Fast PCR is a technique that cut down the running time of conventional PCR process and decrease PCR run times from hours down to minutes. Many labs use fast PCR to save time. The speed of a PCR process is determined by a lot of factors, including the extension rate of the polymerase used, the ramp speed of the thermal cyclers and the complexity of the DNA template. Standard extension rates of Taq DNA Polymerase is about 1 kb/min. However, enzymes that are able to perform fast PCR have extension rates that are typically 2-4 kb/min. Fast PCR can be achieved in three ways: (1) Escalate the temperature quicker. (2) Change protocol to save time (sometimes even combine steps). (3) Enhance the function of enzyme. In these ways, a PCR process may run within 30 minutes, which may run 90 minutes originally.
Enzymes capable of rapid PCR can help achieve faster turnaround time from sample to result and provide improved throughput on existing thermal cyclers. By reducing the hold time of the denaturation, annealing, and extension steps or using a two-step cycling scheme (at 60 ℃) that combines the annealing and extension steps, shorter PCR run times could be achieved. The run time of PCR is limited by the kinetic properties of the PCR enzyme. Significantly reduces denaturation temperature and cycle time while maintaining the sensitivity and specificity of the original PCR conditions. Recently, the amplification time of short PCR amplicons has been increased by increasing the concentration of primers and polymerases to about 20 times the typical concentration, increasing the annealing extension temperature to about 75 ℃, and reducing the denaturation temperature to 90 ℃. Less than 15 seconds, realizing ultra-fast PCR reaction time.
Laser PCR is a fast PCR technology developed on functionalized nanoparticles using pulse-controlled amplification (PCA) principles. Laser-activated PCA locally triggers instantaneous heating and cooling ramps, while most reactions remain at a constant temperature. Because the laser wavelength matches the plasma absorption characteristics of gold nanoparticles, colloidal nanoparticles (preferably made of gold) are irradiated with laser to enable uniformly dispersed nanoparticles to achieve local and ultra-fast heating. The laser heats the nanoparticles (in terms of cortisol concentration) in short pulses (for example, microseconds), which can keep the reaction temperature substantially constant during the nucleic acid amplification process. By limiting thermal cycling to the nanoparticles and attaching primers to the surface of the nanoparticles effectively, the heating and cooling cycles of laser PCR can be a million times faster than traditional PCR (Figure 1).
Fig. 1 Pulse controlled amplification of nucleic acids by laser PCR. The principle of Laser PCR is pulse controlled amplification (PCA). High power laser beam can generate pulses by PCR reaction to irradiate gold nanoparticles selectively and specifically. The heat generated by nanoparticles in the process of laser pulse is dissipated directly into the solution through a steep temperature gradient, and only the primers or amplicons which are effectively combined on the surface of nanoparticles are heated. Therefore, the reaction liquid is used as a built-in cooling container. A, B and C show the principle of nanoscale pulse controlled amplification. (A) In the laser PCR reaction mixture, the gold nanoparticles (yellow) function by target specific primers (light green); (B) The DNA target (red) is annealed to a specific primer on the gold nanoparticles, and the DNA polymerase binds to extend; (C) The laser beam scanned on the sample activates all nanoparticles, leading to local heating and denaturation of the amplicon. This reaction can achieve near instantaneous thermal gradient and super fast PCR reaction time. (Ullerich L, et al, 2017)
Here, the increase in speed does not sacrifice sensitivity. In the current developmental state of laser PCR, it can generate pulse controlled amplification of 10 labeled target DNA copies within 10 minutes (Fig. 2). Here, laser PCR is combined with report probe and real-time fluorescence detection. The existing commercial hydrolysis probe assay and other probe based assays can be used in combination with laser PCR.
Fig. 2 Real-time detection of the bacterial resistance gene mecA by Laser PCR. (Ullerich L, et al, 2017)
The advantage of fast PCR is obvious, it is time-saving and keep its sensitivity when operate properly. However, some complications may arise. Combining the fast PCR reagent with the new fast thermal cycler might be disastrous for an experiment. The majority of fast PCR reagents fail when the protocols are transferred to fast thermal cyclers. This is a challenge for determining what protocols are required for specific assays on specific instruments.