The difference and application of ordinary PCR machine, gradient PCR machine, in situ PCR machine and fluorescence quantitative PCR machine
PCR: that is, the polymerase chain reaction (Polymerase Chain Reaction), which uses DNA to unwind (denature) at 95°C in vitro, and the primers and single strands bind (anneal) according to the principle of base complementary pairing at 55°C, and then adjust the temperature to At around 72°C, DNA polymerase synthesizes complementary strands (extensions) in the direction from phosphate to five-carbon sugar (5′-3′).
The PCR machine is actually a temperature control device, which can control the temperature well between 95°C, 55°C, and 72°C.
According to the purpose of DNA amplification and the standard of detection, PCR machine can be divided into: ordinary PCR machine, gradient PCR machine, in situ PCR, real-time fluorescence quantitative PCR machines, etc.
①Ordinary PCR machine
Generally, a PCR machine that can only run a specific annealing temperature for a PCR amplification is called an ordinary PCR machine, that is, a traditional PCR machine. Multiple runs are required if you want to use it for different annealing temperatures.
Common PCR machine are mainly used in scientific research, teaching, clinical medicine, inspection, quarantine, etc.
②Gradient PCR machine
A series of different annealing temperature conditions (usually 12 temperature gradients) can be set for one-time PCR amplification, which is called a gradient PCR machine. Different DNA fragments have different optimal annealing temperatures. By setting a series of gradient annealing temperatures for amplification, one-time PCR amplification can screen out the most suitable annealing temperature with high expression for effective amplification.
Gradient PCR machine is mainly used to study the amplification of unknown DNA annealing temperature, which saves both time and cost. It can also be used for ordinary PCR without setting gradients. Gradient PCR machines are mostly used in scientific research, teaching institutions, inspection, quarantine and so on.
③In situ PCR machine
PCR is to extract DNA in cells or tissues for gene amplification reaction, while in situ PCR is to maintain the integrity of cells or tissues, so that the PCR reaction system penetrates into tissues and cells, and the gene is carried out at the location of the target DNA of cells. Amplification. Not only can the target DNA be detected, but also what kind of cells the target DNA exists in can be known, which is more conducive to exploring the relationship between the target DNA and cells.
In situ PCR machine is mainly used for: (1) Detecting exogenous gene fragments, improving the detection rate, focusing on the inspection of viral infections, such as HIV, HPV, HBV, CMV, etc.; (2) Observing the distribution of pathogens in the body Rule (3) Endogenous gene fragments, such as human monogenic diseases, recombinant genes, translocated chromosomes, Ig mRNA fragments, oncogene fragments, etc. (4) Detection of imported genes; (5) Detection of genetic diseases such as β-thalassemia.
④ Real-time fluorescence quantitative PCR machine
The fluorescent signal acquisition system and computer analysis and processing system are added on the basis of the common PCR machinedesign, forming a PCR machine with fluorescence quantitative function. The PCR amplification principle is the same as that of ordinary PCR amplification. The primers added during PCR amplification are labeled with isotopes, fluorescein, etc., and the primers and fluorescent probes are used to specifically bind to the template for amplification. The amplified results are connected to the computer analysis and processing system through the real-time acquisition signal of the fluorescence signal acquisition system, and the quantified real-time result output is obtained.
Fluorescence quantitative PCR machine is divided into single channel, dual channel and multi-channel. When only one fluorescent probe is used for labeling, use a single channel; when there are multiple fluorescent probes, use multiple channels. Single-channel can also detect multi-fluorescent markers and target gene expression products, because only the amplification of one target gene can be detected at a time, and multiple amplifications are required to detect the amount of different target gene fragments. Multi-channel facilitates multiplex PCR to achieve the function of detecting multiple target genes at one time.
Fluorescence quantitative PCR machine is mainly used in clinical medical testing, biomedical research and development, food industry, scientific research institutions, etc. Fluorescence quantitative detection technology is widely used in clinical diagnosis, mainly focusing on the clinical diagnosis of diseases caused by various pathogens, such as hepatitis-like diseases, venereal diseases, diseases related to prenatal and postnatal care, and tuberculosis.
Post time: Sep-15-2022