Use of DNA-dependent RNA polymerase transcripts as reporter molecules for signal amplification in nucleic acid hybridization assays
AssigneeChiron Corporation
InventorMichael S. Urdea
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Abstract
A polydeoxynucleotide construct is disclosed for use, in conjunction with a DNA-dependent RNA polymerase, as a signal amplifier in nucleic acid hybridization assays. The construct contains a recognition sequence for a target oligonucleotide, a promoter sequence for a DNA-dependent RNA polymerase, and a polymerase template. A method of use for this construct in hybridization assays is also disclosed. The method involves formation of a hybridization complex comprising the construct and the target sequence; addition of a polymerase which is specific for the promoter in the construct; and quantification of the resulting RNA transcripts.
Claims43
An isolated polydeoxynucleotide construct for use as a signal amplifier in hybridization assays to detect a target, the construct comprising three domains:
- (a) a first domain (A) which is single-stranded and has a nucleotide sequence complementary to a target sequence;
- (b) a second domain (B) which is double-stranded and capable of functioning as a promoter for a DNA-dependent RNA polymerase enzyme activity;
- and
- (c) a third domain (C) which is either single- or double-stranded and adjacent to said second domain, such that said third domain is capable of functioning as a template for the promoter activity of said second domain,
- wherein the domains of the construct are oriented A-B-C or B-C-A, and further wherein the third domain capable of functioning as a template consists of a nucleotide sequence not found in the target.
Dependencies are inferred from the claim text.