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.
3 Assignments
0 Petitions
Accused Products
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.
96 Citations
87 Claims
-
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. - 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)
-
-
26. A method of detecting the presence of a DNA sequence comprising the steps of:
-
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)
-
-
49. A method of quantifying a nucleic acid comprising the steps of:
-
(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)
-
-
60. A method of quantifying a DNA sequence comprising the steps of:
-
(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)
-
-
71. A method for determining the identity of a single nucleotide in a nucleic acid sequence comprising the steps of:
-
(a) conducting a nucleic acid polymerase reaction in the presence of 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;
(c) detecting the presence of said detectable species; and
(d) identifying the nucleoside incorporated. - View Dependent Claims (72, 73, 74)
-
-
75. A method of detecting the presence of a nucleic acid sequence comprising the steps of:
-
(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; and
(b) detecting said labeled polyphosphate. - View Dependent Claims (76)
-
-
77. A method of detecting the presence of a nucleic acid sequence comprising the steps of:
-
(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 (78, 79, 80)
-
-
81. A method for determining the identify of a single nucleotide in a nucleic acid sequence comprising the steps of:
-
(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 labeled polyphosphate;
(b) permitting said labeled polyphosphate to react with a phosphatase to produce a detectable species;
(c) detecting the presence of said detectable species; and
(d) identifying the nucleoside incorporated. - View Dependent Claims (82)
-
-
83. A nucleic acid detection kit comprising:
-
(a) at least one or more terminal-phosphate-labeled nucleotide according to the formula;
wherein P=phosphate (PO3) and derivatives thereof, n is 2 or greater;
Y is an oxygen or sulfur atom;
B is a nitrogen-containing heterocyclic base;
S is an acyclic moiety, carbocyclic moiety or sugar moiety;
L is an enzyme-activatable label containing a hydroxyl group, a sulfhydryl group or an amino group suitable for forming a phosphate ester, a thioester or a phosphoramidate linkage at the terminal phosphate of a natural or modified nucleotide;
P—
L is a phosphorylated label which preferably becomes independently detectable when the phosphate is removed.(b) at least one of the enzymes selected from the group consisting of DNA polymerase, RNA polymerase and reverse transcriptase; and
(c) phosphatase. - View Dependent Claims (84, 85, 86, 87)
-
Specification