Microarray techniques for nucleic acid expression analyses

摘要

Provided are DNA microarray techniques that allow hybridization without RNA amplification, without using cDNA, and without labeling the nucleic acid prior to hybridization. Referred to as the Double-stranded Exonuclease Protection (DEP) assay, the technique permits the sample RNA to be used directly for hybridization, without manipulation in any way. Further provided is a microarray technique for high-throughput miRNA gene expression analyses, termed the RNA-primed, Array-based, Klenow Enzyme (RAKE) assay. The RAKE assay is a sensitive and specific technique for assessing single-stranded DNA and RNA targets, and offers specific advantages over Northern blots.

主权项

1. An assay method for analyzing a nucleic acid sample, the assay method comprising;
obtaining a single-stranded nucleic acid sample comprising at least one target RNA having an unknown expression profile; immobilizing onto a substrate at least one known single-stranded oligonucleotide hybridization probe, to provide at least one immobilized probe template, the probe comprising a nucleotide sequence complementary to the at least one target RNA in the single-stranded nucleic acid sample, wherein the nucleotide sequence is followed 5′ by consecutive thymidine nucleotides; hybridizing the at least one target RNA to the complementary nucleotide sequence of the at least one immobilized probe template under hybridization conditions, to provide at least one hybridized target RNA, thereby producing at least one double-stranded substrate-immobilized hybridized nucleic acid comprising the at least one hybridized target RNA and the at least one immobilized probe template, wherein the at least one hybridized target RNA is a at least one hybridized target RNA primer and the at least one immobilized probe template is a at least one template for template directed synthesis; then washing the substrate containing the substrate-immobilized hybridized nucleic acid; incubating the at least one substrate-immobilized hybridized nucleic acid with polymerase to catalyze the addition of at least one labeled complementary nucleotide onto the 3′ end of the at least one hybridized target RNA primer, producing a primed extension product comprising at least one incorporated nucleotide, wherein the at least one incorporated nucleotide is the at least one labeled complementary nucleotide; then washing to remove non-hybridized and unincorporated nucleotides; incubating the washed, primed extension product with a fluorescent, luminescent or radioactive detection reagent that binds to the at least one labeled complementary nucleotide on the primed extension product; imaging the resulting extension product and signals from the detection reagent bound thereto; and identifying image intensities to determine if a recognition image intensity is present, indicating hybridization between the at least one target RNA and the at least one immobilized probe template.