The time required to complete a PCR reaction on a PCR machine is determined by several factors:
1, module heating and cooling time
2, module and PCR tube temperature balance time
3, extension time
4, the number of cycles
Let's do some analysis on the above points so that everyone can understand the difference between fast PCR technology and fast PCR instrument.
1, module heating and temperature reduction time
The PCR instrument generally indicates the heating rate and the cooling rate, but the target is mostly the maximum temperature changing speed, that is, the speed reached instantaneously. The average temperature rise and fall associated with the experiment is only indicated by a very small number of manufacturers. Calculated from an average of 2 degrees and 4 degrees (the instrument can achieve an average temperature of 4 degrees), from 95 degrees to 55 degrees, respectively, 20 seconds and 10 seconds, each cycle difference of 20 seconds. Calculated in general 35 cycles, affecting the experimental time of 700 seconds, that is, less than 12 minutes. Actually, we must consider the overshoot problem, and the impact time will be less.
2, module and PCR tube temperature balance time
After the module temperature is reached, the temperature inside the PCR tube is far from reaching (the thermal conductivity of plastic and water is more than 300 times worse than that of aluminum), and it takes time to balance the two. There are generally four ways to reduce this time. One is to overshoot the module temperature and drop to the target temperature when the temperature in the tube reaches fast, but too high overshoot may cause the actual temperature overshoot, affecting the PCR reaction result; Reduce the volume of the PCR reaction solution, but the reliability of the PCR reaction will decrease when the volume is small; the third is to reduce the thickness of the PCR tube, but the strength is not enough when it is too thin; the fourth is to increase the degree of attachment of the PCR tube to the module.
3, extension time
The extension time is related to the length of the PCR product and the rate of synthesis of the DNA polymerase. If the PCR product is less than 100 bp in length, it may be considered to change the extension time to 0 (ie, a two-step method); the use of high-speed DNA polymerase is also a common method, but not all PCR experiments can use high-speed DNA polymerase.
4, the number of cycles
The number of cycles is related to the amount of template and the detection requirements.
The so-called rapid PCR technology integrates a fast PCR instrument with ultra-thin tubes (or special attachment technology) and high-speed DNA polymerase technology to achieve a shorter PCR reaction. While some manufacturers are marketing the PCR instrument to users, they do not explain the rapid PCR technology, but only emphasize the speed of the PCR instrument (mostly use the maximum temperature rise and fall to HuYou users). It seems that after buying a fast PCR instrument, the original amplification time of one and a half hours will be shortened to half an hour. After the user purchased the fast PCR instrument, it was found that it was not the case. There are quite a few users who can't do good experimental results in a fast mode. In particular, users who perform multiplex PCR amplification, such as reagents used by the public security system for identification.
Fast PCR technology is a different matter from fast PCR machines. Fast PCR machines can only shorten the PCR reaction time by up to 10 minutes. Other efforts are needed to achieve a true rapid PCR reaction. Most users do not need fast PCR technology at all.
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