Polymerase chain reaction (PCR) is a molecular biological technique used to amplify and amplify specific DNA fragments, which can be regarded as special DNA replication outside the organism.

1.PCR

PCR (Polymerase Chain Reaction) refers to catalyzed by DNA polymerase, using parent strand DNA as the template and specific primers as the starting point for extension. dNTP, Mg2+, extension factors, and amplification enhancement factors are added to the system, and through steps such as denaturation, annealing, and extension, , the process of replicating daughter strand DNA complementary to the parent strand template DNA in vitro, can quickly and specifically amplify any target DNA in vitro.

2. Hot start PCR

When the reaction system is prepared, in the initial heating stage or "hot start" stage of the reaction, the enzyme modifier is released at high temperature (usually higher than 90 ℃), resulting in the activation of DNA polymerase. The specific activation time and temperature depend on the properties of DNA polymerase and hot start modifier.

3. Reverse transcription PCR

Reverse Transcription PCR，is an experimental technique for reverse transcribing from mRNA to cDNA and using it as a template for amplification. The experimental procedure is to extract the total RNA from tissues or cells, use Oligo (dT) as primers, synthesize cDNA by reverse transcriptase, and then PCR with cDNA as template to obtain the target gene or detect gene expression.

4.Fluorescence quantitative PCR

Real-time Quantitative PCR (RT-qPCR) is a method that adds fluorescent groups to the PCR reaction system, uses fluorescence signal accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzes the template through the standard curve. Commonly used qPCR methods include fluorescent dye method (SYBRGreenI) and probe method (TaqMan).

A. Principle of dye method

B.Principle of probe method

5.Nested PCR

Two sets of PCR primers were used for two rounds of PCR amplification, and the second round of amplification products were the target gene fragments. If the mismatch of the first pair of primers (outer primers) leads to the amplification of the non-specific product, the possibility of the same non-specific region being recognized and further amplified by the second pair of primers is very small, so the specificity of PCR has been improved by the second pair of primers.

6. Touchdown PCR

A method to improve the specificity of PCR reaction by adjusting PCR cycle parameters. In the landing PCR, the annealing temperature of the first few cycles is set to several degrees higher than the maximum annealing temperature (Tm) of the primers. Higher annealing temperature can effectively reduce non-specific amplification, but at the same time, higher annealing temperature will aggravate the separation of primers from the target sequence, resulting in a decrease in PCR yield.

7. Direct PCR

Direct PCR amplifies the target DNA directly from the sample without the need for nucleic acid separation and purification. Direct PCR is divided into two categories, direct method: a small number of samples are directly added to PCRMasterMix for PCR identification; cleavage method: after sampling, add to the lysate, release the genome, take a small amount of cleavage supernatant added to PCRMasterMix for PCR identification.

8.SOE PCR

Gene splicing by overlap extension PCR（SOE PCR）is a technology that uses primers with complementary ends to form overlapping strands in the PCR product, so that in the subsequent amplification reaction, the amplified fragments from different sources can be overlapping and spliced together through the extension of the overlapping strands. This technology currently has two main application directions: constructing fusion genes; and gene site-directed mutation.

A. Principle of constructing fusion genes

B. Principle of gene site-directed mutagenesis