Terminal-phosphate-labeled nucleotides and methods of use
First Claim
1. A method of detecting the presence of a nucleic acid sequence comprising the steps of:
- a) conducting a nucleic acid polymerase reaction, said reaction comprising the reaction of at least one nucleotide which is substantially non-reactive to phosphatase and at least one terminal-phosphate-labeled nucleotide, which reaction results in the production of labeled polyphosphate;
b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and
c) detecting the presence of said detectable species.
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Abstract
The present invention 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.
111 Citations
74 Claims
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1. A method of detecting the presence of a nucleic acid sequence comprising the steps of:
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a) conducting a nucleic acid polymerase reaction, said reaction comprising the reaction of at least one nucleotide which is substantially non-reactive to phosphatase and at least one terminal-phosphate-labeled nucleotide, which reaction results in the production of labeled polyphosphate; b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and c) detecting the presence of said detectable species. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of detecting the presence of a DNA sequence comprising the steps of:
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a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide, which reaction results in the production of a labeled polyphosphate; b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and c) detecting the presence of said detectable species. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A method of quantifying a nucleic acid comprising the steps of:
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(a) conducting a nucleic acid polymerase reaction, said reaction comprising the reaction of nucleotide which is substantially non-reactive to phosphatase and at least one terminal-phosphate-labeled nucleotide;
said reaction resulting in production of labeled polyphosphate;(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable by-product species in amount substantially proportional to the amount of said nucleic acid; (c) measuring said detectable species; and (d) comparing said measurements using known standards to determine the quantity of nucleic acid. - View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. A method of quantifying a DNA sequence comprising the steps of:
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(a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide, said reaction resulting in production of labeled polyphosphate; (b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable by-product species in amounts substantially proportioned to the amount of said DNA sequence; (c) measuring said detectable species; and (d) comparing said measurements using known standards to determine the quantity of DNA. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70)
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71. A method of detecting the presence of a nucleic acid sequence comprising the steps of:
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(a) conducting a nucleic acid polymerase reaction in the presence of at least one terminal-phosphate-labeled nucleotide having four or more phosphate groups in the polyphosphate chain, which reaction results in the production of a labeled polyphosphate; (b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species; and (c) detecting the presence of said detectable species. - View Dependent Claims (72, 73, 74)
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Specification