Publish Time: 2022-03-16 Origin: Site
The genome of an organism is stored inside the DNA molecule, but analyzing this genetic information requires large amounts of DNA. In 1985, Kary Mullis invented an efficient method for massively replicating small amounts of DNA in a short period of time. By heating, the two strands of the DNA molecule are separated, and the added DNA building blocks bind to each strand. With the help of the enzyme DNA polymerase, new DNA strands can be formed and the process can then be repeated. PCR is of great importance in both medical research and forensic science.
The full name of PCR is the polymerase chain reaction. It is a very powerful technology that can replicate DNA in a very simple but efficient way. It can be regarded as a special DNA replication outside the organism. The biggest feature of PCR It can greatly increase the trace amount of DNA.
DNA replication is the basis of a lot of research, for example, when we sequence RNA, the RNA must first be converted to DNA and replicated in large numbers. When sequencing the genome of a person, we cannot sequence a cell or a single molecule. The basis of sequencing requires having a large number of identical copies of DNA molecules, therefore, DNA replication is a fundamental tool in biological research. So how do you get so many copies? PCR is like a copier, taking advantage of several properties of DNA to become an excellent method for mass replication of DNA.
1. Initial step: This is necessary for hot-start PCR, heating the reaction mixture to 94-98 degrees to activate the DNA polymerase. The timing of this step depends on the DNA polymerase chosen.
2. Denaturation step: DNA itself is a double-stranded structure, and DNA amplification requires the combination of primers and single-stranded DNA templates. In this step, the reaction mixture is heated to 94-98 degrees for 20-30 seconds to break the hydrogen bonds between the double strands and release the single-stranded DNA. This is the first step in the PCR cycle.
3. Annealing step: After denaturation, the DNA template exists in the reaction mixture as a single strand. Because the primers in the reaction system are complementary to the DNA template, when the reaction temperature drops to 50-65 degrees, the primers form hydrogen bonds with the complementary sequences. The annealing temperature mainly depends on the Tm value of the primer, which is usually 3-5 degrees lower than the Tm value of the primer. This step usually lasts for 20-40 seconds, and the polymerase will bind to the primer-template duplex to start DNA synthesis.
4. Extension step: In this step, DNA polymerase starts DNA synthesis, so the temperature of this step is the optimal temperature of DNA polymerase. Usually we choose 72 degrees, but the optimum temperature for some DNA polymerases is 68 degrees. This step is very similar to DNA replication in vivo: DNA polymerase adds dNTPs to the 3' end of the primer according to the law of base complementation, and finally a new DNA double-stranded fragment is obtained. The extension time depends on the length of the DNA fragment of interest and the synthesis capacity of the DNA polymerase. In general, DNA polymerases can synthesize 1 kb DNA every 60 seconds.
5. Steps 2 to 4 are called a cycle: the amount of DNA after each cycle is double that of the previous one. Typically a PCR reaction is performed for 30-35 cycles. During the first few rounds of PCR cycle, the amount of PCR product increased exponentially, but in the later stage of the reaction, with the reduction of the amount of dNTPs and primers and the inactivation of DNA polymerase, the reaction rate gradually decreased, so the yield of PCR reaction also decreased. restricted.
6. The final extension step: After 30-35 cycles, we usually extend at 68-74 degrees for 5-10 minutes, which is to make all the remaining single-stranded DNA in the reaction system complete the reaction.
7. Maintenance time: Usually we set 4-10 degrees as the storage temperature of PCR products after the reaction.