Universal probe assay methods

Abstract

Reagents and methods are provided for detecting the presence of a target polynucleotide in a sample are disclosed. In one aspect, a method for producing a labeled amplification product by amplifying a target nucleic acid sequence to produce an amplification product comprising the target sequence, a first probe-binding sequence 5′ to the target sequence, and a second probe-binding sequence 3′ to the target sequence, thereby producing an amplification product; and hybridizing a first detection probe to the amplification product, the first detection probe comprising a first segment that hybridizes to the first probe-binding sequence and a second segment that hybridizes to the second probe-binding sequence, thereby producing a labeled amplification product is disclosed.

1 . A method for specific detection of a microRNA, comprising: (a) combining (i) a sample comprising the microRNA, (ii) an artificial DNA template comprising a first sequence that is complementary to the microRNA and a second sequence that is 3′ to the first sequence, (iii) reagents for DNA-dependent extension of an RNA primer including a polymerase; thereby producing an extension combination; (b) exposing the extension combination to conditions under which the microRNA anneals to the DNA template and the microRNA is extended by the polymerase thereby producing a chimeric polynucleotide comprising a 5′ microRNA portion and a 3′ DNA portion complementary to the second sequence; and (c) detecting the production of the chimeric polynucleotide.