Targeted genetic risk-stratification using microarrays
OwnerGerman Pihaninventor
Life of this patent
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Abstract
The invention relates to new expedient and cost-effective assays that are capable of identifying many or all relevant diagnostic and prognostic genetic lesions in cancer or cancer predisposition using multiplex PCR or other nucleic acid amplification or enrichment technology in conjunction with bead microarrays for the purpose of risk-stratifying patients with cancer or cancer predisposition. The new assay methods are referred to herein as BARCODE-MT for Bead ARray COded DEtection of Multiple Targets. These assays are high-throughput, and can be automated for highly accurate diagnoses that can be used to optimize risk-adapted therapy.
Claims34
A method of simultaneously detecting the presence of multiple target nucleic acid molecules in a biological sample, the method comprising
- (a) isolating and enriching target nucleic acid molecules from the biological sample;
- (b) treating the enriched target nucleic acid molecules with Exonuclease I;
- (c) performing linear PCR on the Exonuclease I treated enriched target nucleic acid molecule to produce linear PCR product;
- wherein only a single primer is used;
- (d) obtaining beads coupled to an oligonucleotide molecule complementary to the amplified target nucleic acid molecules;
- (e) forming a mixture by mixing the beads and the enriched linear PCR product nucleic acid;
- (f) forming a reacted sample by incubating the mixture under conditions wherein if the enriched linear PCR product includes the target nucleic acid molecule, the enriched linear PCR product will hybridize to the oligonucleotide molecule;
- (g) analyzing the reacted sample by determining the fluorescence of each bead analyzed;
- and
- (h) detecting a level of fluorescence on the beads, wherein the level of fluorescence corresponds to a level of target nucleic acid molecule in the biological sample.
A method of simultaneously detecting the presence of multiple target nucleic acid molecules in a biological sample, the method comprising
- (a) enriching isolated nucleic acid from the biological sample, wherein enrichment incorporates a detectable label onto a PCR product, wherein the PCR product may comprise a target nucleic acid;
- (b) treating the amplified nucleic acid with Exonuclease I;
- (c) performing linear PCR on the Exonuclease I treated amplified nucleic acid to produce linear PCR product;
- wherein only a single primer is used;
- (d) obtaining addressable beads coupled to at least one oligonucleotide molecule complementary to the target nucleic acid;
- (e) mixing the addressable beads and the linear PCR product to form a mixture;
- (f) incubating the mixture under conditions allowing the linear PCR product to hybridize to oligonucleotide molecules that contain the target nucleic acid;
- (g) analyzing the incubated mixture by determine the address of each bead analyzed by its fluorescence;
- and
- (h) detecting a level of the detectable label on each of the addressable beads, wherein the level of the detectable label corresponds to a level of the target nucleic acid in the biological sample.
Dependencies are inferred from the claim text.