Gene specific arrays, preparation and use

• US 6,716,579 B1
• Filed: 06/09/2000
• Issued: 04/06/2004
• Est. Priority Date: 06/11/1999
• Status: Expired due to Fees

• Current Assignee: Clingenix, Inc.
• Original Assignee: Clingenix, Inc.
• Inventors: Yu, Yie-Teh, Holding, Julie, Baidya, Narayan, Chen, Yii-Der Ida
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/591,366
• Time in Patent Office: 1,397 Days
• Field of Search: 435/6, 435/69.1, 536/23.1, 422/68.1, 702/20
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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

Abstract

The present invention provides arrays comprising a plurality of polynucleotide probes having sequences complementary to the 3′ untranslated region of a gene transcript, whose chromosomal location has been defined. The arrays are particularly useful for conducting comparative gene expression analyses. The present invention also includes a method of preparing these arrays and various methods of using these arrays for detecting differential expression of multiple gene transcripts amongst multiple subjects. Further provided by the invention are computer readable media recorded thereon an array of polynucleotide probes as specified herein, a computer-based system, and kits for detecting differential expression of a multiplicity of gene transcripts.

Claims

1. An array comprising a plurality of polynucleotide probes immobilized on a solid support, wherein:

(a) the plurality of polynucleotide probes corresponds to a multiplicity of gene transcripts and comprises at least 100 polynucleotides that are each complementary to a distinct gene transcript;

(b) each polynucleotide probe of the plurality is localized to a predetermined region on the solid support;

(c) each polynucleotide probe of the plurality is from about 50 to 500 nucleotides in length;

(d) each polynucleotide probe of the plurality is complementary to 3′

untranslated sequence of a gene transcript, the untranslated sequence having a defined chromosomal location; and

(e) each polynucleotide probe of the plurality is prepared by amplification of genomic DNA or cDNA using a primer pair selected from the group consisting of SEQ ID NOs. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64, 65-66, and 67-68.

2. An array of claim 1, wherein each polynucleotide probe of the plurality of polynucleotide probes is from about 50 to 400 nucleotides in length.

3. An array of claim 1, wherein each polynucleotide probe of the plurality of polynucleotide probes is from about 50 to 300 nucleotides in length.

4. An array of claim 1, wherein the predetermined region comprises at least 2 single-stranded polynucleotides that are complementary to the same gene transcript.

5. An array of claim 1, wherein the predetermined region comprises at least 100 single-stranded polynucleotides that are complementary to the same gene transcript.

6. An array of claim 1, wherein the predetermined region comprises at least 2 single-stranded polynucleotides of identical sequences.

7. An array of claim 1, wherein the predetermined region comprises at least 100 single-stranded polynucleotides of identical sequences.

8. An array of claim 1, wherein the predetermined region has an average size ranging from about 0.01 cm² to about 1 cm².

9. An array of claim 1, wherein the plurality of polynucleotide probes is immobilized to the solid support via a covalent linkage.

10. An array of claim 1, wherein the solid support is flexible.

11. An array of claim 1, wherein the solid support is rigid.

12. An array of claim 10, wherein the solid support is made of one or more substances selected from the group consisting of nitrocellulose, nylon, polypropylene, glass, and silicon.

13. An array of claim 11, wherein the solid support is made of one or more substances selected from the group consisting of nitrocellulose, nylon, polypropylene, glass, and silicon.

14. An array of claim 1, further comprising a control probe.

15. An array of claim 14, wherein the control probe is selected from the group consisting of normalization control probe, expression level control probe, and mismatch control probe.

16. An array of claim 14, wherein the control probe has nucleotide sequence complementary to one or more constitutively expressed genes.

17. An array of claim 1, wherein each polynucleotide probe of the plurality is prepared by amplification of genomic DNA or cDNA using a pair of primers that amplify the region corresponding to 3′

untranslated sequence of a gene transcript.

18. An array of claim 1, wherein the array comprises a polynucleotide probe prepared by amplification of genomic DNA or cDNA using a primer pair selected from the group consisting of SEQ ID NOs. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64, 65-66, and 67-68 and at least one control probe.

19. An array of claim 1 further comprising target polynucleotides corresponding to gene transcripts expressed in a subject, wherein the target polynucleotides are bound to the polynucleotide probes in form of stable target-probe hybridization complexes.

20. An array of claim 19, wherein the target polynucleotides are conjugated with a detectable label selected from the group consisting of an enzyme, a radioactive isotope and a luminescent substance.

21. An array of claim 19, wherein the target polynucleotides are DNA or RNA molecules.

22. An array of claim 19, wherein the target polynucleotides are cDNAs.

24. A method of using an array to detect gene expression in a sample, the method comprising:

(a) contacting the polynucleotide probes of the array of claim 1 with labeled target polynucleotides from the sample under conditions to form stable target-probe complexes; and

(b) detecting complex formation, thereby detecting gene expression in the sample.

23. A method of preparing an array of polynucleotide probes corresponding to a multiplicity of gene transcripts, the method comprising:

(a) generating a plurality of gene-specific polynucleotides by amplification of genomic DNA or cDNA using a primer pair selected from the group consisting of SEQ ID NOs. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, 13-14, 15-16, 17-18, 19-20, 21-22, 23-24, 25-26, 27-28, 29-30, 31-32, 33-34, 35-36, 37-38, 39-40, 41-42, 43-44, 45-46, 47-48, 49-50, 51-52, 53-54, 55-56, 57-58, 59-60, 61-62, 63-64, 65-66, and 67-68, wherein each polynucleotide of the plurality is from about 50 to about 500 nucleotides in length, and wherein each polynucleotide is complementary to 3′

untranslated sequence of a gene transcript, the untranslated sequence having a defined chromosomal location;

(b) immobilizing the plurality of polynucleotides in a predetermined region on a solid support; and

(c) repeating steps (a) and (b) to yield an array of polynucleotide probes corresponding to a multiplicity of gene transcripts.