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.