Enzymes are very important biocatalysts with high catalytic efficiency and reaction specificity. Most biochemical reactions require the participation of enzymes, and nucleic acid detection is no exception.
In the process of COVID-19 nucleic acid detection, different types of molecular enzymes play important roles in different experimental stages of nucleic acid detection (nucleic acid extraction & RT-qPCR).
Here, we sorted out the core enzyme raw materials used in the nucleic acid detection process according to the different experimental links in the nucleic acid detection.
Core enzymes in nucleic acid extraction
The process of COVID-19 nucleic acid extraction mainly includes two steps: Lysis is the process of destroying the cell structure of the sample, so that the nucleic acid in the sample can be released. Purification is to completely separate the nucleic acid from other components in the lysis system, such as protein, salt and other impurities. The reaction process requires the participation of Proteinase K, DNase I and RNasin (RNase inhibitor).
Proteinase K
Proteinase K is a serine protease with wide cleavage activity, which can cleave the carboxyl terminal peptide bond of aliphatic and aromatic amino acids. In the process of nucleic acid extraction, proteinase K can degrade histone closely bound with nucleic acid, promote the separation of nucleic acid, and make the nucleic acid better extracted. In addition, proteinase K can degrade the activity of RNA hydrolase (RNase) and inhibit the hydrolysis of template RNA by RNase.
Our Mutant Proteinase K has higher specific activity and is more stable at room temperature compared with wild-type Proteinase K. It is a non-specific serine proteinase with broad substrates. It is active over the pH range from 4 to 12 and can be used in any situation to digest native and denatured proteins.
The application of this Mutant Proteinase K is similar to wild-type Proteinase K. But this mutant one has higher specific activity and is more stable at room temperature. It can be used in any situation to digest native and denatured proteins. For instance, it is used for isolating mRNA or genomic DNA from different tissues and modifying glycoprotein for structure studies. Mutant Proteinase K is active with SDS, urea, and EDTA and active between 15°C and 75°C.
Our Mutant Proteinase K is included in New Products, Science Journal, March 8, 2019.
Mutant Proteinase K latest list price: $400 for 10g
DNase I
DNase I (RNase Free) is an endonuclease that decomposes single- or double-stranded DNA to produce 5'-P-terminal oligonucleotides. As the protease is almost completely removed, the stability of DNase I in pH neutral region is improved. In addition, RNase inhibitor is added to DNase I to inhibit the residual RNase in RNA samples, so it can effectively inhibit RNase degradation of RNA during the extraction.
Generally, DNase I can be inactivated by heating. If it is necessary to remove the residual denatured protein, the RNA sample can be extracted and precipitated with phenol-chloroform after digestion at 37°C (without heating).
DNase I (RNase Free) latest list price 2022: $350/g
RNasin (RNase inhibitor)
RNasin (RNase inhibitor) is a ribonuclease inhibitor extracted from human placenta with a molecular weight 51 kDa. It inhibits the activity of RNase by specifically binding up to RNase with a non-covalent bond. RNasin, free of RNase or Nickase, can maintain its activity at pH from 5 to 8, with the highest activity at pH 7.8. The concentration of RNasin is 20~40 units/µl.
RNasin (RNase inhibitor) can be used in almost any application where RNase contamination may exist. Our high-quality RNasin can inhibit RNase more extensively than many traditional RNase inhibitors, so it has become a better choice of RNase inhibitor at present, which can provide a higher level of protection against RNA degradation.
RNasin (RNase inhibitor) latest list price 2022: $1,000 for 100KU
Core enzymes in RT-qPCR
After the nucleic acid extraction of the sample is completed, the nucleic acid detection can be completed by RT-qPCR. In these experiments, DNA Polymerase, Reverse Transcriptase and Uracil DNA Glycosylase are essential core enzyme materials.
Reverse Transcriptase
After extracting purified RNA, dNTP polymerization catalyzed by reverse transcriptase is required before qPCR reaction to generate cDNA sequence complementary to template RNA. For RT-qPCR reaction, the reverse transcriptase that can withstand high temperature should be selected. At present, the most widely used reverse transcriptase is M-MLV reverse transcriptase. Because it lacks DNA endonuclease activity and has low RNase H activity, it has more advantages in cDNA cloning application.
Thermo-stable M-MLV Reverse Transcriptase (RNase H-) is able to synthesize the complementary DNA strand from a single-strand RNA template. M-MLV Reverse Transcriptase (RNase H-) is a mutant type of M-MLV Reverse Transcriptase, obtained by eliminating the active center of RNase H through multiple point mutations. The alteration decreases the activity of RNase H and reduces RNA degradation in reverse transcription, which increases the yield of first-strand cDNA to get full-length cDNA more easily. In addition, the thermal stability of the reverse transcriptase is improved and the optimal reaction temperature is therefore 50°C.
Ultra Thermo-stable M-MLV Reverse Transcriptase (RNase H-) is a mutant type of M-MLV Reverse Transcriptase, obtained by eliminating the active center of RNase H through multiple point mutations. The alteration decreases the activity of RNase H and reduces RNA degradation in reverse transcription, which increases the yield of first-strand cDNA and gets full-length cDNA more easily. In addition, the thermal stability of the reverse transcriptase is improved and the enzyme is active up to 65°C. Compared with RT-PCR at low temperature, RT-PCR at a high temperature can significantly open the secondary structure of RNA, promote the amplification performance of complex RNA templates, increase the length and yield of cDNA, and improve the sensitivity of subsequent detection. This product has a high concentration (200U/μl) and is glycerol-free, which can be used to establish a freeze-drying system.
Thermo-stable M-MLV Reverse Transcriptase (RNase H-) latest list price 2022: $1,500/MU
Ultra Thermo-stable M-MLV Reverse Transcriptase (RNase H-) latest list price 2022: $300 for 20KU
DNA Polymerase
After the reverse transcription process is completed to generate double stranded cDNA, the DNA polymerase in the PCR reaction is then required. The DNA strand is extended by polymerizing free deoxyribonucleotides (dNTPs), and a large amount of template DNA is amplified in vitro to achieve the purpose of nucleic acid detection.
The commonly used polymerase is Taq DNA Polymerase or U-Taq DNA Polymerase. U-Taq DNA Polymerase consists of a single polypeptide with a molecular weight of 94 kDa. It has a 5'→3' DNA polymerase activity and lacks 3'→5' exonuclease activity. Its extension rate is 2~4 kb/min in standard conditions. The appropriate reaction temperature is 70~75℃, the work concentration of dNTPs is 100~300 μM, the work concentration of Mg2+ is 2~3 mM, and the suitable pH is 8.1~9.1. The enzyme generates PCR products with 3'-dA overhangs, suitable for T-A cloning. The amount of enzyme is 1~1.5 unit for 20μl PCR reaction, while 2~3 unit for 50μl PCR reaction. 6 kb Lambda DNA and 2.1 kb Human genomic DNA can be amplified very well at our laboratory.
HM-Taq DNA Polymerase adopts the latest synthetic affinity ligand technology, which can reversibly block the activity of the Taq DNA Polymerase in a temperature-dependent manner. The difference between the HM-Taq DNA Polymerase and the general hot start Taq DNA Polymerase is that the general hot start Taq DNA Polymerase only blocks the polymerase activity before the temperature rise in the first step, while the HM-Taq DNA Polymerase uses the inhibitory ligand to block the substrate-binding site of the HM-Taq DNA Polymerase through temperature regulation. When the temperature is lower than 40°C, an inactive enzyme-inhibitor complex is formed. When the temperature rises to the annealing temperature, the binding balance moves towards the formation of the template-primer complex, so the production of non-specific amplification products in the whole process of PCR amplification is minimized and the accuracy of PCR reaction is greatly improved. The 3' end of PCR product is A, which can be cloned directly with the TA vector.
HS-Taq DNA Polymerase (also known as HotStart-Taq DNA Polymerase) contains engineered Taq polymerase and Taq monoclonal antibody, thereby preventing DNA synthesis at room temperature. During the initial DNA denaturation step, the antibody is denatured, releasing the polymerase and allowing DNA synthesis to proceed. HS-Taq DNA Polymerase prevents nonspecific amplification due to mispriming and/or the formation of primer-dimers during PCR assembly.
U-Taq DNA Polymerase latest list price 2022: $1,200 for 200KU
HM-Taq DNA Polymerase latest list price 2022: $500 for 5KU
HS-Taq DNA Polymerase latest list price 2022: $500 for 5KU
Uracil DNA Glycosylase
In the process of COVID-19 nucleic acid detection, aerosol pollution in the operating environment is the most common factor causing false positive PCR results. Adding UDG enzyme (uracil DNA glycosylase) to the amplification system can effectively eliminate the amplification residual contamination (mostly in the form of aerosol) mixed in the PCR system and ensure the accuracy of the amplification results.
Heat-Labile Uracil DNA Glycosylase can effectively hydrolyze uracil-glycosidic bonds in single- or double-stranded DNA, excising uracil and creating alkali-sensitive abasic sites in the DNA, which can be hydrolyzed by endonuclease, heat, or alkali treatment. The enzyme has no activity to RNA and is mainly used to prevent contamination in the PCR reaction system.
The uracil DNA glycosylase from E.coli is relatively heat-resistant, and a small amount of uracil DNA glycosylase activity remains after being treated at 95°C for 10 min, which leads to the degradation of PCR products containing dU base. While the HL-UDG from psychrophilic marine bacteria is completely inactivated at 55°C for 5 min.
Before PCR amplification, HL-UDG is added to the PCR reaction system, and the contamination in the system could be eliminated at 25°C for 10 min. HL-UDG will then be inactivated in the first step of denaturation (at 94°C) of the PCR cycle, preventing the degradation of the synthesized PCR products containing dU.
Heat-Labile Uracil DNA Glycosylase latest list price 2022: $500 for 500U