US 2003/0207269 A9 · Preferential display

Abstract

There is a tremendous need for high throughput gene expression technology which can efficiently and cheaply identify and accurately isolate different genes expressed between diseased and normal tissues for use in discovering new drugs. The present invention utilizes a combination of biomolecular chemistry methods to eliminate/degrade redundant sequences and fluorescence dye assay to identify these unique sequences from two cell or tissue populations. cDNA from normal or diseased cells or tissues are hybridized with the RNA of the complement normal or diseased cells or tissues. The hybridized cDNA/RNA is incubated with exonucleases, resulting in degradation of all but the single stranded RNA and DNA. RNA are then elimated using RNase and the remaining DNA which are unique to the sample are amplified. This technique may be used to isolate differentially expressed genes or gene fragments and will provide a means to isolate and identify medium to low gene expressions that may otherwise be buried under gene “noise”.

Claims25

Dependent claims

A method to eliminate redundant sequences which are common between two or more samples using bimolecular degrading enzymatic reagents and radiolabeled assay to identify the remaining sequences which are uniquely expressed or over and under expressed in these samples, comprising of the steps:

a). isolating RNAs or DNAs from samples which are of interest,
b). generating cDNA from RNAs using RT-PCR methods and technologies,
c). cross mixing complementary cDNAs with RNAs of samples of interest,
d). degrading the hybridized complements using degrading reagents,
e). amplifying resultant cDNAs or RNAs by PCR amplification,
f). displaying and reading resultant cDNAs or RNAs, and
h). sequencing unique cDNAs or RNAs.

4 more elements

Dependencies are inferred from the claim text.

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Abstract

Claims25