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The difference and application of four common PCR instruments

Technical Knowledge 2021-08-17 11:11:57
PCR (polymerase chain reaction) is an extremely important tool in molecular biology research. It has been widely used by laboratories all over the world for a variety of experimental applications, such as molecular cloning, gene expression analysis, genotyping, sequencing, and mutation. The four types of PCR instruments commonly used in the laboratory are: ordinary PCR, gradient PCR, in-situ PCR, and fluorescence quantitative PCR. What are the differences between them? The following content will help you answer:

PCR: Polymerase Chain Reaction, which uses DNA to unwind (denature) in vitro at 95°C. At 55°C, primers and single strands are combined (annealing) according to the principle of base complementary pairing, and then the temperature is adjusted to Around 72°C, DNA polymerase synthesizes complementary strands (extends) along the direction from phosphoric acid to five carbon sugars (5'-3').

The PCR instrument is actually a temperature control device, which can perform temperature control well between 95°C, 55°C, and 72°C.

According to the purpose of DNA amplification and detection standards, PCR instruments can be divided into: ordinary PCR instruments, gradient PCR instruments, in-situ PCR, real-time fluorescent quantitative PCR instruments, 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, which is also a traditional PCR machine. If you want to use it for different annealing temperatures, you need to run it multiple times.

Ordinary PCR machines are mainly used in scientific research, teaching, clinical medicine, inspection, quarantine, etc.

②Gradient PCR instrument

One-time PCR amplification can set a series of different annealing temperature conditions (usually 12 temperature gradients) called gradient PCR instrument. 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 level for effective amplification.

Gradient PCR instrument is mainly used to study the amplification of unknown DNA annealing temperature, which saves time and cost. It can also be used as an ordinary PCR without setting a gradient. Gradient PCR instrument is mostly used in scientific research, teaching institutions, inspection, quarantine, etc.

③In-situ PCR machine

PCR is to extract DNA from cells or tissues for gene amplification reaction, while in situ PCR is to maintain the integrity of cells or tissues, make the PCR reaction system penetrate into tissues and cells, and perform genes on the location of the cell's target DNA. Amplification. Not only can the target DNA be detected, but also the type of cell the target DNA exists in, which is more conducive to exploring the relationship between the target DNA and the cell.

The in-situ PCR instrument is mainly used for: (1) Detecting exogenous gene fragments, increasing 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 Law (3) Endogenous gene fragments, such as human monogenic diseases, recombinant genes, translocated chromosomes, Ig mRNA fragments, oncogene fragments, etc. (4) Detection of introduced genes; (5) Detection of genetic diseases such as β-thalassemia.

Real-time fluorescent quantitative PCR instrument

The fluorescent signal acquisition system and computer analysis and processing system are added on the basis of the ordinary PCR instrument design to form a PCR instrument with fluorescence quantitative function. The principle of PCR amplification 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 at the same time for amplification. The amplified result is 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 machines are 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 labels, use multiple channels. A single channel can also detect multi-fluorescence markers and target gene expression products, because only one target gene amplification amount can be detected at a time, and multiple amplifications are required to detect the amount of different target gene fragments. Multi-channels are conducive to multiplex PCR and realize the function of detecting multiple target genes at one time.

Fluorescence quantitative PCR instrument is mainly used in clinical medical testing, biomedicine research and development, food industry, scientific research institutions, etc. Fluorescence quantitative detection technology has many aspects of clinical diagnosis, mainly focusing on the clinical diagnosis of diseases caused by various pathogens, such as hepatitis diseases, venereal diseases, diseases related to prenatal and postnatal care, and tuberculosis.

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