What are the characteristics and applications of the various enzymes of IVD raw materials?
Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, which is regarded as DNA replication outside of the organism, whereby minute amounts of DNA fragments are amplified to such an extent that they can be easily identified and recognized.
For PCR reactions, “enzymes” play a crucial role, and each type of “enzyme” has a different activity and function. So what kinds of enzymes are there? And what are the applications in the IVD field?
DNA polymerase
Definition: It is a type of enzyme that uses DNA as a template, replicates from the 5-terminal to the 3-terminal end of DNA, and catalyzes the polymerization of the substrate dNTP molecules to form daughter DNA.
Main activities and roles: catalyze DNA synthesis. 1, polymerization. 2, 3, → 5, exonuclease activity – proofreading role 3, 5, → 3, exonuclease activity – excision repair role 4, pyrophosphate hydrolysis role 5, pyrophosphate exchange.
Common DNA polymerases in the IVD field include: Taq hot-start enzyme, which uses chemical modification to close the activity of Taq enzyme before the reaction system is heated to 70°C, thereby inhibiting non-specific amplification. High-fidelity DNA polymerase, containing a polymerization center and cleavage center, the polymerization center has a 5′-3′ polymerase activity, responsible for polymerization; cleavage center has a 3′-5′ exonuclease activity (also known as proofreading activity), responsible for the unpaired nucleotides to be excised. (also known as proofreading activity), which is responsible for the excision of unpaired nucleotides.
reverse transcriptase
Definition: An enzyme that uses RNA as a template and synthesizes a single strand of DNA complementary to the RNA template in the 5′-3′ direction.
Major activities and roles: 1. RNA-directed DNA polymerase activity. 2. RNase H activity 3. DNA-directed DNA polymerase activity.
Application in the field of IVD: 1, reverse transcription polymerase chain reaction (RT-PCR) 2, cDNA library construction 3, RNA sequencing 4, RT-qPCR , 2019 New Crown Nucleic Acid Detection configured system reagents, will inevitably join the reverse transcriptase. This is because RNA viruses must be reverse transcribed to DNA in order to perform PCR reactions.
RNA polymerase
Definition: a DNA or RNA chain as a template polymerase, because in the cell with the genetic information of DNA transcription, also known as transcriptase.
Main activities and roles: 1, with DNA as a template; 2, catalyzes the synthesis of nucleic acids through polymerization reactions; 3, causes nucleotides to form 3,→5,-phosphodiester bonds; 4, transcribes the template sequence in the 5′-3′ direction according to the principle of base complementary pairing.
Common RNA polymerases in the IVD field include T7 RNA polymerase, which specifically catalyzes the process of RNA formation in the 5’→3′ direction. Currently in in vitro diagnostics, SAT technology is the application of T7 RNA polymerase for RNA amplification. This technology has been widely used in pathogen detection, blood virus screening, and environmental microbial detection.
Proteinase K
Definition: Proteinase K is a powerful proteolytic enzyme isolated from Candida albicans and is a key reagent for DNA extraction.
Main function: to degrade membrane proteins and DNA-bound proteins, and promote DNA freeing. Application in IVD field: for the isolation and extraction of plasmid, genomic DNA, RNA. In viral nucleic acid detection, proteinase K can cleave and inactivate viral capsid proteins, while releasing the viral genome to complete nucleic acid extraction.
UDG Enzyme
Definition: alias uracil-DNA glycosylase also known as uracil -N- glycosylase or UNGase. It is the chemical structure that forms DNA and RNA monomers and encodes genetic information.
Function: Efficient control of residual PCR contamination. Application in IVD: PCR reagents for clinical tests contain UDG enzyme to prevent contamination.
Endonuclease
Definition: a class of enzymes that recognizes a specific nucleotide sequence and cleaves the phosphodiester bond between two deoxyribonucleotides at a specific site in each chain, referred to as restriction enzymes.
Role: there are 3 types: type 1: both modification and recognition cleavage, the cleavage site is far away from the recognition sequence. type 2: only recognition cleavage, the cleavage site is the recognition sequence. type 3: both modification and recognition cleavage, the cleavage site and the recognition sequence closer.
Applications in IVD field: 1, gene localization and gene isolation, 2, DNA molecular base sequence analysis, 3, genetic engineering editing, 4, the formation of DNA physical map.
Deconjugating enzyme
Definition: use the energy provided by ATP hydrolysis to unwind the hydrogen bonds formed by pairing of double-stranded DNA nucleotides to form single-stranded DNA.
Role: plays a role in catalyzing the deconvolution of DNA or RNA during DNA or RNA replication.
Application in the field of IVD: Isothermal amplification technology, using heat-stabilized deconjugating enzyme instead of the thermal denaturation process in qPCR, to unravel the double strand and extend the amplification. This technique is less prone to non-specific amplification, and also avoids the process of elevating and lowering the temperature in the PCR cycle, requiring only simple equipment for detection.
Modifying Enzyme
Definition: An enzyme that catalyzes the incorporation of rare bases into RNA or DNA, or modifies the original bases to prevent destruction by restriction endonucleases.
Role: Covalent modification of the structure of other enzymes so that they transform into each other between a highly active form and a relatively less active form.
Application in IVD: 5mC/m6A nucleic acid modifying enzyme activity assay HTMR method provides strong evidence for the discovery and activity evaluation of small molecule modulators targeting DNA/RNA methylation.
Pyrophosphatase
DEFINITION: An enzyme that catalyzes the conversion of one molecule of pyrophosphate to two molecules of phosphate ions.
Function: Heat-stable inorganic pyrophosphatase is still 100% active when heated at 100°C for 4h, so it can be used in PCR reactions to lift the inhibition of PCR by the generated inorganic pyrophosphate and enhance DNA replication.
taq antibody
Definition: Antibody against Taq DNA polymerase.
Function: During PCR amplification, Taq Antibody binds to Taq enzyme to inhibit DNA polymerase activity before denaturation at high temperature, and can effectively inhibit non-specific annealing of primers and non-specific amplification caused by primer dimer under low temperature.
Application in IVD: The high containment efficiency of Taq antibody can improve the detection sensitivity of the new coronavirus nucleic acid detection kit and help the new coronavirus detection.
The standard PCR process is divided into three steps: DNA denaturation, annealing, and extension. The inactivation reaction can occur at the beginning of PCR by warming up to 50°C for 10min or 95°C for 2min, and the UDG enzyme eliminates the amplification generated by the contaminating DNA in the reaction system. Subsequently, the template DNA is denatured to single-stranded DNA by high temperature. two primers anneal with two template DNA strands respectively under DNA polymerase and suitable temperature. The primers are again extended under DNA polymerase.Taq Antibody binds to Taq enzyme to inhibit DNA polymerase activity and inhibit non-specific amplification at low temperatures. Addition of inorganic pyrophosphatase to the PCR reaction hydrolyzes pyrophosphate to orthophosphate, increasing the amplification efficiency of the reaction.
Raw material enzyme is the upstream industry chain of molecular testing industry.In 2020, in front of the huge demand for nucleic acid testing, the market demand for molecular diagnostic reagents increased dramatically, and the raw material enzyme industry has been developed rapidly as a result. In terms of production, due to the high technical threshold of molecular testing raw material enzyme production, the current domestic market is mainly dependent on imports. In recent years, along with the rise of the domestic production process, a number of raw material enterprises listed, product categories are also gradually enriched, but it is undeniable that the domestic IVD raw materials industry is still in the early stages of development, the market share is also relatively small, the future is bound to exist in a larger room for improvement.
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| Compound Glucoamylase | 9032-08-0 |
| Pullulanase | 9075-68-7 |
| Xylanase | 37278-89-0 |
| Cellulase | 9012-54-8 |
| Naringinase | 9068-31-9 |
| β-Amylase | 9000-91-3 |
| Glucose oxidase | 9001-37-0 |
| alpha-Amylase | 9000-90-2 |
| Pectinase | 9032-75-1 |
| Peroxidase | 9003-99-0 |
| Lipase | 9001-62-1 |
| Catalase | 9001-05-2 |
| TANNASE | 9025-71-2 |
| Elastase | 39445-21-1 |
| Urease | 9002-13-5 |
| DEXTRANASE | 9025-70-1 |
| L-Lactic dehydrogenase | 9001-60-9 |
| Dehydrogenase malate | 9001-64-3 |
| Cholesterol oxidase | 9028-76-6 |