| Name | TAQ DNA POLYMERASE 5U|UL |
| Use | The application of PCR technology has been continuously developed. In recent years, real-time PCR(real-time PCR) technology has achieved a leap from qualitative to quantitative PCR, with strong specificity, high sensitivity, automation, good repeatability, quantitative accuracy, closed reaction has become an important technical platform for molecular biology and molecular diagnosis, and is widely used in the detection and diagnosis of genetic diseases, pathogens and tumors. It is widely used in gene quantification, genotyping and SNPs detection. When used for DNA polymorphism genotyping, it is often necessary to apply to the probe. At present, various labeled probe technologies based on real-time PCR mainly include Taq Man probe, FRET probe, molecular beacon probe and novel fluorescent double-stranded displacement probe. Among these probes, the fluorescent double-stranded displacement probe has a very good ability to recognize single base mutations, and its specificity can be achieved by adjusting the length of the probe and the base number of the difference between the two strands, it has strong specificity and sensitivity, and is suitable for genotyping. However, sometimes the fluorescence signal of the probe is detected at the beginning of the PCR cycle, and the S-type signal is not seen to rise, which affects the interpretation of the result. Considering that the fluorescent group labeled at the 5 'end of the probe was cleaved by Taq DNA polymerase, we tried to solve the above problems with Taq DNA polymerase that eliminated the 5' → 3 'exonuclease activity. |
polymerase (Polymerase), also known as DNA polymerase. A collective term for a class of enzymes that biocatalytically synthesize deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polymerase is the most known enzyme. For all organic organisms, high-fidelity replication of the genome by polymerase is the key to maintaining genome integrity and stable inheritance. The polymerase is synthesized along the template in the direction of 5 '-3. Through evolutionary biology and protein sequence alignment, polymerase can be divided into six families, namely A, B, C, D and families X and Y. Family A DNA polymerases include such as E. coli polymerase I,T7 DNA polymerase, and Tag DNA polymerase, which is widely used in PCR. B family DNA polymerase includes E. coli DNA polymerization II, and a series of DNA polymerases including human DNA polymerase. C family polymerase includes the alpha subunit of polymerase III holoenzyme. D family DNA polymerase is mainly some DNA polymerases in archaea, such as DNA polymerase D. X family DNA polymerase includes many enzymes. The last Y family polymerase is different in prokaryotes, such as Escherichia coli and archaea. In eukaryotes, there are multiple polymerases.
So far, the protein structure of many high-fidelity DNA polymerases has been successfully resolved. They all have similar tertiary structures and protein folding. Since the domain of the entire DNA polymerase is composed of a right-hand shape, each domain is also named thumb domain, index finger domain, palm domain, and additional exonuclease with error correction function The domain has the activity of 3 '- 5' exonuclease. Among all DNA polymerase molecular structures, the catalytic center located in the palm domain is quite conservative, usually 3 acidic amino acids and 2 metal ions, usually magnesium ions). At the same time, the new deoxyribonucleotide also binds to this site.
Under normal circumstances, various DNA damages will be specifically and effectively repaired by various repair mechanisms. But when the damaged DNA is too late to be repaired or newly produced in the replication process before entering the S phase of the cell replication phase, the high-fidelity DNA polymerase cannot replicate through, which will eventually cause the termination of the replication fork. The termination of the replication fork for a long time will eventually lead to the collapse of the entire replication machine, which in turn induces the initiation of the cell apoptosis program, resulting in the final death of the cell. In order to effectively solve this problem, cell evolution has produced a mechanism of cross-damage synthesis.
Human DNA polymerase & eta;, first discovered from yeast, has a very high ability to replicate through. Further research shows that this is a replication process without error tendency. Therefore, DNA polymerase & eta;, is associated with UV-induced pyrimidine dimer replication. Under UV-induced conditions, knockout of DNA polymerase gene in yeast can increase the mutation rate of yeast genome. However, after the deletion of DNA polymerase & eta; gene, it did not show a strong phenotype for ultraviolet. The reason is that there are many other CPDs repair pathways in yeast cells. Unlike other XP patients who lack the key enzyme of NER repair pathway, XPV patients have a complete NER pathway, but they cannot effectively replicate CPDs through UV crosslinking products. XPV patients are super sensitive to ultraviolet radiation and have a probability of getting skin cancer hundreds of times higher than ordinary people. The patient's symptoms include erythema, brown spots and patches on the face and other exposed parts, accompanied by capillary dilation, and depigmentation spots. Patients cannot go out normally during the day and need all-round protection. A large number of studies have studied human DNA polymerase from different angles and found that it participates in many important physiological activities.
[1] Guo Jia. Structural modification and characterization of Taq DNA polymerase [D]. Xiamen University, 2014.
[2] Zhao Ye. Human DNA Polymerase & eta; Study on Structure and Catalytic Mechanism [D]. Zhejiang University, 2012.
Zhang Min, Cai Junpeng. Effects of Different DNA Polymerases on PCR-DGGE Technology [J]. Anhui Agricultural Sciences, 2011,(14):8231-8233.
[4] Lin Qing. Modification and Application of Taq DNA Polymerase [D]. Xiamen university, 2007.
| Overview | Polymerase, also known as DNA Polymerase. A class of enzymes that catalyze the synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The polymerase is one of the most widely known enzymes. For all organic organisms, high-fidelity replication of the genome by polymerase is the key to maintaining genomic integrity and stable inheritance. The polymerase is synthesized in a 5 '-3' direction along the template. Through Evolutionary Biology and protein sequence alignment, the polymerase can be divided into six families, namely A, B, C, D and family X, Y. Family A DNA polymerases include, for example, E. Coli polymerase I,T7 DNA polymerase, and Tag DNA polymerase, which are widely used in PCR. The B- family DNA polymerases include a range of DNA polymerases including E. Coli DNA polymerization II, and human DNA polymerases. The C- family polymerase includes the alpha subunit of the polymerase III holoenzyme. D family DNA polymerases are mainly some DNA polymerases in archaea, such as DNA polymerase D. Family X DNA polymerases include many enzymes. The final Y-family polymerase, in prokaryotes, such as E. Coli and archaea, are different. In eukaryotes, a variety of polymerases are present. |
| Introduction | to date, the protein structures of many high-fidelity DNA polymerases have been successfully resolved, they all have similar tertiary structures as well as protein folding. Since the domains of the entire DNA polymerase are composed to resemble the shape of the right hand, each domain is also named the thumb domain, the index finger domain, and the palm domain, respectively, and an additional exonuclease domain having an error correction function, which has the activity of a 3 '- 5' exonuclease. Among the molecular structures of all DNA polymerases, the catalytic center located in the palm domain is fairly conserved, usually three acidic amino acids are conjugated with two metal ions, usually magnesium ions). At the same time, the new deoxyribonucleotides, also bind to the site. |
| Picture | Tag DNA polymerase 3D structure and human DNA polymerase beta active center. |
| efficacy | under normal circumstances, a variety of different DNA damage will be a variety of different repair mechanisms specific, effective repair. However, when the damaged DNA has no time to be repaired or is newly produced in the replication process before entering the S phase of the cell replication phase, the high-fidelity DNA polymerase cannot replicate through, and will eventually cause the termination of the replication fork. The termination of the replication fork for a long time will eventually lead to the collapse of the whole replication machine, and then induce the initiation of the cell apoptosis program, resulting in the final death of the cell. In order to effectively solve this problem, cell evolution has produced a mechanism of cross-damage synthesis. |
| disease-related | Human DNA Polymerase η, first found in yeast, has an extremely high ability to replicate through. Further studies have shown that this is an error-free replication process. Thus, DNA polymerase N, is associated with UV-induced pyrimidine dimer replication. Under UV-induced conditions, knockout of DNA polymerase gene in yeast can increase the mutation rate of yeast genome. However, after the deletion of the DNA polymerase N gene, it does not show a strong phenotype for UV, which is due to the existence of other CPDs repair pathways in yeast cells. Unlike other XP patients due to missing key enzymes in the NER repair pathway, XPV patients have an intact NER pathway but are unable to efficiently replicate the product CPDs via UV cross-linking. XPV patients are super sensitive to UV radiation, with hundreds of times more chance of getting skin cancer than normal people. The patient's symptoms included erythema, brown spots and patchy spots on the face and other exposed areas, with telangiectasia, and depigmented spots. The patient was unable to go out normally during the day and required full protection. A large number of studies have studied human DNA polymerase from different angles and found that it is involved in many important physiological activities. |
| the application of | PCR technology has been continuously developed. In recent years, real-time PCR(real-time PCR) technology has achieved a leap from qualitative to quantitative PCR, with strong specificity, high sensitivity, automation, good repeatability, quantitative accuracy, closed reaction has become an important technical platform for molecular biology and molecular diagnosis, and is widely used in the detection and diagnosis of genetic diseases, pathogens and tumors. It is widely used in gene quantification, genotyping and SNPs detection. When used for DNA polymorphism genotyping, it is often necessary to apply to the probe. At present, various labeled probe technologies based on real-time PCR mainly include Taq Man probe, FRET probe, molecular beacon probe and novel fluorescent double-stranded displacement probe. Among these probes, the fluorescent double-stranded displacement probe has a very good ability to recognize single base mutations, and its specificity can be achieved by adjusting the length of the probe and the base number of the difference between the two strands, it has strong specificity and sensitivity, and is suitable for genotyping. However, sometimes the fluorescence signal of the probe is detected at the beginning of the PCR cycle, and the S-type signal is not seen to rise, which affects the interpretation of the result. Considering that the fluorescent group labeled at the 5 'end of the probe was cleaved by Taq DNA polymerase, we tried to solve the above problems with Taq DNA polymerase that eliminated the 5' → 3 'exonuclease activity. |
| references | [1] Guo Jia. Structural modification and characterization of Taq DNA polymerase [J]. Xiamen University, 2014. [2] Zhao Ye. Study on the structure and catalytic mechanism of human DNA polymerase [D]. Zhejiang University, 2012. [3] Zhang Min, Cai JunPeng. Effect of different DNA polymerases on PCR-DGGE technology [J]. Journal of Anhui Agricultural Sciences, 2011,(14):8231-8233. [4] Lin Qing. Modification and application of Taq DNA polymerase [D]. Xiamen University, 2007. |