Thermally multiplexed polymerase chain reaction.
Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Christopher R
CR
Phaneuf
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Nikita
N
Pak
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
D Curtis
DC
Saunders
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Gregory L
GL
Holst
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Joav
J
Birjiniuk
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Nikita
N
Nagpal
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Stephen
S
Culpepper
Emily
E
Popler
Andi L
AL
Shane
Robert
R
Jerris
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, USA.
Craig R
CR
Forest