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Radioactive multiplexing analytical methods for biomarkers discovery

a biomarker and radioactive multiplexing technology, applied in the field of radioactive multiplexing analytical methods for biomarker discovery, can solve the problems of label interference with the analyte, protein cannot be used for protein array analysis, and dye may alter the structure of dna or rna significantly, so as to achieve the effect of higher sensitivity

Active Publication Date: 2006-04-20
PROTEOMYX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for analyzing biomolecules using radioactive isotopes labeling. This method allows for the simultaneous analysis of multiple samples, saving time and cost. The labels are unique for each sample, allowing for the separation and quantification of molecules. The method also allows for the study of post-translational modifications and DNA methylation. The use of radioactive isotopes labeling provides higher sensitivity and is compatible with various analytical methods. Overall, this invention provides an improved method for biomarker discovery and genomic and proteomic analysis."

Problems solved by technology

While DNA and RNA often can accommodate structural modifications caused by covalent linkage with dyes for DNA array analysis, proteins cannot for protein array analysis.
As a result, the label interferes with the analyte and often prevents an antibody from binding normally to labeled proteins the same way it would to unlabeled proteins.
Furthermore, dye may alter the structure of DNA or RNA significantly, such that proteins, e.g., transcription factors, won't be able to recognize and bind as they would do with native sequences.
Direct detection with devices such as Geiger counter is also possible; however, the cost for making such instruments has limited its use.

Method used

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  • Radioactive multiplexing analytical methods for biomarkers discovery
  • Radioactive multiplexing analytical methods for biomarkers discovery
  • Radioactive multiplexing analytical methods for biomarkers discovery

Examples

Experimental program
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example 1

Biomarker Discovery and High-Throughput Assay Development

[0058] Because of the limited antibodies availability, biomarkers must first be discovered and then antibodies are made against them to build useful antibody arrays for high throughput screening. Current methods use 2-D gel electrophoresis to separate proteins. This example presents an alternative to 2-D gel electrophoresis.

[0059] Samples from two sources such as drug-treated and vehicle treated cells are used to look for biomarkers. These samples are prepared so that one is labeled with 3H while the other is labeled with 14C by chemically similar labels or by metabolic incorporation and then mixed together for analysis. A small aliquot is counted on scintillation counter capable of distinguishing between 3H and 14C to establish the ratio of samples mixture. The mixture is then separated by tandem chromatography. The fractions resulting from one type of chromatography are further separated by another form of chromatography....

example 2

Post-Translational Modification Studies

Protein Phosphorylation Study

[0061] Post-translational modifications of proteins are very important regulatory mechanism in eukaryotic organisms. One such important modification is phosphorylation: a phosphate added to an enzyme or receptor can turn it on or off depending on the type of enzyme or receptor. Thus a system that enables the comparison of protein phosphorylation with high degree of sensitivity can be a very powerful tool for research and diagnostic use. A dual labeling system using chemically similar labeling agents that comprises either 32P or 33P is used to determine the degree of phosphorylation in two set of cells or cells undergoing two different treatments.

[0062] Cellular drug response can be studied as followed: (1) Cells are grown equally in two culture dishes. Prior to labeling, the cells are starved of phosphate by washing and replacing growth media with phosphate-deficient media for 1 hour. (2) Labeling media are add...

example 3

Genomic / RNA Expression Studies

[0068] For tissue samples where RNA is readily extracted, the RNA can be labeled with either 32P or 33P at their end terminals and then applied to a complementary DNA array. The array is then exposed to the two-screen phosphorescent imaging system so signals specific to either 32P or 33P could be quantified and compared after normalized with internal controls' signals. Alternatively, total signals can be quantified before and after a few days and used to calculate signal unique to 32P or 33P. The half-life of 32P is shorter than that of 33P (14.3 days compared to 25.3 days) thus makes the calculation possible. For higher sensitivity in smaller tissue sample, a Reverse Transcriptase reaction can be performed with either 32P or 33P labeled poly dT or labeled nucleotides. The poly dT is hybridized to the poly A tail of mRNA and reverse transcriptase synthesizes the rest of the complementary sequence to form a single stranded cDNA. The RNA is then digeste...

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Abstract

A novel analytical method involves labeling samples with different radioactive labeling agents, mixing and subjecting the mixture to any separation technique, and then differentially detecting and quantifying subcomponents from each sample for comparison. The novel technique exploits the differences in radiation energy or half-life between isotopes to make differential detection and quantification of labels possible. Detailed methods for differential detection and quantification are also described as well as the construction and application of hardware and software to enable and enhance such a process. This method is useful in finding molecular differences between two samples in differential proteomics, phosphor-proteomics, glycomics, metabolomics, transcriptomics, genomics and diagnostic applications.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority of provisional application U.S. Ser. No. 60 / 443,017, titled multiplexing analytical techniques, filed Jan. 28, 2003, the content of which is incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] Many analytical methods require side-by-side or sequential comparison to quantitatively compare differences between two samples. For example, to compare proteins on a Western blot, samples are run on adjacent lanes; to compare small molecules on HPLC, samples are run sequentially one next to the others. This format of analysis requires twice the effort in sample preparation; in addition, variability can be introduced into the system if the treatments of one sample slightly deviated from another. For these reasons, many repetitions plus statistical analysis are required before any differences found become credible. A method that allows two samples to be mixed together for analysis and quantitative co...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/53
CPCG01N33/60G01N33/6848G01N2030/77G01N2458/15
Inventor TRAN, NATHANIEL TUE
Owner PROTEOMYX
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