Rapid thermocycling methods
Inventors
Jones, Charles • Hemmert, Andrew • Crisp, Robert • Campbell, Elizabeth Diana • Kirk, Karina S. • Hatch, Andrew Carter
Assignees
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Publication Number
US-12270072-B2
Publication Date
2025-04-08
Expiration Date
Abstract
Instruments, methods, and kits are disclosed for performing fast thermocycling.
Core Innovation
The invention performs polymerase chain reaction (PCR) using thermocycling in an amplification container with an amplification zone. A PCR mixture is thermocycled with a first volume in the one amplification zone for a first number of cycles, where the cycles include an annealing temperature and a denaturation temperature with corresponding annealing and denaturation holds.
The PCR method then reduces the volume of the PCR mixture in the same one amplification zone to a second volume smaller than the first volume, and thermocycling continues with the second volume for at least a second number of cycles. The second number of cycles includes a second cycle time, a second annealing temperature, and a second denaturation temperature and hold, where the second cycle time is shorter than the first cycle time and the second denaturation temperature is lower than the first denaturation temperature after reducing the volume.
In additional implementations, cycle parameters are adjusted across multiple thermocycling phases, including differing cycle time, annealing temperature/hold, and denaturation temperature/hold across the thermocycling sets before and after reducing the volume. The method can also include an initial cycle prior to thermocycling for the first number of cycles, where an initial denaturation hold is longer than the first denaturation hold and an initial denaturation temperature is lower than the first denaturation temperature.
Claims Coverage
The independent claims cover three principal thermocycling/volume-reduction PCR workflows, each using one amplification zone of an amplification container and changing thermocycling parameters over cycle sets in relation to reducing the PCR mixture volume. Across the independent claims, the inventive features are the staged thermocycling of different cycle parameter sets, the reduction of PCR mixture volume within the same amplification zone, and parameter relationships that shorten cycle times and adjust denaturation temperature and hold after volume reduction, including optional initial-cycle parameter constraints.
Staged thermocycling with volume reduction and shorter cycle time
Thermocycling a PCR mixture having a first volume in one amplification zone of an amplification container for a first number of cycles, reducing the volume of the PCR mixture in the same one amplification zone to a second volume smaller than the first volume, and thermocycling the second volume of PCR mixture in at least a portion of the one amplification zone for a second number of cycles, where the second cycle time is shorter than the first cycle time and the second denaturation temperature is lower than the first denaturation temperature after reducing the volume.
Initial cycle with longer denaturation hold and lower initial denaturation temperature
Performing an initial cycle in the one amplification zone prior to thermocycling for a first number of cycles, where the initial cycle comprises an initial cycle time, an initial annealing temperature, an initial denaturation temperature, and an initial denaturation hold, the initial denaturation hold is longer than the first denaturation hold, and the initial denaturation temperature is lower than the first denaturation temperature.
Multiple thermocycling cycle sets with differing parameters and volume reduction
Thermocycling a PCR mixture with a first volume in one amplification zone for at least a first cycle, thermocycling for a second number of cycles in the same one amplification zone with one or more differing cycle parameters relative to the first cycle, reducing the volume of the PCR mixture in the same one amplification zone to a second volume smaller than the first volume, and thermocycling for a third number of cycles in the same one amplification zone with one or more parameters differing from the second cycle set.
Three-phase denaturation parameter progression around volume reduction
Thermocycling a PCR mixture with a first volume in an amplification zone for at least a first cycle comprising a first denaturation temperature and a first denaturation hold, thermocycling for a second number of cycles with a second denaturation temperature higher than the first denaturation temperature and a second denaturation hold shorter than the first denaturation hold, reducing the volume of the PCR mixture in the amplification zone to a second volume smaller than the first volume, and thermocycling for a third number of cycles with a third denaturation temperature lower than the first or the second denaturation temperatures and a third denaturation hold shorter than the second denaturation hold.
Across the independent claims, the core claim coverage centers on thermocycling a PCR mixture in a single amplification zone in a staged manner with changing cycle parameters, reducing the PCR mixture volume within the same amplification zone, and enforcing comparative relationships that shorten cycle time and adjust denaturation temperature/hold after volume reduction, with optional initial-cycle constraints and multi-stage denaturation progression.
Stated Advantages
Documented Applications
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