Little could Jean Charles Peltier envisage in 1834 that his discovery of the Peltier effect would become an indispensable driver of the 21st century molecular biology laboratories. More recently, the invention of the polymerase chain reaction (PCR) by Kary Mullis in 1985 catapulted a biotechnology revolution. PCR today has become a ubiquitous molecular tool. With PCR came the need for robust systems to automate the assays, using what came to be known as PCR thermal cyclers or thermocyclers. The early models used three water baths at pre-set temperatures and a mechanical arm to move samples between baths. The water baths gave way to resistive water heaters, electric fans, and electric coils. But, shortfalls in each system eventually galvanized the Peltier technology with PCR. Most modern thermal cyclers have at their heart the Peltier thermoelectric modules composed of bismuth telluride semiconductor couples. These Peltier thermocyclers have established their popularity because of absence of any moving parts, no use of freon refrigerants, freedom from noise and vibration, compact size, long shelf life, and precision temperature control. For over two decades, Peltier thermocyclers have dominated the market. Today, a new generation of Peltier thermocyclers is emerging with enhanced capabilities for high throughput molecular labs. Some of these emerging advancements are discussed here.

Thermal blocks. Modular nickel-plated anodized aluminium blocks are industry standard and give uniform PCR yields. New age cyclers also offer electroplated solid silver- or gold-plated blocks which though more expensive, are faster, uniform, and give high yield performance. Primus has introduced high temperature range (HTR) Peltier blocks which can attain temperatures ranging from -50ºC to +200ºC, at heating rates up to 4ºC/second which are 3-4 times faster than conventional blocks with 1ºC-2ºC/second heating rates.

Throughput. Modern cyclers offer the option of interchangeable thermal blocks between 96 x 0.2 ml, 60 x 0.5 ml, and 384-well plate formats. The throughput capabilities of available instruments were so far limited for routine laboratories to 384 reactions per run. Recently, the LightCycler 1536 (Roche) instrument was launched with a high-throughput capability of performing 1536 qPCR reactions simultaneously in approximately 45 minutes. The LightCycler 1536 supports the combination of two excitation filters with two detection filters, which are optimized for detecting green intercalating dyes as well as monocolor and dual-color hydrolysis probes. New age cyclers also support dual blocks (Gene Technologies, GS482), quadruple blocks (QuattroCycler, VWS), going up to 16-28 parallel blocks (Intelligent Bio-Instruments TC 1600; Primus Multiblock) to raise throughput capacities.

User Interface and Software. Standard software are capable of regulating fixed ramp speeds, time and temperature auto-extend/auto-decrease settings, execute programmable pauses, manage auto-restart after power disruption, in-built Tm calculator, real time temperature verification, and handling a wide range of PCR formats (gradient PCR, touch down PCR, high throughput PCR, and in-situ PCR) besides storing 100 or more pre-set programs even in the simplest machines. New generation software offer flexibility to either operate a stand-alone system or to simultaneously network and regulate up to 30 satellite cycler units (Techne TC-PLUS; Esco Healthcare Swift MaxPro). Modern cyclers today are increasingly incorporating intuitive and user friendly VGA color touch-screen graphical display. Real-time machines (Bio-Rad CFX384) are now driven by software with multiple data acquisition modes to suit any test format and application. In addition, the convenience of a USB port to simplify data transfer and storage is being added in upcoming models.

Notwithstanding the success of Peltier thermocyclers, they suffer from disadvantages like high thermal mass, high power consumption, slow heating/cooling rates and thus slow ramping, and large reaction volumes inefficient for analysis of sub-microliter samples. With growing technological leaps, novel PCR formats are becoming popular norms including, pathogen detection in whole-blood samples, PCR-mediated genomic walking methods (random PCR, inverse PCR, panhandle PCR, cassette PCR, and rapid amplification of genomic ends), single-cell PCR, and electrochemical PCR (EC-PCR) for point-of-care applications. Successes of such applications depend on rapid analysis time utilizing least amount of reaction volume (often in pico-to-nano liters). Peltier thermocyclers fall short on these fronts and hence evolved the technology of miniaturized PCR.

Miniaturization. The first miniaturized PCR system appeared in 1993 with micro-machined chemical reactors and integrated heaters. Since then, the technology has undergone revolutionary advancements. Indium-tin oxide (acts as a heater and temperature sensor) coated microfluidic capillary tubes with forced air convection for thermal management came next, followed by infrared (IR) heating and water impingement cooling technique for PCR in thin glass capillary tubes. Northrup et al. devised the first miniature analytical thermal cycling instrument (MATCI) which brought down total PCR cycling times to seven minutes.

The development of micro-electro-mechanical systems (MEMS) technology has recently allowed drastic miniaturization and complete migration of PCR on to what is known as lab-on-a-chip systems. The proven advantages of the lab-on-a-chip system are its compact size, low sample volume to nanoliters, short analysis time of less than 10 seconds to complete one PCR cycle and 370 seconds for completing the whole quantification process, and low energy consumption. These PCR chips are micro-fabricated on silicon, glass, or polydimethylsiloxane (PDMS) platforms, with arrays of picoliter volume PCR microchambers etched into the platform. High precision thermal cycling is achieved using resistive heating elements, infrared heating, thin film heaters, platinum resistors, and/or forced air convection.

The PCR chip can be designed for 1-4 chambers stationary PCR to dynamic continuous flow PCR for high-throughput settings. Typical dimensions of these chips are 59.5 mm long, 10.5 mm wide and 1 mm thick. Since, the reduced size brings down the thermal mass to negligible, the temperature transition times depend only on sample flow rate and it's time to reach thermal equilibrium and can reach up to 90¬?C/second or higher. In continuous flow system the reaction speed is even faster and limited only by the synthesis rate of DNA polymerase. Recently, micro-fabrication engineers have gone a step ahead and brought in total functional integration and convergence of sample pre-conditioning, DNA extraction, reagent addition, PCR amplification, capillary electrophoresis, and product detection by LED/fluorescence systems onto a single PCR chip. Hence, the lab-on-a-chip systems are also known as micro total analysis systems (µTAS).

The lab-on-a-chip technology, however, is currently refining its detection limits, quantification uncertainties, melting analysis, and real-time diagnostic accuracy, before they can enter the mainstream PCR market. But, if the technological advancements over the past decade are an indication to go by, it's only a matter of time before these systems score over traditional PCR cyclers for point-of-care testing and rapid high-throughput genomic research.