNA isoforms from at least two mouse multi exon genes.

23. The method of claim 22 wherein the labeled nucleic acid hybridized to the array consists essentially of DNA.

24. The method of claim 22 wherein the labeled nucleic acid hybridized to the array consists essentially of RNA that is complementary to the target mRNA.

25. The method of claim 22 wherein the labeled nucleic acid hybridized to the array consists essentially of RNA that is in the sense orientation relative to the target mRNA.

26. The method of claim 22 wherein the labeled nucleic acid is hybridized to the array in a single reaction.