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AssigneeGE Healthcare Bio-Sciences AB
InventorsAnup Sood, Shiv Kumar, John Nelson, Carl Fuller, Anuradha Sekher
Estimating the term…
The present invention relates generally to the use of terminal-phosphate-labeled nucleotides having three or more phosphates as substrates for nucleic acid polymerases and their use in DNA amplification. The labels employed are chemiluminescent, fluorescent, electrochemical and chromogenic moieties as well as mass tags and include those that are directly detectable, detectable after enzyme activation or feed into other processes to generate a different signal. The signal generated from the attached dyes may also be used to quantify the amount of amplification. Further provided are stabilizers that enhance the stability of terminal-phosphate labeled nucleoside polyphosphates in aqueous solutions and are useful for reducing non-enzymatic hydrolysis of these nucleotides, hence decrease background.
In a method for nucleic acid amplification, which includes a polymerase reaction of a nucleic acid template, a primer, a nucleic acid polymerase, and at least one nucleoside polyphosphate, the improvement comprising conducting said polymerase reaction in the presence of at least one terminally labeled nucleoside polyphosphate, and wherein the amplification is conducted in the presence of a stabilizer that stabilizes the terminal-phosphate labeled nucleoside polyphosphate against non-enzymatic hydrolysis.
In a method for nucleic acid amplification, which includes a polymerase reaction of a nucleic acid template, a primer, a nucleic acid polymerase, and at least one nucleoside polyphosphate, the improvement comprising conducting said polymerase reaction in the presence of at least one terminally labeled nucleoside polyphosphate, and wherein said terminal-phosphate labeled nucleoside polyphosphate comprises four or more phosphate groups in the polyphosphate chain.
A method of detecting the presence of a nucleic acid sequence in a sample comprising the steps of:
The method of claim , wherein said enzyme-activatable label is a fluorogenic moiety selected from the group consisting of 2-(5′-chloro-2′-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, fluorescein diphosphate, fluorescein 3′(6′)-O-alkyl-6′(3′)-phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate, and 6,8-difluoro-4-methylumbelliferyl phosphate.
A kit for amplifying or quantifying a nucleic acid target comprising:
A kit for amplifying or quantifying a nucleic acid target comprising:
A method of detecting the presence of a nucleic acid sequence in a sample comprising the steps of:
Dependencies are inferred from the claim text.