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This paper presents the design, fabrication, and characterization of a miniaturized multichamber thermal cycler that is independently controllable with multiplex thermal protocols for the polymerase chain reaction (PCR) of nucleic acids. Thermal isolation between multiple chambers is achieved by an etch-through slot on a silicon membrane containing the reaction chambers, while keeping the silicon substrate unheated by directly contacting the substrate with a bottom heat sink. The thermal response is very fast due to reduced parasitic thermal mass. Typical ramping and cooling rates achieved are 15-100°C/s and 10-70°C/s, respectively. In contrast to uniform heating, as reported by other research groups, a side-heating scheme is used in this study to improve the temperature uniformity inside the reaction chamber. Finite-element-analysis (FEA) is used to predict and optimize the thermal performance. A temperature uniformity of <± 0.15°C in the reaction chamber filled up with water has been measured at 95°C, while keeping the substrate temperature as low as 0.4°C above the room temperature. This ensures independent controllability of multichamber thermal multiplexing on the same chip with low thermal cross talk between chambers. Experiments are in agreement with FEA predictions. The developed device can be used for quick optimization of PCR protocols and multiplexing of numbers of PCR reactions simultaneously in a short time.