Introduction of PCR technology:
PCR is also known as a polymerase chain reaction. It's a technique used to amplify specific DNA fragments so that the number of NDA fragments increases exponentially. Before PCR, nucleic acid samples were often used with great care when studying DNA, because until PCR was developed for in vitro amplification, each sample of DNA was precious and could only be used sparingly. The advent of PCR technology broke this limitation. The principle is similar to DNA replication in vivo, which can be regarded as special DNA replication outside the organism. Wherever we can isolate a little bit of DNA, we can amplify it with PCR technology.
The PCR process can be roughly divided into three steps: denaturation, annealing and elongation.
1. Denaturation (90℃-96℃): Under the action of heat, the hydrogen bond of the double-stranded DNA template breaks, forming single-stranded DNA.
2. Annealing: (60℃-65℃): The system temperature decreases, and the primer binds to the DNA template to form a local duplex.
3. Extension: (70℃-75℃): Under the action of Taq enzyme (around 72℃, the best activity), the DNA strand complementary to the template was synthesized by using dNTP as raw material, starting from the 3 'end of the primer and extending in the direction from 5' to 3 'end.
This is the normal process of a conventional PCR. So we need to test the amplified DNA after the amplification is done, and the conventional PCR technique is usually only done by gel electrophoresis. Electrophoresis is a technique that separates, identifies, and purifies DNA according to its molecular weight. With the help of gel electrophoresis, we can judge whether the size of the amplified DNA fragment is consistent with the size of the target band, so as to know whether the desired product is obtained. To determine whether the amplification has specificity by looking at whether there is a hybrid band; See if there is a primer dimer to judge the design of the primer and so on.
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