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Analysis of gene expression profiles using sequential hybridizationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidAnalysis of gene expression profiles using sequential hybridization description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070148690, Analysis of gene expression profiles using sequential hybridization. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional of U.S. Ser. No. 10/222,459 filed Aug. 16, 2002, and now allowed, which claims priority of U.S. Provisional Application Ser. No. 60/312,696, filed Aug. 16, 2001. The contents of these applications are incorporated herein by reference. TECHNICAL FIELD [0003] This invention relates generally to analysis of gene expression profiles. In particular, the present invention provides a method for analyzing gene expression profiles of a cell, which method comprises: a) providing for isolated mRNA or cDNA target sequences from a cell; b) sequentially hybridizing said isolated mRNA or cDNA target sequences with a plurality of nucleotide probes; and c) assessing the sequential hybridization between said isolated mRNA or cDNA target sequences and said plurality of nucleotide probes to analyze gene expression profiles of said cell. Systems for analyzing gene expression profiles are also provided. Optical devices for detecting hybridization signal are further provided. BACKGROUND OF THE INVENTION [0004] The functional states and the specific genotype of a cell are predominantly determined by the set of genes expressed in that cell, not only by the number of genes but also by their relative level of expression. In general, both of these critical information can be obtained by an analysis of the level of messenger RNA (mRNA) expressed in the cell. An accurate characterization of the mRNA levels in various cells not only is necessary for a thorough understanding of the fundamental principles governing the determination of each cell type in the body, but can also be used to determine whether a cell has been transformed into a harmful, disease causing state. [0005] Several different methods have been developed to quantify mRNA abundance in biological samples. But, so far, the most reliable, convenient and economical method for the quantitation of mRNA levels is based on the principle of hybridization, which uses target nucleic acid sequences to incubate with a probe sequence under conditions where the complementary probe and its target can form stable hybrid duplexes through base pairing, which is highly sequence specific. By measuring the hybridization results of probes to different mRNA or their cDNA, the expression level of each mRNA species in original sample can be quantified. Blot assays have been used for some time to hybridize nucleic acid material from biological samples to specifically designed probes for analyzing expression level of specific genes in the original sample. Although the traditional blot hybridization assays are well developed, it is not suitable for screening large number of genes and the sensitivity of this technique is relatively low, which requires a large amount of material. This technique further suffers from its low accuracy in quantitative analysis of expression profiles. [0006] The more recent array technology, such as oligonucleotide array or cDNA array, is suited for the analysis of large number of genes, which is made possible by the use of parallel detection of hybridization signals. Both array methods involve physically immobilizing many different hybridization probes on a solid surface in a small array format. Target mRNA or cDNA in solution are hybridized with the tens of thousands different probes defined as "spots" on the surface. The amount of each target species is quantified by scanning individual "spot" in the whole array after targets have hybridized to the probes. This technology has been applied to both basic research and clinical applications. However, a major limitation of this approach is its need for large amount of target material, which is often impossible to obtain. In particular, it has been estimated that even under most ideal conditions, 10.sup.6-10.sup.7 of cells would be required for an acceptable signal to noise ratio. The usefulness and the power of this approach notwithstanding, we must recognize that the ability of being able to analyze gene expression at large scale in single or just a few cells is an overwhelming need in both basic research in biology and clinical medicine. In fact, there are many important areas of application where the available materials for analysis cannot be more than a few cells. For example, to understand the principles governing the development of the embryo, we must be able to analyze the first differentiated cells that have a particular spatial relationship. Similarly, to understand how cancer is developed, it is preferable to analyze the early stages of the development, which may or may not exhibit identical traits as mature tumors. Even in the case of biopsy, the small amount of materials available makes it difficult to perform an in-depth analysis of the genes expressed that may be used as reliable markers for tumorigenesis. Therefore, the development of such a powerful technology for both fundamental research and medical diagnosis has constituted a major focus of biotechnology. [0007] Although PCR amplification schemes have been employed to improve upon this lower limit, the large pools of target material generated by amplification have not been shown to faithfully represent the relative abundance of different targets in the original material, especially for those low copy number target species. Apparently, this technology is not applicable to single cell analysis with acceptable reliability. Furthermore, the accuracy of this method is also limited due to detection sensitivity. [0008] A very important point to make, however, is the fact that in most practical applications, there is no need or even the desire to monitor the expression levels of the entire genome which involves hundreds of thousands of genes. Since usually there are up to few hundred genes involved in specific cellular pathways and certain disease such as hypertension, in which it is believed that there are about 150 target genes' expression are modified or changed, information related to these specific groups are of most interests. Another example is the prostate cancer where no more than 500 genes are believed to be involved in this disease. [0009] To date, methods of analyzing gene expressions, which can be applied to single or a limited number of cells, employ fluorescence in situ hybridization, or hybridization based on reverse transcription coupled PCR (RT-PCR) using specific primers. In situ hybridization method has been used to directly visualize the expression of a few particular genes in individual cells, but the procedure involved is rather laborious and the sensitivity of this technique limited its application in gene species. Furthermore, the number of genes that can be analyzed in a sample is very limited. When the later RT-PCR based method is employed with specific primers for amplification, the complexity of the analysis limited the applicable number of genes, which is not realistic for monitoring expression profiles and quantifying the changes in expression levels. [0010] In order to achieve the required sensitivity for single cell analysis and to be able to analyze a large number of genes, a different approach than the current array technology must be pursued. In this application, we describe a practical system and method thereof that is based on sequential detection of hybridization signals that not only is sufficiently sensitive for the analysis of mRNA levels from single cells, but can also allow the quantitation of up to several thousand gene products in a single analytical cycle. Therefore, the applicable range of this technology fulfills the gap between the array technology (suitable for hundreds of thousands of genes by parallel analysis) and the more conventional approaches (only adequate for analyzing small number of gene products). This technology will open the possibility to analyze groups of genes involved in specific pathways, such as development of the embryo and the neuro network, or processes involved in the development of certain diseases, such as hypertension, cancer, with minute amount of materials from few cells and even down to a single cell, as well as applications in clinical medicine, such as the detection of cancerous cells at an early few cell stage, and other genetically determined diseases. BRIEF SUMMARY OF THE INVENTION [0011] In one aspect, the present invention is directed to a method for analyzing gene expression profiles of a cell, which method comprises: a) providing for isolated mRNA or cDNA target sequences from a cell; b) sequentially hybridizing said isolated mRNA or cDNA target sequences with a plurality of nucleotide probes; and c) assessing the sequential hybridization between said isolated mRNA or cDNA target sequences and said plurality of nucleotide probes to analyze gene expression profiles of said cell. [0012] In another aspect, the present invention is directed to a system for analyzing gene expression profiles of a cell, which system comprises: a) means for providing isolated mRNA or cDNA target sequences from a cell; b) means for sequentially hybridizing said isolated mRNA or cDNA target sequences with a plurality of nucleotide probes; and c) means for assessing the sequential hybridization between said isolated mRNA or cDNA target sequences and said plurality of nucleotide probes to analyze gene expression profiles of said cell. [0013] In still another aspect, the present invention is directed to an optical device for detecting hybridization signal, which device comprises: a) a microneedle comprising a mRNA or cDNA target sequence immobilized on its tip and in optical connection with a light source; b) a light concentrator; c) a filter system; and d) a photomultiplier tube (PMT) unit, wherein in operation, hybridization of said mRNA or cDNA target sequence to a complementary probe brings a fluorescent label to the close proximity to said tip of said microneedle, provision of light to said tip from said light source generates fluorescent light from said tip, and said fluorescent light is reflected by said light concentrator to become parallel light passing through said filter system and detected by said PMT unit. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0014] FIG. 1 shows a block diagram of this method, illustrating the steps in realizing sequential gene expression analysis. [0015] FIGS. 2A and 2B illustrate 2 stages in handling, extracting, and immobilizing mRNA, cDNA material from single or a small cluster of cells. The procedures referred to in FIG. 2B include: (a) Cell lysate of cellular extracts are transferred into a micro well, and mRNA are captured by oligo-dT sequences covalently linked to the wall, and then subsequently eluted. Alternately, cDNAs are synthesized with oligo-dT primer. Using covalently linked first strand cDNAs as templates, second strand cDNAs are synthesized for the use as target sequences; (b) mRNA eluted from step (a) can be reverse transcribed into first strand cDNA with oligo-dT primer; (c) Using micro well coated with selected complimentary sequences to hybridize with mRNA or cDNA, unwanted species are removed from the total population; and (d) Hybridize mRNA, second strand cDNA, or first strand cDNA to oligo-dT or oligo-dA sequences linked on the micro probe (needle), cDNA is synthesized with oligo-dT as the primer or crosslinked to oligo-dA to achieve immobilization. [0016] FIGS. 3A and 3B show a block diagram, illustrating the procedures in target material extraction, preparation and immobilization. [0017] FIGS. 4A and 4B show an illustration for the design of the microneedle used for immobilizing the target sequences, and the chemistry of covalently linking oligonucleotide sequences to the surface of the needle. [0018] FIG. 5 shows an example of synthesizing a uniform fluorescent label with fixed number of fluorophores and tagging the hybridization probe with this label. [0019] FIG. 6 shows a plot of hybridization kinetics of bead immobilized oligo-dT sequence and poly-dA tailed single strand DNA sequence. In this case, the probe is in vast excess of the target. Therefore, the process becomes first order. FIGS. 7A and 7B illustrate a representative hybridization station for the analytical cycle. The probes are contained in the "matrix" and the entire gene group is pre-fabricated precisely to allow an automated analysis with robotic control. [0020] FIGS. 8A, 8B, and 8C further illustrates the optical detection system. It is noted that for the single wavelength analysis (A) and (B), the detection efficiency is up to 90%, and an imaging system is not required. Continue reading about Analysis of gene expression profiles using sequential hybridization... 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