Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-10-27
2002-10-22
Marschel, Ardin H. (Department: 1631)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C435S006120, C435S069100, C702S019000, C702S020000
Reexamination Certificate
active
06468476
ABSTRACT:
TABLE OF CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION
5.1. Introduction
5.1.1. Definition of Biological State
5.1.2. Repredentation of Biological Responses
5.1.3. Overview of the Invention
5.2. Specific Embodiment: Defining Basis Genesets
5.2.1. Co-regulated Genes and Genesets
5.2.2. Geneset Classification by Cluster Analysis
5.2.3. Geneset Classification Based upon Mechanisms of Regulation
5.2.4. Refinement of Geneset and Geneset Definition Database
5.3. Representation of Gene Expression Profiles Based upon Basis Genesets
5.4. Application of Projected Profiles
5.4.1. Advantage of the Projected Profile
5.4.2. Profile Comparison and Classification
5.4.3. Illustrative Drug Discovery Applications
5.4.4. Illustrative Diagnostic Applications
5.4.5. Response Profile Classification by Cluster Analysis
5 5.4.6. Removal of Profile Artifacts
5.4.7. Projected Titration Curves
5.4.8. Use of Genesets in Microarrays
5.5. Computer Implementation
5.6. Analytic Kit Implementation
5.7. Methods for Determining Biological Response
5.7.1. Transcript Assay Using DNA Array
5.7.1.1. Preparing Nucleic Acids for Microarrays
5.7.1.2. Attaching Nucleic Acids to the Solid Surface
5.7.1.3. Target Polynucleotide Molecules
5.7.1.4. Hybridization to Microarrays
5.7.1.5. Signal Detection and Data Analysis
5.7.2. Pathway Response and Genesets
5.7.3. Measurement of Graded Perturbation Response Data
5.7.4. Other Methods of Transcriptional State Measurement
5.7.5. Measurement of other Aspects of Biological State
5.7.5.1. Embodiments Based on Translational State Measurements
5.7.5.2. Embodiments Based on other Aspects of the Biological State
5.8. Method for Probing Cellular States
5.8.1. Titratable Expression Systems
5.8.2. Transfection Systems for Mamalian Cells
5.8.3. Methods of Modifying RNA Abundances or Activities
5.8.4. Methods of Modifying Protein Abundances
5.8.5. Methods of Modifying Protein Activities
6. EXAMPLES
6.1. Example 1
Clustering Genesets by Coregulation
6.1.1. Materials and Methods
6.1.2. Results and Discussion
6.2. Example 2
Enhancing Detection of Response Pattern Using Geneset Average Response
6.3. Example 3
Improved Classification of Drug Activity
6.4 Experiment 4
Improved Classification of Biological Response Profiles
6.5. Example 5
Projecting out Profile Artifacts
7. REFERENCES CITED
1. FIELD OF THE INVENTION
The field of this invention relates to methods for enhanced detection of biological responses to perturbations. In particular, it relates to methods for analyzing structure in biological expression patterns for the purposes of improving the ability to detect certain specific gene regulations and to classify more accurately the actions of compounds that produce complex patterns of gene regulation in the cell.
2. BACKGROUND OF THE INVENTION
Within the past decade, several technologies have made it possible to monitor the expression level of a large number of transcripts at any one time (see, e.g. Schena et al., 1995, Quantitative monitoring of gene expression patterns with a complementary DNA micro-array,
Science
270:467-470; Lockhart et al., 1996, Expression monitoring by hybridization to high-density oligonucleotide arrays,
Nature Biotechnology
14:1675-1680; Blanchard et al., 1996, Sequence to array: Probing the genome's secrets,
Nature Biotechnology
14, 1649; U.S. Pat. No. 5,569,588, issued Oct. 29, 1996 to Ashby et al. entitled “Methods for Drug Screening”). In organisms for which the complete genome is known, it is possible to analyze the transcripts of all genes within the cell. With other organisms, such as human, for which there is an increasing knowledge of the genome, it is possible to simultaneously monitor large numbers of the genes within the cell.
Such monitoring technologies have been applied to the identification of genes which are up regulated or down regulated in various diseased or physiological states, the analyses of members of signaling cellular states, and the identification of targets for various drugs. See, e.g., Friend and Hartwell, U.S. Provisional Patent Application Serial No. 60/039,134, filed on Feb. 28, 1997; Stoughton, U.S. patent application Ser. No. 09/099,722, filed on Jun. 19, 1998, now U.S. Pat. No. 6,132,969; Stoughton and Friend, U.S. patent application Ser. No. 09/074,983, filed on May 8, 1998, now U.S. Pat. No. 5,965,352; Friend and Hartwell, U.S. Provisional Application Serial No. 60/056,109, filed on Aug. 20, 1997; Friend and Hartwell, U.S. application Ser. No. 09/031,216, filed on Feb. 26, 1998, now U.S. Pat. No. 6,165,709; Friend and Stoughton, U.S. Provisional Application Serial Nos. 60/084,742 (filed on May 8, 1998), No. 60/090,004 (filed on Jun. 19, 1998) and No. 60/090,046 (filed on Jun. 19, 1998), all incorporated herein by reference for all purposes.
Levels of various constituents of a cell are known to change in response to drug treatments and other perturbations of the cell's biological state. Measurements of a plurality of such “cellular constituents” therefore contain a wealth of information about the effect of perturbations and their effect on the cell's biological state. Such measurements typically comprise measurements of gene expression levels of the type discussed above, but may also include levels of other cellular components such as, but by no means limited to, levels of protein abundances, or protein activity levels. The collection of such measurements is generally referred to as the “profile” of the cell's biological state.
The number of cellular constituents is typically on the order of a hundred thousand for mammalian cells. The profile of a particular cell is therefore typically of high complexity. Any one perturbing agent may cause a small or a large number of cellular constituents to change their abundances or activity levels. Not knowing what to expect in response to any given perturbation will therefore require measuring independently the responses of these about 10
5
constituents if the action of the perturbation is to be completely or at least mostly characterized. The complexity of the biological response data coupled with measurement errors makes such an analysis of biological response data a challenging task.
Current techniques for quantifying profile changes suffer from high rates of measurement error such as false detection, failures to detect, or inaccurate quantitative determinations. Therefore, there is a great demand in the art for methods to enhance the detection of structure in biological expression patterns. In particular, there is a need to find groups and structure in sets of measurements of cellular constituents, e.g., in the profile of a cell's biological state. Examples of such structure include associations between the regulation of the expression levels of different genes, associations between different drug or drug candidates, and association between the drugs and the regulation of sets of genes.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
This invention provides methods for enhancing detection of structures in the response of biological systems to various perturbations, such as the response to a drug, a drug candidate or an experimental condition designed to probe biological pathways as well as changes in biological systems that correspond to a particular disease or disease state, or to a treatment of a particular disease or disease state. The methods of this invention have extensive applications in the areas of drug discovery, drug therapy monitoring, genetic analysis, and clinical diagnosis. This invention also provides apparatus and computer instructions for performing the enhanced detection of biological response patterns, drug discovery, monitoring of drug therapies, genetic analysis, and clinical diagnosis.
One aspect of the invention provides methods for classifying cellular constituents (measu
Friend Stephen H.
He Yudong
Stoughton Roland
Marschel Ardin H.
Pennie & Edmonds LLP
Rosetta Inpharmatics, Inc.
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