Polymerase Chain Reaction (PCR) is a method for synthesizing double-stranded DNA in vitro. Its principle is similar to the replication process of natural DNA, and its specificity mainly depends on specific primers complementary to two segments of its target fragment. A typical PCR reaction has three steps: denaturation, annealing, and extension, and after several cycles of reaction, the target fragment is obtained.

　　The principle of hot-start Taq enzyme

　　In the development history of PCR, the discovery of hot-start enzymes is an important factor restricting the development of PCR. The enzyme initially used in PCR is the Klenow fragment of E. coli DNA polymerase I. Klenow enzyme needs to be re-added every cycle. The process is cumbersome. Since then, scientists have successfully extracted thermostable DNA polymerase from Thermobacter aquaticus, the enzyme still has 70% activity at 90°C for 2 hours, which avoids the process of re-adding the enzyme every cycle and improves the performance of the enzyme. Amplification efficiency is therefore widely used in PCR amplification.

　　The effect of temperature on the activity of the enzyme showed a dual effect, inactivation at high temperature and inhibition at low temperature. The optimum temperature of common Taq DNA polymerase is 72℃, the activity of the enzyme is good at this time, and the enzyme has weak activity below this temperature. In this way, in the process of PCR amplification from low temperature to high temperature (that is, when the temperature reaches the denaturation temperature), the enzyme exerts a weak activity, and the system is very easy to mismatch or form primer dimers, especially when the designed primer 3' The ends are G or C, and mismatched chains are difficult to unravel. Therefore, in order to improve the specificity of PCR amplification and reduce the mismatch rate of the reaction, the activity of the enzyme can be controlled artificially, and the enzyme can be inactive before 72°C, thus avoiding the occurrence of chain chain during the heating process (or configuring the reaction system). mismatches, thereby ensuring the specificity of amplification. Relia hot-start enzyme uses chemical modification to block the active center of the enzyme: chemical small molecules bind to the active center of the enzyme at low temperature, the enzyme is inactive, the temperature rises to 95 °C, the two are separated, the active center of the enzyme is exposed, and the system begins to guide Amplification (as shown in Figure 1) greatly improves the specificity and sensitivity of amplification. Compared with antibody-blocked enzymes, chemically modified enzymes maintain more stable activity (antibody blocking is a dynamic equilibrium) without any foreign DNA contamination.

　　Figure 1: The principle of hot start

　　Application of Hot Start Taq Enzyme

　　PCR can rapidly and specifically amplify any known DNA fragment, so it is widely used in various fields of molecular biology. Relia hot start enzyme can be used in real-time quantitative PCR reaction because of its 5'-3' exonuclease activity. Amplification by hot-start method is a common method to improve the specificity of PCR, and hot-start enzyme is a good choice. In addition, because of its high specificity and high sensitivity, it is widely used in various aspects: construction of cDNA library, generation of large-scale DNA sequencing, analysis and construction of mutant analysis, gene isolation, genetic disease diagnosis, forensic identification, etc.