Terminal-phosphate-labeled nucleotides and methods of use
First Claim
1. A method of detecting a target using a labeled substrate or substrate analog comprising reacting said substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled polyphosphate that has independently detectable signal only when said substrate or substrate analog reacts, and wherein, when the target is a nucleic acid, the labeled polyphosphate has three or more phosphates.
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Abstract
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.
91 Citations
33 Claims
- 1. A method of detecting a target using a labeled substrate or substrate analog comprising reacting said substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled polyphosphate that has independently detectable signal only when said substrate or substrate analog reacts, and wherein, when the target is a nucleic acid, the labeled polyphosphate has three or more phosphates.
- 26. A method of detecting a target nucleic acid sequence using a labeled nucleotide analog comprising incorporating said analog by a nucleic acid polymerase into a polynucleotide complimentary to the target sequence thereby, producing a species which is neither pyrophosphate nor a mono-substituted pyrophosphate that undergoes a chemical change to produce a detectable signal.
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32. A labeled phosphate containing compound comprising the formula
Label-(P)n— - P
wherein the label is a detectable moiety selected from the group consisting of fluoresent, colored, fluorogenic, chromogenic, luminogenic moiety or an electrochemical label and n is 2-5.
- P
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33. A labeled phosphate containing compound comprising the formula
Label-(P)n— - P
wherein the label is a detectable moiety selected from the group consisting of fluorogenic, luminogenic moiety or an electrochemical label and n is 1-5.
- P
Specification