Some like it hot – DNA amplification using thermal cyclers
Thermal cycler, also known as PCR machine or thermocycler, provides a thermally controlled environment for the PCR-reaction solutions. It precisely controls and changes the temperature repeatedly between the three stages. In the early days, the PCR technique was laborious and slow, requiring manual transferring between water baths at different temperatures. Later on, conventional bench-top PCR thermal cyclers were introduced, based on proportional-integral-derivative (PID) temperature controller, based on Peltier thermoelectric units, achieving simple and reliable PCR amplification that is widely used today. Further, multiplex PCR machine offers simultaneous amplification of more than one DNA targets in the same reaction. Similarly, real-time PCR machine offers additional capability of monitoring DNA amplification in real time, using a camera or a detector and micro-machined/nano-machined chip PCR that requires minute amount of sample, and simultaneously analyzes several samples with enhanced integration ability.
The amplification process in a traditional thermal cycler is time consuming because of the relatively low temperature change rates. To further increase the speed of the thermal cycler, two strategies are required: (i) improving the thermal ramp rate to reduce the time between temperature transitions, which could affect amplification efficiency; and (ii) reducing cycling time by simplifying three-step PCR to two steps using VPCR technique. Recently, dPCR instrument (QX100 and QX200 by Bio-Rad; Rain Drop by RainDance and Quant Studio 20K chip by Thermofisher) has been developed where nucleic acid sample is partitioned into different compartments or fabricated microwell array chips or soft-lithography-based multilayer-channels or microfluidic chips (termed as integrated fluid circuit by Fluidigm). The amplicons are evaluated individually; hence, the outcome is unaffected by variations in the amplification efficiency, leading to a more precise, sensitive, and reproducible target quantification. All these cycling systems can be classified into two systems, based on the position between the sample and the thermal block during amplification, i.e., stationary system (no movement occurs during the PCR amplification process) and spatial system (position change between the sample and the individual thermostatical temperature blocks, i.e., microfluidic chips or rotary PCR systems).
Recently, various heating techniques have been integrated into microfluidic systems for PCR thermal cycling, including Joule heating (electric current passes through an ohmic conductor and heat energy generated is proportional to the square of the electric current and the resistance of the ohmic conductor), thermoelectric heating (electric current passes through the junction of two dissimilar metals, one junction experiences cooling while the other junction experiences heating), acoustic heating (heat is generated because of energy dissipation by surface acoustic waves (SAWs)), photonic heating (converting light energy form laser or light-emitting diodes (LEDs) into heat), induction heating (electrically conductive metal is heated by eddy currents produced through electromagnetic induction), and microwave heating.
In addition, the traditional thermal cyclers are bulky, expensive, and consume high power, hindering their extensive utilization in diagnostics, especially for developing countries and rural areas. Thus, there remains an unmet need for a rapid, low-cost, compact, portable thermal cycler with improved heating/cooling rates and reduced amplification time, which could mark a revolutionary step forward in global public healthcare. Recently, commercial portable thermal cyclers have been developed to perform on-site quantitative and qualitative DNA analysis, such as Mini8 Plus real-time PCR system, Mic qPCR system, MyGo mini real-time PCR system, and Chai real-time PCR system. Further integration of isothermal amplification and lens-free imaging in PCR systems will improve their applications in biomedical area, especially in rural areas and developing countries in a rapid and low-cost manner. Extensive research is still needed to design and develop simple, inexpensive, and portable PCR systems with a reasonable accuracy, precision, and high throughput.
