Method for determining the amount of template nucleic acid present in a sample
First Claim
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1. A method for determining the amount of template nucleic acid present in a sample comprising the steps of:
- i) bringing into association with the sample all the components necessary for nucleic acid amplification, and all the components necessary for a bioluminescence assay for nucleic acid amplification including;
a) a nucleic acid polymerase,b) the substrates for the nucleic acid polymerase,c) at least two primers,d) a thermostable luciferase,e) luciferin,f) an enzyme that converts PPi to ATP, wherein the enzyme is not ATP sulphurylase, andany other required substrates or cofactors of the enzyme of part f), wherein the substrate or cofactor is not PPi,wherein when the enzyme of part f) is pyruvate orthophosphate dikinase, the substrates added in part g) are phosphoenolpyruvate and AMP; and
subsequently;
ii) performing a nucleic acid amplification reaction of the template nucleic acid, wherein generated copies are themselves further copied;
iii) coupling the production of PPi to light output from the bioluminescence assay by converting generated PPi to ATP and using luciferase to follow the changes in the concentration of ATP by monitoring the intensity of light output from the bioluminescence reaction; and
iv) determining the amount of template nucleic acid present in the sample.
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Abstract
A method for determining the amount of template nucleic acid present in a sample comprising the steps of: i) bringing into association with the sample all the components necessary for nucleic acid amplification, and all the components necessary for a bioluminescence assay for nucleic acid amplification and subsequently: ii) performing the nucleic acid amplification reaction; iii) monitoring the intensity of light output from the bioluminescence assay; and iv) determining the amount of template nucleic acid present in the sample.
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39 Claims
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1. A method for determining the amount of template nucleic acid present in a sample comprising the steps of:
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i) bringing into association with the sample all the components necessary for nucleic acid amplification, and all the components necessary for a bioluminescence assay for nucleic acid amplification including; a) a nucleic acid polymerase, b) the substrates for the nucleic acid polymerase, c) at least two primers, d) a thermostable luciferase, e) luciferin, f) an enzyme that converts PPi to ATP, wherein the enzyme is not ATP sulphurylase, and any other required substrates or cofactors of the enzyme of part f), wherein the substrate or cofactor is not PPi, wherein when the enzyme of part f) is pyruvate orthophosphate dikinase, the substrates added in part g) are phosphoenolpyruvate and AMP; and
subsequently;ii) performing a nucleic acid amplification reaction of the template nucleic acid, wherein generated copies are themselves further copied; iii) coupling the production of PPi to light output from the bioluminescence assay by converting generated PPi to ATP and using luciferase to follow the changes in the concentration of ATP by monitoring the intensity of light output from the bioluminescence reaction; and iv) determining the amount of template nucleic acid present in the sample. - 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, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39)
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35. A method for determining the amount of template nucleic acid present in a sample comprising the steps of:
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i) bringing into association with the sample all components necessary for a nucleic acid amplification reaction, which releases pyrophosphate (PPi) for each nucleotide incorporated into a growing chain of nucleotides, and all components necessary for a bioluminescence assay, which converts PPi into ATP, including; a) a nucleic acid polymerase, b) substrates for the nucleic acid polymerase, c) at least two primers, d) a thermostable luciferase, e) luciferin, f) an enzyme that converts PPi to ATP which is neither ATP sulphurylase nor pyruvate orthophosphate dikinase, and g) any other required substrates or cofactors of the enzyme of part f) which is not PPi; and subsequently; ii) performing the nucleic acid amplification reaction of the template nucleic acid, which generates copies of the template nucleic acid that are themselves further copied, coupled to the bioluminescence assay for quantitating PPi produced as a consequence of nucleic acid polymerization; iii) monitoring the light output from the bioluminescence assay; and iv) determining the amount of template nucleic acid present in the sample from the light output. - View Dependent Claims (36)
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Specification