The purpose of the PCR steps of reverse transcription PCR

Reverse transcription PCR: A widely used variant of polymerase chain reaction.

Denaturation (90-96): The double-stranded DNA template breaks the hydrogen bonds under the action of heat, forming single-stranded DNA

2.Annealing (refolding) (40-65): The system temperature decreases and the primers bind to the DNA template to form a local double strand.

3.Extension (68-75): Under the action of Taq enzyme (optimal activity around 72), DNTP is used as raw material to extend from the 5 ends and 3 ends of the primer to synthesize DNA strands that are complementary to the template.

Each cycle is denatured, annealed, and elongated, and the DNA content is doubled.

Nowadays, some PCR can be changed to a two-step method, i.e., annealing and elongation are performed at the same time between 60 and 65 because the amplification region is very short, and the Taq enzyme activity can be replicated in a very short time, so as to reduce the ramp and increase the reaction speed.

Method. In an ice bath, add the components to a sterile centrifuge tube in the following order.

10×pcr buffer

5 μl dntp mix （2mm）

4 l primer 1 (10 pm).

2 l primer 2 (10 pm).

2 L Taq enzyme (2 U L).

1 L DNA template (50 ng-1 g L) 1 L plus DDH2O to 50 L

Depending on whether the PCR instrument has a hot lid or not, no paraffin oil is added or added.

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Adjust the reaction procedure. Centrifuge the mixture and immediately place it on a PCR machine for amplification. General:

At 93 pre-denaturation for 3-5 min, enter the cyclic amplification stage: 93 40s 58 30s 72 60s, 30-35 cycles, and finally hold warm at 72 for 7 min.

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At the end of the reaction, the PCR product is placed for 4 years to be detected by electrophoresis or -20 years of storage.

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PCR electrophoresis detection: if paraffin oil is added to the reaction tube, 100 L chloroform is used to extract the reaction mixture to remove the paraffin oil; Otherwise, take 5-10 L electrophoresis directly.

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1. The temperature is different, PCR is carried out in vivo, and it has to go through three different high temperatures, while DNA replication in vivo is carried out under mild temperature conditions.

2. Different enzymes, DNA replication requires ordinary DNA polymerase and helicase. The PCR technique requires a heat-stable DNA polymerase and does not require helicase.

3. Different primers: PCR technology pure primers are artificially added DNA single strands, while in vivo DNA replication is a piece of RNA chain synthesized in cells.

If it is, there are many differences with transcription.

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