Devices and methods for rapid PCR
Inventors
Ririe, Kirk Max • Jones, David E. • Pasko, Christopher Paul • Chamberlain, Anson Cole • David, Derek • Wernerehl, Aaron • Bruns, Jonathan Allen
Assignees
Publication Number
US-10875026-B2
Publication Date
2020-12-29
Expiration Date
2037-02-21
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Instruments, methods, and kits are disclosed for performing fast thermocycling.
Core Innovation
The invention provides devices, methods, and systems for rapid amplification of nucleic acids using a compressible or flexible sample vessel, such as a flexible sample container or pouch, equipped for use in a rapid thermocycling protocol. The flexible sample container typically comprises distinct zones or chambers (for example, a first-stage chamber fluidly connected to a second-stage reaction zone with multiple reaction wells) and is compatible with an external instrument that includes a temperature control element providing at least two fixed or narrowly cycled temperature zones. In operation, the container and temperature control element (such as a heater assembly) are aligned so that portions of the sample are cycled between constant high and low temperature zones, or the container is alternately contacted with these zones, enabling fast and efficient PCR amplification.
The method involves thermocycling a PCR mixture in the sample vessel for an initial number of cycles at a first volume, then compressing the vessel to reduce the internal volume and expelling a portion of the PCR mixture. This reduction in volume allows subsequent cycles to be performed more rapidly due to decreased thermal mass, enabling significantly shorter cycle times in later PCR stages. Further reductions in volume and additional rapid cycling steps can be performed as needed. Sample movement between thermal zones, mixing, and precise temperature control are achieved through specialized components such as wipers, blades, or mixers integrated into the instrument, and the compression or expulsion processes are performed primarily by mechanical or pneumatic means.
The problem being addressed is the slow speed and inefficiency in traditional and even rapid PCR protocols, particularly when ramping between temperatures and managing larger sample volumes. Standard methods require significant time for temperature transitions and are limited by the thermal mass of the system, resulting in longer cycle times and extended total assay durations. Furthermore, existing systems require cycling entire heater assemblies, which is less energy efficient. This invention overcomes these limitations by leveraging fast temperature transitions, reducing sample volume during PCR, and using constant temperature zones, thereby achieving rapid PCR with high specificity and yield, even in closed and self-contained systems.
Claims Coverage
The independent claims define methods for achieving rapid PCR by combining thermocycling in a compressible vessel with volume reduction steps and sample movement relative to temperature zones. There are two main inventive features described in the claims.
Rapid PCR using a compressible sample vessel with staged volume reduction
A method for performing PCR comprising: - Providing a PCR mixture in a compressible sample vessel. - Thermocycling the PCR mixture for an initial number of cycles at a first volume, each with a first cycle time. - Compressing the vessel to reduce its internal volume and expel a portion of the sample, resulting in a second, smaller volume of PCR mixture. - Thermocycling the reduced volume for a subsequent number of cycles at a shorter cycle time. This staged reduction in sample volume, achieved by physical compression and expulsion, allows for accelerated thermocycling as the thermal mass decreases.
Thermocycling by moving or aligning portions of the sample vessel with fixed temperature zones
A method in which the sample vessel is contacted with at least a first temperature zone and a second temperature zone (where one is hotter than the other), and the thermocycling step includes moving the sample between these constant (or narrowly cycled) temperature zones. - The movement may include alternatingly aligning the vessel with each zone, or simultaneously controlling different portions of the sample with different zones. - This feature permits rapid temperature transitions without requiring the heaters themselves to be cycled, improving speed and efficiency.
In summary, the inventive features are focused on methods that accelerate PCR by combining sample volume reduction within a compressible vessel and rapid thermocycling enabled by moving the sample between fixed temperature zones, thereby permitting shorter cycle times and efficient nucleic acid amplification.
Stated Advantages
The temperature changes for nucleic acid amplification can be accomplished more quickly due to fixed or narrow-range cycled temperature zones and alignment of the sample container with these zones.
Holding the temperature of the thermal zones constant (or in a limited range) eliminates the need to ramp the heater itself, reducing total run time and increasing electrical efficiency.
Reduction in sample volume during the PCR process allows for further decrease in cycle times as thermal mass is reduced, enabling PCR to proceed faster at each stage.
The flexible or compressible container and methods allow PCR to take place in a closed, self-contained system, reducing contamination risk.
Accelerated PCR protocols and device configurations can complete all processing steps in 20 minutes or less, with cycle times of 8 seconds or less, and preferably 4 seconds or less per cycle.
The techniques can enhance specificity and yield of amplification compared to traditional slow PCR processes, as precise temperature control and rapid cycling are possible.
Automated sample preparation, PCR amplification, and analysis can be integrated within the same device using the described methods and devices.
Documented Applications
Rapid diagnosis of infectious agents in human, veterinary, industrial, and environmental samples, including detection and sequencing of a wide variety of nucleic acid sequences.
Multiplex PCR panels in self-contained flexible pouches, such as detection of multiple pathogens (e.g., respiratory viruses and bacteria) using high-density reaction well arrays.
Automated, closed-system nucleic acid assay systems integrating sample preparation, first-stage multiplex PCR, dilution, and second-stage single-plex PCR (nested or otherwise), followed by detection or further analysis.
PCR in sample containers compatible with automation, including microfluidic devices, arrays, well plates, and other reaction containers for high-throughput environments.
Interested in licensing this patent?