Simply put, PCR is the use of DNA polymerase for the specific synthesis of a specific gene in vitro or in vitro In Vitro, basically it is the use of DNA polymerase for specific linkage replication. At present, the commonly used technology can copy a gene into the original 10 billion to 100 billion times. According to the purpose of DNA amplification and the standards of detection, the PCR instrument can be divided into four types: ordinary PCR instrument, gradient PCR instrument, in situ PCR instrument, and real-time fluorescence quantitative PCR instrument.
Basic elements
Basic PCR must be available
1. DNA template to be copied
2. Define Primers for the ends of the replication range.
3.DNA polymerase Taq. Polymearse
4. Synthetic raw materials (four deoxynucleotides) and water.
working principle
The DNA is denatured by the temperature rise, and the single strand is double-stranded by the action of the polymerase, thereby achieving the purpose of gene duplication.
Reaction step
They are 1. Denaturation 2. Annealing of primers, 3. Extension of primers. Denaturing is the process of denaturation of DNA (to 90-95 ° C), heating double-stranded DNA into single-stranded DNA as a template for replication. Annealing is to make Primers at a certain temperature (cooling to 55). ~60 ° C) attached to both ends of the template DNA. Finally, the extension of primers and the synthesis of another strand were carried out under the action of DNA polymerase eg Taq-polymerase (heated to 70-75 ° C).
The earliest envisaged nucleic acid research of PCR has been more than 100 years old. In the late 1960s and early 1970s, people devoted themselves to the study of in vitro separation of genes. Korana first proposed the in vitro amplification of nucleic acids in 1971: "DNA denaturation." Hybridization with a suitable primer, extension of the primer with a DNA polymerase, and repeated procedures to clone the tRNA gene".
achieve
In 1985, Mullis et al. of the Human Genetics Laboratory of PE-Cetus in the United States invented the epoch-making polymerase chain reaction. The principle is similar to the in vivo replication of DNA, but the improvement and perfection of the DNA polymerase originally used by Mullis is the Klenow fragment of E. coli DNA polymerase I. The disadvantages are:
1Klenow enzyme is not resistant to high temperatures, will be denatured and deactivated at 90 °C, and will be re-added every time.
2 The primer strand extension reaction was carried out at 37 ° C, and the base mismatch between the template and the primer was prone to occur, the PCR product was poor in specificity, and the synthesized DNA fragment was not uniform. This Klenow-catalyzed PCR technique has many outstanding advantages over traditional gene amplification. However, because the Klenow enzyme is not heat-resistant, the enzyme is passivated when the DNA template is thermally denatured. The ability to complete an amplification reaction cycle adds a lot of difficulty to the PCR technology operating procedure. This has made PCR technology less attractive to the biomedical community for a period of time.
At the beginning of 1988, Keohanog switched to T4 DNA polymerase for PCR. The amplified DNA fragments were uniform and highly authentic, with only one DNA fragment expected. But every cycle, you still need to add new enzymes.
In 1988, a thermostable DNA polymerase was extracted from a thermophilic aquaticus thermomus isolated from a hot spring, such as Saiki. The enzyme has the following characteristics: 1 high temperature resistance, the residual activity after reaction at 70 ° C for 2 h is greater than the original 90%, the residual activity after the reaction at 93 ° C for 2 h is 60%, after reacting at 95 ° C for 2 h The residual activity is 40% of the original. 2 It is not passivated during thermal denaturation, and it is not necessary to add a new enzyme after each amplification reaction. 3 greatly improved the amplification fragment specificity and amplification efficiency, and increased the amplification length of 2.0Kb. The sensitivity is also greatly improved by increasing the specificity and efficiency of amplification. To distinguish it from the E. coli polymerase IKlenow fragment, this enzyme was named Taq DNA polymerase Taq DNA Polymerase. The discovery of this enzyme allows PCR to be widely used.
classification
Ordinary PCR instrument
A PCR instrument that can only run a specific annealing temperature by a PCR amplification is called a conventional PCR instrument, also called a common PCR instrument. If you want to do different annealing temperatures, you need to run multiple times. Mainly to do simple, amplification of the annealing temperature of the target gene. The instrument is mainly used in scientific research, teaching, medical clinical, inspection and quarantine institutions.
Gradient PCR instrument
One-time PCR amplification can be set up with a range of different annealing temperature conditions (temperature gradients), typically with 12 temperature gradients. Such an instrument is called a gradient PCR instrument. Because the different annealing DNA fragments have different annealing temperatures, by setting a series of gradient annealing temperatures for amplification, one-time PCR amplification can select the optimal annealing temperature with high expression level. Perform effective amplification. It is mainly used to study the amplification of unknown DNA annealing temperature, which saves cost and saves time. Mainly used in scientific research, teaching institutions. Gradient PCR instrument can also perform ordinary PCR amplification without setting a gradient. There are not many double-slot gradients, and this feature is available.
In situ PCR
An intracellular gene amplification instrument for localization analysis of target DNA from cells, such as the location of a pathogenic gene at a cell or the position of a target gene in a cell. Is to maintain the integrity of the cell or tissue, so that the PCR reaction system penetrates into the tissues and cells, and the gene is amplified at the position of the target DNA of the cell, which can not only detect the target DNA, but also mark the target sequence in the cell. The location, at the molecular and cellular levels, studies the pathogenesis of the disease and the clinical process and pathological changes have significant practical value.
Real-time PCR
Adding a fluorescent signal acquisition system and a computer analysis processing system based on the ordinary PCR instrument becomes a fluorescence quantitative PCR instrument. The principle of PCR amplification is the same as that of ordinary PCR instrument, except that the primers added during PCR amplification are labeled with isotopes, fluorescein, etc., and primers and fluorescent probes are used to simultaneously bind to the template for specific amplification. The results of the amplification are transmitted to the computer analysis processing system through a real-time acquisition signal connection of the fluorescence signal acquisition system to obtain a quantized real-time result output. Call this PCR instrument a real-time PCR instrument (qPCR instrument)
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