Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-09
2004-04-06
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C422S068100, C536S023100
Reexamination Certificate
active
06716579
ABSTRACT:
TECHNICAL FIELD
This invention is in the field of genetic analysis. Specifically, the invention relates to the generation of an array of polynucleotide probes comprising sequences complementary to the 3′ untranslated region of a gene transcript, whose chromosomal location has been defined. The compositions and methods embodied in the present invention are particularly useful for high throughput screening of differential gene expression patterns among multiple subjects.
BACKGROUND OF THE INVENTION
The structure and biological behavior of a cell is determined by the pattern of gene expression within that cell. Each human cell contains approximately three billion base pairs encoding between 50,000 to 100,000 genes (Schuler et al. (1996)
Science
274:540-546; Guyer et al. (1995)
Proc. Natl. Acad. Scie
. USA 92:10841-10848; Rowen et al. (1997)
Science
278:605-607). In any given cell only a fraction of these genes is being actively transcribed. Deciphering the fundamental structure and biological behavior of any given cell requires knowledge of which genes are transcribed and the relative abundance of those transcribed genes.
Perturbations of gene expression have long been acknowledged to account for a vast number of diseases including, numerous forms of cancer, vascular diseases, neuronal and endocrine diseases. Abnormal expression patterns, in form of amplification, deletion, gene rearrangements, and loss or gain of function mutations, are now known to lead to aberrant behavior of a disease cell. In the case of cancer, a deviated expression profile from that of a normal progenitor cell may result in dysfunction of cellular processes, which ultimately lead to dysregulated growth, lack of anchorage inhibition, genomic instability and propensity for cell metastasis.
Monitoring the expression profile of a panel of genes to determine the role of genes in regulating any cellular process has until now been a daunting task. Traditional approaches for identifying transcripts unique to a particular cell type are generally highly focused, targeting only one specific gene or chromosome region at a time. Conventional techniques such as cDNA subtraction, differential display (Liang et al. (1992)
Science
257:967-971), expressed sequence tag (EST) isolation, provide valuable tools for comparative gene expression analysis, but they have pronounced limitations. Whereas these approaches to certain extent yield quantitative information about the abundance of the gene transcripts of particular interest, they do not provide insight systematically into global gene expression patterns. Recently, a new technique, array-based analysis has emerged in the study of genome-wide expression.
The array-based technology involves hybridization of a pool of target polynucleotides corresponding to gene transcripts of a test subject to an array of tens and thousands of probe sequences immobilized on the array substrate. The technique allows simultaneous detection of multiple gene transcripts and yields quantitative information on the relative abundance of each gene transcript expressed in a test subject. By comparing the hybridization patterns generated by hybridizing different pools of target polynucleotides to the arrays, one can readily obtain the relative transcript abundance in two pools of target samples. The analysis can be extended to detecting differential expression of genes between diseased and normal tissues, among different types of tissues and cells, amongst cells at different cell-cycle points or at different developmental stages, and amongst cells that are subjected to various environmental stimuli or lead drugs.
Currently employed arrays including oligonucleotide arrays and cDNA arrays bear a number of intrinsic limitations. WO 97/10365 describes an oligonucleotide array made of synthetically generated oligonucleotides of 20-500 nucleotides in length; each of the following references WO 98/53103, Duggan et al. (1999)
Nature Genetics Supplement
21: 10-14, Wang et al. (1999)
Gene
229: 101-108, Khan et al. (1999)
Electrophoresis
20: 223-229, and Chen et al. (1998)
Genomics
51: 313-324, describes a DNA microarray for monitoring changes in gene expression profile of one or multiple test subjects. Neither of these references discloses arrays necessarily contains probes having minimum secondary structure and lacking internal sequence homology. These are necessary criteria for achieving optimal hybridization efficiency and signal
oise ratio. The wide range of oligonucleotide length (20-500 bases as disclosed in WO 97/10365, 120-1000 bases as specified in WO 98/53103), and hence the thermal stability profile of the probes, inevitably introduces intrinsic variability to the hybridization efficiency of the arrays. There thus remains a considerable need for arrays of probes that are more uniform, highly specific, and more applicable for genome-wide study of expression patterns.
SUMMARY OF THE INVENTION
A principal aspect of the present invention is the design of arrays of polynucleotide probes having reduced secondary structures. Such arrays are highly specific for simultaneous detection of differential expression of multiple genes. Accordingly, the present invention provides an array comprising a plurality of polynucleotide probes immobilized on a solid support, which exhibits the following characteristics: (a) the plurality of polynucleotide probes corresponds to a multiplicity of gene transcripts; (b) each polynucleotide probe of the plurality is localized to a predetermined region on a solid support; (c) each polynucleotide probe is from about 50 to about 500 nucleotides in length; and (d) each polynucleotide probe is complementary to 3′ untranslated sequence of a gene transcript, said untranslated sequence having a defined chromosomal location.
In one aspect of this embodiment, the arrays of the present invention further comprise control probes which can be normalization control probes, expression level control probes, and/or mismatch control probes. In another aspect, the arrays comprise 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 complexes.
In a separate aspect, the plurality of polynucleotide probes immobilized on the arrays may comprise at least about 10 polynucleotides, each being complementary to a distinct gene transcript. Preferably, the plurality of polynucleotide probes comprises at least about 100 polynucleotides. In a preferred embodiment, an array comprises a plurality of sequence-tagged site (STS) tags.
In yet another separate aspect, the predetermined region of the invention array comprises at least 10 single-stranded polynucleotides that are complementary to the same gene transcript. The predetermined region may also comprise at least 100 single-stranded polynucleotides that are complementary to the same gene transcript. In a preferred embodiment, the predetermined region comprises single-stranded polynucleotides of identical sequences.
The solid support on which the probes are arrayed can be flexible or rigid. Preferably, the solid support is made of one or more substances selected from the group consisting of nitrocellulose, nylon, polypropylene, glass, and silicon.
The present invention also provides a method of preparing and using an array having the above-mentioned characteristics.
In one embodiment, the present invention provides a method of simultaneously detecting expression of a multiplicity of gene transcripts of a subject. The method comprises the steps of: (a) contacting more than one labeled target polynucleotides corresponding to gene transcripts of said subject with an array of polynucleotide probes as disclosed herein under the conditions sufficient to produce stable target-probe complexes; and (b) detecting the formation of the stable target-probe complexes, thereby detecting expression of a multiplicity of gene transcripts.
In another embodiment, the invention provides a method of detecting differential expr
Baidya Narayan
Chen Yii-Der Ida
Holding Julie
Yu Yie-Teh
Clingenix, Inc.
Marschel Ardin H.
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