Amplification of enzymatic reactions for use with an enthalpy array
AssigneesPalo Alto Research Center Incorporated, The Scripps Research Institute
InventorsFrancisco E. Torres, Richard H. Bruce, James R. Williamson, Peter Kuhn, Ray Stevens
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Abstract
Provided is a method of detecting characteristics of a reaction of interest, including instituting the reaction of interest, obtaining an amplified heat related to the reaction of interest, measuring the amplified heat, and determining the characteristics of the reaction of interest, using the signal obtained in the step of measuring.
Claims27
A method of detecting characteristics of an enzymatic reaction of interest comprising:
- initiating a primary enzymatic reaction wherein a product and a first amount of heat are generated in a volume of approximately 30 microliters or less;
- activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein the product of the primary enzymatic reaction is converted to a species that activates the amplifying enzyme, wherein the species is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme;
- measuring the amplified amount of heat;
- and
- determining, the characteristics of the reaction of interest using the measured amplified heat.
A method for monitoring a primary enzymatic reaction comprising:
- conducting a primary enzymatic reaction wherein a product and a first amount of heat are produced in a volume of 30 microliters or less;
- producing a detectable amplified amount of heat by activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein the product of the primary enzymatic reaction is converted to a specie that activates the amplifying enzyme wherein the specie is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme;
- and detecting the amplified amount of heat.
A method for monitoring ATP kinase enzymatic reactions, the method comprising:
- conducting an ATP kinase enzymatic reaction, the ATP kinase reaction including reacting an ATP kinase with one or more substrates to produce one or more ATP kinase-specific products, the ATP kinase reaction of interest generating a first amount of heat, wherein the ATP kinase and one or more substrates have a combined volume of less than 30 microliters;
- and
- activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein a product of the ATP kinase enzymatic reaction is converted to a species that activates the amplifying enzyme, wherein the species is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme;
- and
- detecting the heat generated.
Dependencies are inferred from the claim text.