The present invention relates to improved methods of detecting a target using a labeled substrate or
substrate analog. The methods comprise reacting the substrate or
substrate analog in an
enzyme-catalyzed reaction which produces a labeled
moiety with independently detectable
signal only when such substrate or
substrate analog reacts. The present invention, in particular, describes methods of detecting a
nucleic acid in a sample, based on the use of terminal-
phosphate-labeled nucleotides as substrates for
nucleic acid polymerases. The methods provided by this invention utilize a
nucleoside polyphosphate, dideoxynucleoside
polyphosphate, or deoxynucleoside
polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent
moiety, a
mass tag or an electrochemical tag attached to the terminal-
phosphate. When a
nucleic acid polymerase uses this analogue as a substrate, an
enzyme-activatable
label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via
phosphatase leads to a detectable change in the
label attached thereon. When the
polymerase assay is performed in the presence of a
phosphatase, there is provided a convenient method for real-time monitoring of
DNA or
RNA synthesis and detection of a target nucleic acid.