Views:14 Author:Site Editor Publish Time: 2021-09-14 Origin:Site
Principle of PCR thermal cycler
The essence of PCR technology is in vitro nucleic acid amplification, heating to unscrew double-stranded DNA, hybridization of primers with template DNA under annealing temperature conditions, extension of primers in the presence of Taq DNA polymerase, dNTPs, Mg2+ and a suitable pH buffer, Repeat the process of "denaturation→annealing→primer extension" to 25-40 cycles, exponentially expanding the number of nucleic acid copies in the sample to be tested.
A PCR amplification can only run a PCR machine with a specific annealing temperature, which is also called a traditional PCR machine.
Purpose: Mainly to do some simple amplification of target genes with a single annealing temperature.
(1) Gradient PCR instrument: A series of different annealing temperature conditions (temperature gradient) can be set for one PCR amplification, usually a common PCR instrument with 12 temperature gradients.
Purpose: To study the amplification of unknown DNA annealing temperature.
(2) In situ PCR: The in situ hybridization technology with cell localization capability is applied to the localization analysis of the target DNA in the cell, and the common PCR instrument that realizes the gene amplification in the cell.
Purpose: used for gene amplification at the location of the cell's target DNA
The primers added during PCR amplification are labeled with fluorescein, so that the primers and fluorescent probes are specifically combined with the template at the same time. The amplification results are collected in real time by the fluorescence signal acquisition system and sent to the computer analysis and processing system, and the quantitative results are output in real time. , Such a PCR machine is called a real-time quantitative PCR machine.
(1) Metal plate type real-time quantitative PCR instrument
Can be used as an ordinary PCR machine
Can be equipped with gradient function
Large sample size that can be accommodated, no special consumables required
The temperature uniformity is not good, there are edge effects, and the reaction conditions of the standard curve are difficult to be completely consistent with the sample
(2) Centrifugal real-time quantitative PCR instrument
Better temperature uniformity
The same excitation light source and detector are used to detect samples rotating to the front at any time, effectively reducing system errors
It can hold a small amount of sample, and some need special capillary as sample tube, which increases the cost of use and does not have gradient function.
(3) Quantitative PCR instrument with independent temperature control for each well
Different sample tanks have independent intelligent heating and cooling modules, and the temperature of each hole is independently controlled, which is suitable for rapid detection of multiple indicators.
The operation of the ordinary PCR amplification instrument is very simple, just turn on the power, self-check the instrument, set the temperature program or recall the stored program to run.
The operation of the quantitative PCR thermal cycler is basically the same as that of the ordinary thermal cycler.
① Fluorescent dye pollutes the sample hole;
②The PCR tube melts, the reason may be a problem with the temperature sensor or a problem with the thermal cover;
③The amplification efficiency of individual holes varies greatly. The possible reason is that the semiconductor module has defective spots;
④ The fluorescence intensity is weakened or unstable. The cause is moldy filter or moisture, or loss of light source (with halogen lamp as light source), the light source needs to be replaced; or the sensitivity of the detection element needs to be adjusted;
⑤ Noise occurs when the instrument is working.
The above faults can be checked by yourself, and some need to be repaired by engineers