How to choose the right PCR function when facing different samples

As we all know, the PCR instrument, also known as the thermal cycler, is a special temperature-variable instrument that cooperates with the PCR process. The thermal cover is a part of the upper part of the instrument to prevent the sample from evaporating and condensing on the tube cover in a high-temperature environment to reduce the risk of reaction sample volume.

  PCR, also known as polymerase chain reaction, is a method to amplify target DNA fragments in vitro and is widely used in many aspects. It is mainly divided into three steps: high temperature denaturation, low temperature annealing, and temperature extension. Add primers, templates, Taq enzymes, dNTPs, buffers and other reactants to mix, and the double-stranded DNA hydrogen bond breaks and unwinds into a single strand at about 95°C. At a temperature of about 60°C, the single-stranded primers are combined with artificially designed primers according to the principle of base complementary pairing; and the temperature is raised to about 72°C, and a new semi-reserved copy complementary to the template DNA strand is synthesized by Taq enzyme extension chain.

During this process, the temperature of a certain section will be close to or higher than 100℃, and some samples will evaporate upwards with the high temperature. When encountering a PCR tube cover with a lower temperature, it will condense on the tube cover, and the sample in the tube will have a certain amount Loss, a small part does not participate in the cycle, resulting in a decrease in sample yield.

Prior to this, the instrument did not have this device. This problem was solved by adding paraffin oil to the sample, but in fact, the subsequent treatment of paraffin oil is more cumbersome. The emergence of subsequent hot lids can effectively avoid this risk. The temperature of the hot lid is generally 5-10°C higher than that of the module, so that the temperature of the upper end of the PCR tube in the module is always greater than the temperature of the reaction liquid. Will not evaporate upwards, so the volume of all sample reaction liquids will not change.

However, due to the different heights of the sample tubes, the non-adjustable thermal lid of the early instruments can only be used for sample tubes of uniform height. When using a shorter sample tube, an aluminum block needs to be added, which is more troublesome; the current adjustable thermal lid is divided into Knob type and self-adjusting type, etc. For some instruments, the height and pressure of the knob-type hot lid can be adjusted according to the sample tube, but you need to turn the knob back to the original position before changing the sample. In addition, there is a problem that the pressure is not well controlled. Too tight will denature the sample tube, etc. If it is too loose, it will not be able to achieve the corresponding temperature effect. Self-adjusting can adjust the height according to the height of the sample tube.

The so-called hot start is the step of adding Taq enzyme to the sample tube for temperature extension. However, Taq enzyme can also play a role in the room temperature environment. Therefore, it is necessary before adding reactants or denaturing at high temperature. Prevent the Taq enzyme from starting to work at an inappropriate temperature to avoid non-specific products in this situation. A certain number of templates and enzymes produce non-specific products, which means that the content of specific products will be reduced. Therefore, samples will be added on ice, or the PCR machine will be preheated first, and then quickly put into the PCR tube.

 You can also mix the primer, Mg2+, dNTP, and buffer with the solidified and cooled wax and isolate the upper and lower layers of the remaining reaction components. In the denaturation stage, the wax is heated and melted, all ingredients are mixed, and the liquid wax floats to the upper part of the sample, acting as a thermal cover. But the subsequent wax treatment is more troublesome. The use of chemical modification or preparation of antigen-antibody complexes allows the enzyme activity to be controlled according to the temperature.

In the face of high-throughput samples, manual hot-start protection will be very time-consuming. Therefore, the PCR machine will set the temperature of the hot lid. Before the hot cover is heated to a certain temperature, the temperature of the module remains unchanged. When the temperature of the hot cover reaches the set value, the module heats up rapidly to achieve the effect of hot start.

 In addition, many PCR machines have two options for heating mode: Tube and Block. This is because the temperature sensor of the instrument is in the lower part of the module, and the temperature in the sample tube cannot actually be measured. Under normal circumstances (Block), when entering the set temperature and running time, it means that the module will continue to reach the specified temperature for a corresponding time, and the temperature of the sample tube cannot quickly reach the specified temperature; while in Tube mode, the module temperature is first set If the temperature is higher than the set temperature, wait for the temperature of the sample tube to reach the specified temperature, and then lower the temperature of the module to the specified temperature, so that the temperature in the tube can reach the set value quickly. Different reaction volumes and requirements can use different modes: normal PCR or Tube mode for larger volume, Block mode can be used when holding for a long time and requiring higher temperature overshoot or smaller volume.