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This article mainly shows you the "sample Analysis of PCR Polymerase chain reaction", which is easy to understand and clear. I hope it can help you solve your doubts. Let me lead you to study and learn the article "sample Analysis of PCR Polymerase chain reaction".

Define

Polymerase chain reaction, Polymease Chain Reaction (PCR) is a method of enzymatic synthesis of specific DNA fragments in vitro, which consists of high temperature denaturation, low temperature annealing and temperature extension to form a cycle, so that the target DNA can be amplified rapidly, which has the characteristics of high specificity, high sensitivity, simple operation, time saving and so on. It can be used not only for basic research such as gene isolation, cloning and nucleic acid sequence analysis, but also for disease diagnosis or wherever there is DNA,RNA. Polymerase chain reaction is also called acellular molecular cloning or in vitro primer directed enzyme amplification of specific DNA sequence.

working principle

Similar to the natural replication process of DNA, its specificity depends on oligonucleotide primers that complement both ends of the target sequence. PCR consists of three basic reaction steps: denaturation-annealing (renaturation)-extension: denaturation of ① template DNA: after the template DNA is heated to about 93 ℃ for a certain period of time, the template DNA double strand or the double strand DNA formed by PCR amplification is dissociated to form a single strand, so that it can combine with primers to prepare for the next round of reaction. Annealing (renaturation) of ② template DNA and primers: after the template DNA was denatured into a single strand by heating, the temperature dropped to about 55 ℃, and the primers paired with the complementary sequences of the template DNA single strand. Extension of ③ primers: DNA template-primer conjugate, under the action of TaqDNA polymerase, at about 72 ℃, using dNTP as raw material and target sequence as template, according to the principle of base pairing and semi-reserved replication, a new semi-reserved replication chain complementary to template DNA chain was synthesized by repeated denaturation-annealing-extension three processes, and more "semi-reserved replication chains" could be obtained. And this new chain can serve as a template for the next cycle. It takes 2-4 minutes to complete each cycle, and it takes 2-3 hours to amplify the genes to be amplified millions of times.

Technical principle

Semi-preserved replication of DNA is an important way for biological evolution and passage. Double-stranded DNA can be denatured and unstranded into a single strand under the action of a variety of enzymes, and replicated into the same two molecules in accordance with the principle of base complementary pairing with the participation of DNA polymerase and promoter. In the experiment, it is found that DNA can also be denatured and unstranded at high temperature, and can be renatured into double strands when the temperature decreases. Therefore, the denaturation and renaturation of DNA can be controlled by temperature change, primers are designed as promoters, and specific genes can be replicated in vitro by adding DNA polymerase and dNTP.

However, DNA polymerase will be inactivated at high temperature, so new DNA polymerase has to be added in each cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology. It is found that the thermostable DNA polyenzyme-Taq enzyme is a milestone in the application of PCR. The enzyme can withstand the high temperature of more than 90 ℃ without losing its activity, and does not need to add enzyme in each cycle, which makes the PCR technology very simple and greatly reduces the cost. PCR technology has been widely used and gradually applied in clinic.

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