The difference between rapid PCR technology and rapid PCR instrument

The time required to complete a PCR reaction on the PCR machine depends on the following factors:

1. Module heating and cooling time

2. The temperature equilibration time between the module and the PCR tube

3. Extended time

4. Number of cycles

Let's do a little analysis on the above points, so that everyone can understand the difference between fast PCR technology and fast PCR instrument.

1. Module heating and cooling time

PCR machines generally indicate the heating rate and cooling rate, but most of the targets are the maximum temperature change rate, that is to say, the speed reached in an instant. The average temperature rise and fall speed related to the experiment is only indicated by a very small number of manufacturers. Calculated with an average of 2 degrees and 4 degrees (very few instruments can achieve an average temperature rise and fall of 4 degrees), the reduction from 95 degrees to 55 degrees is 20 seconds and 10 seconds, respectively, with a difference of 20 seconds for each cycle. Calculated with 35 cycles in general, it affects the experiment time of 700 seconds, which is less than 12 minutes. In fact, the overshoot problem must be considered, and the impact time will be less.

2. The temperature equilibration time between the module and the PCR tube

After the module temperature is reached, the temperature in the PCR tube is far from reaching (the thermal conductivity of plastic and water is more than 300 times worse than that of the aluminum block), and it takes time to balance the two. Generally, there are four ways to reduce this time. One is to overshoot the temperature of the module and drop to the target temperature when the temperature in the tube is about to reach, but too high an overshoot may cause the actual temperature to overshoot and affect the PCR reaction results; Reduce the volume of the PCR reaction solution, but when the volume becomes smaller, the reliability of the PCR reaction will be reduced; 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. Extended time

The extension time is related to the length of the PCR product and the synthesis speed of the DNA polymerase. If the length of the PCR product is less than 100bp, you may consider changing the extension time to 0 (ie, two-step method); using high-speed DNA polymerase is also a common method, but not all PCR experiments can use high-speed DNA polymerase.

4. Number of cycles

The number of cycles is related to the amount of template and testing requirements.

The so-called fast PCR technology is the integration of fast PCR instrument with ultra-thin tube (or special attachment technology) and high-speed DNA polymerase technology, which can complete the PCR reaction in a short time. When some manufacturers sell PCR machines to users, they do not explain the fast PCR technology, but only emphasize the high speed of the PCR machines (mostly, HuYou users use the maximum temperature rise and fall speed). It seems that after buying a fast PCR machine, the original 1.5-hour amplification time will be shortened to half an hour. However, after purchasing a fast PCR machine, the user realized that it was not so. There are many users who adopt the fast mode and cannot produce good experimental results at all. Especially for users who perform multiplex PCR amplification, such as reagents used by public security systems for identification.

Fast PCR technology and fast PCR instrument are two different things. The fast PCR instrument can only shorten the PCR reaction time by about 10 minutes at most. To achieve a truly fast PCR reaction, some other efforts are needed. Most users do not need rapid PCR technology at all.