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What is the role of “enzymes” for various Polymerase chain reaction (PCR)?
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What is the role of various Polymerase chain reaction (PCR) “enzymes”?
Polymerase chain reaction (PCR) is a molecular biology technique used to amplify and amplify specific nucleotide fragments. It can be regarded as the replication of special nucleotides in vitro.
The biggest feature of PCR is that it can The nucleotides of the product are greatly amplified, so as to achieve the degree of easy identification and recognition.
What kinds of enzymes are needed for various PCRs? Look at the central law, the central dogma of molecular biology.
Various polymerase chain reactions in vitro are the perfect application of the central law.
1. DND polymerase-also known as DNA-dependent DNA polymerase
It is a type of enzyme that catalyzes the polymerization of substrate dNTP molecules to form progeny DNA using DNA as a template.
DNA polymerases can be divided into many types according to their polymerase activity characteristics and exonuclease activity characteristics.
The more common enzymes in the IVD field are as follows:
Taq DNA polymerase
Taq DNA polymerase is the first thermostable DNA polymerase discovered, with a molecular weight of 65kD, isolated from Thermus aquaticus, and is currently the most widely used polymerase in scientific research and molecular diagnostic kits.
In order to optimize the PCR reaction system, Taq DNA polymerase has undergone a series of performance enhancements and optimizations, among which the most well-known is the hot-start Taq enzyme.
Hot-start Taq enzyme:
The enzyme has been chemically modified, antibody modified, or ligand modified.
Regardless of the modification, the principle is: before the reaction system is heated to a high temperature, the Taq DNA polymerase activity is “modified” to inhibit, thereby inhibiting non-specific amplification under low temperature conditions.
In addition, a series of mutant Taq DNA polymerases have been screened to meet the requirements of magnesium ion tolerance, salt tolerance, and high fidelity.
Bst strand displacement DNA polymerase
Bst DNA polymerase is derived from DNA Polymerase I of Bacillus stearothermophilus, which has been genetically engineered to remove its 5′-3′ exonuclease activity, but retains 5′-3′ DNA polymerase Activity and strong chain displacement activity.
At the same time, the enzyme has been greatly improved in terms of amplification speed, yield, salt tolerance and thermal stability, and increased dUTP tolerance, which is very suitable for anti-pollution isothermal amplification reactions, such as LAMP.
Due to the impact of the COVID-19, Bst has become another DNA polymerase darling in the IVD field.
In addition to Bst, there are other strand-displacement DNA polymerases with similar functions, such as Bca best polymerase, Klenow polymerase, and phi29 DNA polymerase.
Tth DNA polymerase
Tth DNA polymerase comes from Thermus thermophilus HB8. Its most interesting phenomenon is that under Mg2+ conditions, Tth DNA polymerase has strong DNA polymerase activity.
Under Mn2+ conditions, Tth DNA polymerase has stronger reverse transcription activity. This feature allows Tth DNA polymerase with the corresponding buffer to simultaneously amplify DNA template and RNA template.
2. Reverse transcriptase-also known as RNA-dependent DNA polymerase
The enzyme uses RNA as a template to synthesize a single strand of DNA complementary to the RNA template in the 5′-3′ direction.
This single strand of DNA is called complementary DNA (CDNA). The most commonly used reverse transcriptases are M-MLV and AMV.
3. RNA polymerase-a polymerase with a DNA strand or RNA as a template
Also called transcriptase. The most common is T7 RNA polymerase, with a molecular weight of about 99kDa.
Specially catalyze the process of RNA formation in the 5’→3′ direction. In the current in vitro diagnosis, RNA polymerase will be seen in the SAT technology.
RNA amplification methods such as Rendu and Zhongzhi require the use of RNA polymerase.
4. Proteinase K-an enzyme that can digest all kinds of proteins in the sample
Proteinase K is a powerful proteolytic enzyme isolated from Candida albicans. It has high specific activity and is active in a wide pH range (4~12.5) and high temperature (50~70°C).
Chelating agents such as EDTA or detergents such as SDS cannot inactivate it. Used for isolation and extraction of plasmid or genomic DNA and RNA.
In the detection of viral nucleic acid, proteinase K is one of the important components in the virus sampling solution.
Proteinase K can cleave and inactivate the coat protein of the virus, and at the same time release the viral genome for subsequent nucleic acid extraction.
5. UDG enzyme-efficient control of PCR residual pollution
Uracil DNA glycosylase (UDG enzyme), this enzyme is also called uracil-N-glycosylase or UNG enzyme.
Because PCR is an extremely sensitive amplification technique, it is susceptible to contamination.
A small amount of foreign DNA contamination can be amplified together with the target template.
The Ministry of Health clearly stipulates that all PCR reagents used for clinical testing should have UNG enzyme technology to prevent contamination. Since the decontamination effect of UV irradiation is not good for fragments below 500bp, and the PCR amplified fragments used in clinical detection are usually about 300bp, the preventive effect of UNG has been paid more attention and affirmed.
The working principle of UDG enzyme:
replace dT or dU with dU in PCR products or primers. This dU-ylated PCR product is incubated with UNG, because UDG can cleave the N-glycosyl bond between the uracil base and the sugar phosphate backbone, remove dU and prevent the extension of TaqDNA polymerase, thereby losing the re-amplified ability. UNG has no effect on templates without dU.
UNG can eliminate uracil from single or double-stranded DNA, but has no effect on uracil and single uracil molecules in RNA.
In addition to conventional UDG, heat-sensitive UDG has also been developed. Heat-sensitive uracil-DNA glycosylase (UDG) is a recombinant protein derived from psychrophilic marine bacteria expressed and purified by E. coli, also known as Antarctic heat-sensitive UDG, heat-sensitive UDG is completely inactivated at 50°C for 5 minutes.
Uracil-DNA glycosylase derived from Escherichia coli is relatively heat-resistant, and a small amount of uracil-DNA glycosylase activity will still remain after treatment at 95°C for 10 minutes, leading to degradation of PCR products containing dU bases.
6. Restriction endonucleases-recognize and cut specific deoxynucleotide sequences
Restriction endonucleases are a class of enzymes that can recognize and attach specific deoxynucleotide sequences, and cleave the phosphodiester bond between two deoxyribonucleotides at specific positions in each chain, referred to as Restriction enzymes.
Commonly used enzymes, such as BsoB I, Hinc II, Nt.BstNBI cutting endonuclease.
Among them, the enzyme selected in the strand displacement amplification (SDA) isothermal PCR system of BD Company needs to be specific to the cleavage site and sensitive to chemical modification of the recognition site.
In the COVID-19 epidemic, Abbott’s star product ID NOW isothermal PCR system uses Nt.BstNBI endonuclease.
7. Helicase-makes double-stranded DNA into single-stranded DNA
Use the energy provided by ATP hydrolysis to untie the hydrogen bonds formed by the pairing of double-stranded DNA nucleotides, thereby forming single-stranded DNA;
Commonly used helicases: UvrD of Escherichia coli and gp4 of T7 phage; Escherichia coli helicase II (UvrD), whose melting speed is 20bp/s, has a great restriction on the reaction speed. The helicase gp4 helicase of T7 phage has a faster melting speed, up to 400bp/s, which can greatly increase the reaction speed.
At present, this enzyme is used in the Quidel isothermal PCR system HAD.
Of course, in addition to the enzymes mentioned above, there are various other enzymes that are widely used.
What is the role of “enzymes” for various Polymerase chain reaction (PCR)?
(source:internet, reference only)