Recently the polymerase chain reaction (PCR) turned 30, marking an important birthday for molecular biologists. PCR has transformed molecular research and diagnostics, both in a recognisable form such as single end-point PCR or real-time PCR reactions but also present at the heart of the latest technologies. Digital PCR, molecular diagnostic and even Next Generation Sequencing sample preparation all rely on PCR. The principle behind all these applications is always the same and depends on the original three thermal steps and their repetition: DNA denaturation, primer annealing and primer extension.
The extraordinary longevity of the PCR technique can be explained by its ability to continuously absorb new technologies in its equipment (hardware) and chemistry (DNA polymerase). For example, technologies such as thermoelectric cooling or microfluidics have given a major boost in improving the specificity of the reaction as well as its speed. Simultaneously, the scope and performance of PCR has been improved by the development of new DNA polymerases, with the original enzyme from Thermus aquaticus (Taq) being supplemented by Pyrococcus furiosus family B polymerase (Pfu) as well as many other natural variants or mutants.
The growth in the diversity of applications has generated a demand for PCR reagents with additional properties and better performance such as high-fidelity, higher yield or higher specificity. As a consequence, new thermostable DNA polymerases with different properties have been introduced. The improvement of the PCR fidelity was mainly achieved by the discovery that Pfu could be used in PCR, and possessed a valuable proofreading 3’-5’ exonuclease activity. This was followed by the discovery of other high-fidelity enzymes such as that from Thermococcus kodakaraensis (KOD) and Thermococcus litoralis (Vent DNA polymerase) all aiming at increasing the yield of the PCR up to the level observed with Taq DNA polymerase. In addition to the search of potentially interesting enzymes in natural environments, new variants of thermostable DNA polymerases have been engineered using rational protein engineering or directed molecular evolution approaches. Another major improvement in PCR performance has been achieved by the development of “hot-start” polymerases, where enzyme function is inhibited either by a thermolabile element (such as an antibody or aptamer) or by chemically crosslinking the enzyme. In both cases, the polymerase only becomes fully activated during an initial denaturation step of the PCR, carried out at relatively high temperature (usually above 94°C).The inhibition of the polymerase at room temperature avoids modifications of the primers by the polymerase, which not only facilitates the experimental set-up, but also increases the specificity of the PCR.
Today, the challenges for PCR are even bigger: a higher level of sensitivity is required across a vast range of applications ranging from single cell analysis to fast real-time PCR directly from crude material. A very low amount of available template per PCR, the presence of PCR inhibitors or the introduction of ultra-fast thermocyclers, makes the stakes even higher and some limitations in the technology abilities may soon be reached. So, in order to overcome these current challenges and deliver higher sensitivity or faster cycling abilities it is necessary to improve performance of PCR further.
In order to address these challenges, Bioline has adopted a very simple but global approach based on a careful optimization of all the components of PCR. This strategy is centred on harnessing the best performance of ultra-pure components and placing them in a finely optimized physico-chemical environment. The purity of the components does not stop at the DNA polymerase protein itself but extends to the dNTP, additives and other chemicals involved, including, where possible, the template. Highly pure components allow a fine control of the enzyme/buffer system properties which enables the PCR reagent to be tailored for a particular application. This strategy by Bioline benefits PCR performance and minimizes contamination issues, non-specific amplification and false positives as well as decreases the batch to batch variation. In combination this increases the robustness and reliability of all Bioline's kits.
To achieve higher PCR performance levels, the particularities of specific applications are considered and the enzyme/buffer system is totally reformulated and adapted to suit assay requirements. Each and every component of the PCR reaction is carefully selected and its concentration optimized. Furthermore, the interactions between components and their effects on the PCR performance are also considered and the best concentrations and ratios are selected. This tailored approach to PCR also takes into account the preparation of the template. A finely tuned PCR would not deliver maximum sensitivity or yield without a template of good quality. Removal of any contaminants from the sample or carry-over of the extraction and purification process is very important as it avoids any uncontrolled effects of the template addition on the physico-chemical environment of the DNA polymerase, keeping the system perfectly optimized for the application.
In conclusion, through the careful combination of pure enzymes and chemicals, ultra-high purity nucleotides and strict quality standards, Bioline has pushed the PCR performance further, delivering a reliable and powerful amplification platform at the service of today’s molecular biologists.