Comparative gene transcript analysis

• US 5,840,484 A
• Filed: 01/27/1994
• Issued: 11/24/1998
• Est. Priority Date: 07/17/1992
• Status: Expired due to Fees

• Current Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Original Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Inventors: Seilhamer, Jeffrey J., Scott, Randal W.
• Primary Examiner(s): Martinell, James
• Application Number:
  US08/187,530
• Time in Patent Office: 1,762 Days
• Field of Search: 435/6, 364/413.02
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/20 Screening of libraries
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

Abstract

A method and system for quantifying the relative abundance of gene transcripts in a biological sample. One embodiment of the method generates high-throughput sequence-specific analysis of multiple RNAs or their corresponding cDNAs (gene transcript imaging analysis). Another embodiment of the method produces a gene transcript imaging analysis by the use of high-throughput cDNA sequence analysis. In addition, the gene transcript imaging can be used to detect or diagnose a particular biological state, disease, or condition which is correlated to the relative abundance of gene transcripts in a given cell or population of cells. The invention provides a method for comparing the gene transcript image analysis from two or more different biological samples in order to distinguish between the two samples and identify one or more genes which are differentially expressed between the two samples.

Claims

1. A method of analyzing a specimen containing gene transcripts, comprising the steps of:

obtaining a first mixture of mRNA;

making cDNA copies of the mRNA;

isolating a representative population of the cDNA copies and producing therefrom a first cDNA library, wherein a selected set of random primers is used in the generation of the first cDNA library;

identifying a first set of gene transcripts from the first library and determining cDNA sequences corresponding to the gene transcripts;

processing the cDNA sequences corresponding to the first gene transcripts in a programmed computer in which a database of reference transcript sequences indicative of reference cDNA sequences is stored, to generate a first identified sequence value for each of the first gene transcripts, where each said identified sequence value is indicative of a sequence annotation and a degree of match between one of the first gene transcripts and at least one of the reference cDNA sequences; and

processing each said identified sequence value to generate first final data values indicative of a number of times each first identified sequence value is present in the first cDNA library.

2. The method of claim 1, wherein the first mixture of mRNA is obtained from a human cell.

3. The method of claim 1, wherein the mixture of mRNA is obtained from a combination of two or more samples.

4. The method of claim 1, wherein the mRNA obtained is partial mRNA sequences.

5. The method of claim 1, further comprising:

obtaining a second mixture of mRNA from a human cell;

making cDNA copies of the second mixture of mRNA;

isolating a representative population of cDNA copies and producing therefrom a second cDNA library, wherein a selected set of random primers is used in the generation of the second cDNA library;

identifying a second set of gene transcripts from the second library and determining cDNA sequences corresponding to the gene transcripts;

processing the cDNA sequences corresponding to the second gene transcripts in a programmed computer in which a database of reference transcript sequences indicative of reference biological sequences is stored, to generate a second identified sequence value for each of the second gene transcripts, where each said second identified sequence value is indicative of a sequence annotation and a degree of match between one of the second gene transcripts and at least one of the reference gene transcripts; and

processing each said second identified sequence value to generate second final data values indicative of a number of times each second identified sequence value is present in the second cDNA library.

6. The method of claim 5, wherein the second library comprises at least 5,000 cDNAs.

7. The method of claim 5, further comprising:

processing the first final data values and the second final data values to generate ratio values of gene -transcripts, each of said ratio values indicative of differences in numbers of gene transcripts between the first mixture of mRNA and the second mixture of mRNA.

8. The method of claim 7, further comprising:

subtracting the first final data values from the second final data values to identify one or more genes that are differentially expressed.

9. The method of claim 7, wherein the first mixture of mRNA is obtained from a sample extracted from a healthy human patient and the second mixture of mRNA is obtained from a sample extracted from an unhealthy human patient.

10. The method of claim 1, wherein the first library comprises at least 5,000 cDNAs.

11. A method of producing a gene transcript image, comprising the steps of

obtaining a mixture of mRNA from a biological specimen;

making cDNA copies of the mRNA, wherein the cDNA copies of mRNA are made using a selected set of random primers;

inserting the cDNA copies into a suitable vector and transfecting suitable host strain cells with the vector and growing clones, each clone representing a unique mRNA;

isolating a representative population of at least 5,000 recombinant clones;

identifying amplified cDNAs from each clone in the population by a sequence-specific method which identifies a gene from which the unique mRNA was transcribed;

determining a number of times each gene is represented within the population of clones as an indication of relative abundance; and

listing genes and their relative abundance in order of abundance, thereby producing a gene transcript image

comparing the gene transcript image with a gene transcript image derived from a diseased biological specimen and/or a normal biological specimen.