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Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms

a technology of expression and eukaryotic organisms, applied in the field of gene expression analysis, can solve problems such as requiring significant resources to identify expressed genes

Inactive Publication Date: 2006-05-18
KANE MICHAEL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is a system and method for identifying and analyzing genes in RNA samples. It can identify any or all genes expressed in a sample and compare the expression levels of genes between different samples. The invention uses a special sequence of DNA called an identimer to detect the presence of specific genes. The identimer has a fluorescent molecule attached to it, which allows for easy detection of the resulting fragments. The invention can also identify novel genes that are not previously known. Overall, the invention provides a useful tool for research and clinical scientists to study gene expression and regulation.

Problems solved by technology

However, current genomic tools and techniques continue to require significant known genomic sequence information for the organism or tissue under investigation, or require that the investigators derive libraries of clones from the particular organism or tissue.
However, these methods also require significant resources to identify expressed genes and related expression products, such as mRNAs.

Method used

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  • Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms
  • Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms
  • Compositions and systems for identifying and comparing expressed genes (mRNAs) in eukaryotic organisms

Examples

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example 1

[0032] First and second strand cDNA synthesis. First strand synthesis is performed by means known to those of ordinary skill (using any experimental cell / tissue type) on the total RNA population utilizing a four-base identimer of sequence NNNVT21, where each N=A, C, T, or G, and V=A, C, or G but not T (SEQ ID NO: 2). In practical application, the total number of unique identimer tags theoretically required to span the total estimated mRNA population (in a eukaryotic organism) would be 192 (thus 192 unique subsets). Compared with most differential display protocols, which typically utilize a two-base anchored primer for first strand synthesis, a four-based identimer has advantages by: (1) significantly reducing the complexity of the mRNA pool by a factor of 16 (192 / 12=˜16), thereby reducing the number of bands displayed per fingerprint (or subset); (2) providing a more accurate prediction of the candidate mRNA(s) of interest through the additional two nucleotide sequence at the 3′-en...

example 2

[0042] In one embodiment, the invention is a system whereby the identity and relative expression level of all eukaryotic mRNAs (messenger RNAs) are determined. Some components of the invention include, without limitation (1) primer design & reverse transcription, (2) production of double-stranded cDNA, (3) sequence-specific cleavage of ds cDNA, and (4) fragment detection & analysis.

[0043] Primer Design. The invention takes advantage of the polyadenylation of eukaryotic mRNAs by utilizing an anchored oligo-T primer. The basic primer design includes an oligo-T nucleotide sequence (5′ end) linked to a 5-nucleotide sequence (3′ end) where the bases immediately adjacent to the oligo-T stretch is not a T. This sequence can be written (5′ to 3′) as Tn-VNNNN (n=any single integer of 8 or greater representing how many T bases are present). The 5′ end of the primer contains one or more reporter molecules or markers (e.g. fluorescent molecule, hapten, biotin, radioisotope, etc.) that allows f...

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Abstract

The invention comprises compositions and systems to identify and compare expressed genes in a given in vivo or in vitro RNA sample, as well as the relative difference in mRNA expression between two or more samples, where desired. Furthermore, the invention comprises compositions and systems to identify novel genes. The invention comprises, without limitation, one or more mRNA specific identimers for use in reverse transcription that themselves comprise an oligo-T nucleotide sequence (at the 5′ end) linked to a nucleotide sequence VNx (at the 3′ end) where the V nucleotide immediately adjacent to the oligo-T segment is not a T.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of U.S. patent application Ser. No. 10 / 002,536, filed on Nov. 1, 2001, which claims priority based on U.S. provisional patent application No. 60 / 244,933 filed Nov. 1, 2000.FIELD OF THE INVENTION [0002] The present invention relates to the fields of genomic and proteomic analysis. In particular, the present invention relates to the field of gene expression analysis. BACKGROUND OF THE INVENTION [0003] With progress in sequencing many genomes, including among them the human genome, there is additional interest in understanding the significance of changes in gene expression. The ability to correlate changes in gene expression, for example, with specific treatments and phenotypes in clinical and non-clinical biological systems, allows scientists to understand the underlying cell biology and identify the roles of specific genes, receptors and signaling pathways. One objective, among many, of this research is ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34C12N15/10C12Q1/6809
CPCC12N15/1096C12Q1/6809C12Q2531/143C12Q2525/173C12Q2525/143
Inventor KANE, MICHAELNAGEL, AARONDOMBKOWSKI, ALAN
Owner KANE MICHAEL