What Is The Function Of Taq Polymerase – DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the DNA template in living cells. Multiple enzymes from each organism were identified, and the shared functions of these enzymes were investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other purposes. The basic ability of DNA polymers to synthesize a deoxyribonucleotide chain is preserved. However, more specific properties, including processivity, fidelity (accuracy of synthesis), and substrate nucleotide selectivity differ among the enzymes. The specific properties of each DNA polymerase may lead to the potential development of unique reactions, so the search for novel DNA polymerases is one of the main focuses in this research area. In addition, protein engineering techniques to create mutant or artificial DNA polymers are successfully developing potent DNA polymers, suitable for specific purposes among the many types of DNA manipulations. Thermocoupled DNA polymers are of particular importance for PCR-related techniques in molecular biology. In this review, we summarize the history of research into the development of thermostatic DNA polymers as reagents for genetic manipulation and discuss the future of this research area.

DNA polymerase I from Thermus aquaticus (Taq polymerase) is the best-known representative enzyme among the thermostable DNA polymerases. Taq polymerase was identified from T. aquaticus isolated from Yellowstone National Park in Montana, USA. The report was published by Chien et al. (1976) as a study for his Masters course. At that time, no one predicted how famous the enzyme would be later. In 1985, PCR (polymerase chain reaction) technology was reported using the Klenow fragment of DNA polymerase I from Escherichia coli (Saiki et al., 1985). It was easy to imagine that heat-stable DNA polymerases that are not inactivated at the denaturation step from double-stranded DNA to single-stranded DNA would transform this method of gene amplification into a practical technology. Subsequently, a simple and robust PCR method using Taq polymerase was published (Saiki et al., 1988). Due to the heat stability of Taq polymerase, the reaction tube could remain in the incubator after the preparation of the reaction mixture containing the DNA polymerase, and only temperature changes were required for PCR. An instrument capable of rapid reaction temperature change was developed, and the PCR market opened with a PCR kit (GeneAmp PCR Reagent Kit) and an instrument (Thermal Cycler) provided by Perkin-Elmer Cetus. DNA polymerase from Thermus thermophilus (Tth polymerase) was also developed as a commercial product early in the PCR age, but a scientific report was only an abstract of ASBMB in 1974 from the Mitsubishi-Kasei Institute of Life Sciences, Japan, where this was located. enzyme was first identified. A distinct property of Tth polymerase is that it has specific reverse transcription (RT) activity, and a single-tube RT-PCR method was developed with this enzyme.

What Is The Function Of Taq Polymerase

At the dawn of the PCR era, Taq polymerase was purified from T. aquaticus cells. However, the pol gene was soon cloned from the T. aquaticus genome and expressed in E. coli cells. The recombinant Taq polymerase, named AmpliTaq DNA polymerase, replaced the native Taq polymerase in the commercial field. The amount of recombinant Taq polymerase produced in E. coli cells was very low, probably due to the low expression of the T. aquaticus gene, which has a high GC content (70%), although the protein quality was improved, in comparison. to the native Taq polymerase (Lawyer et al., 1989). We succeeded in constructing an efficient overproduction system by changing the codons around the N-terminal region from the original gene to the AT type at the third letter or the optimal codons for E. coli. These manipulations enhanced Taq polymerase production more than 10-fold, compared to AmpliTaq production (Ishino et al., 1994). Taq polymerase has been used as the standard enzyme for PCR since its inception. A wealth of PCR data has been obtained using accumulated Taq polymerase, providing a valuable resource for the development of new products for useful PCR modifications.

Polymerization And Editing Modes Of A High Fidelity Dna Polymerase Are Linked By A Well Defined Path

Thermophilic organisms use thermophilic DNA polymers, and therefore, thermophiles have become increasingly popular as genetic resources of DNA polymers and other enzymes for industrial use. The heat stability of enzymes is directly related to the temperature at which the organism thrives. Thermophiles are classified into extreme thermophiles, which grow at temperatures higher than 75°C, and moderate thermophiles, which grow at 55–75°C. It is clear that the thermostabilities differ between the DNA polymers from large thermophiles and moderate thermophiles as shown in Figure 1. Taq polymerase is applicable to PCR; however, the DNA polymers from the moderately thermophilic Bacillus species are not suitable for PCR, due to their insufficient stability. Hypothermophiles are particular extreme thermophiles that grow optimally at temperatures above 80°C. Most of the hyperthermophilic organisms are Archaea, although some are bacteria, as shown in (Table 1). In general, hypothermophiles have the potential to provide more heat-stable enzymes than normal thermophiles. In fact, the DNA polymerase from Pyrococcus furiosus (Pfu polymerase) is more stable than Taq polymerase (Figure 1). Hypothermophilic archaea have become popular not only as useful sources of enzymes for application, but also as interesting model organisms for molecular biology. In the early 1990s, the metabolic phenomena in archaeal cells were barely understood, and thus, the molecular biology of Archaea, the third domain of life, became a brand new and exciting field.

FIGURE 1. Heat resistance of DNA polymers. Residual DNA polymerase activities after incubation at the indicated temperature for 30 minutes were plotted. DNA polymerases from Pyrococcus furiosus (open circles), Thermus aquaticus (closed circles), and Bacillus caldotenax (open squares) were used as representatives of hyperthermophiles, large thermophiles, and moderate extremophiles, respectively.

When selecting thermoset DNA polymers as reagents for genetic engineering, research scientists generally do not consider the biology of the source organisms. The properties of the enzyme obtained are important, regardless of the source. For thermostatic DNA polymerases, the growth temperature of the thermophile attracts the most attention. Thermotoga maritima DNA polymerase was the first commercial product (ULTIMA DNA polymerase) from the hyperthermophilic bacteria. This enzyme has an associated 3′–5′ exonuclease activity and is therefore expected to perform a more accurate PCR with its proofreading activity. All PCR enzymes from the domain Bacteria come from family A, whose members usually lack 3′–5′ exonuclease activity, and ULTMA DNA polymerase was an exception, like E. coli Pol I. Despite the selling point this, there was ULTIMA DNA polymerase. it is not a commercial success. One report described no significant differences in the fidelity of ULTIMA and Taq polymerases, when using the optimal buffer conditions for each enzyme, for sequencing purposes (Diaz and Sabino, 1998).

DNA polymerases from the hyperthermophilic archaea were also evaluated as PCR enzymes. We cloned a pol gene from P. furiosus and expressed it in E. coli (Uemori et al., 1993). We thought that this report of ours would be the first report of a full-length sequence of a DNA polymerase of archaeal family B, which was predicted earlier due to an aphidiline-sensitive phenotype of a halophile and a methanogen (Forterre et al., 1984; Zabel et al. al., 1985). However, two papers showing the complete deduced amino acid sequences of DNA polymerases from the hyperthermophilic archaea, Sulfolobus solfataricus (Pisani et al., 1992) and Thermococcus litoralis (Perler et al., 1992) were published during the preparation of our manuscript (Uemori et al., 1993). All these reports clearly showed that the archaeal DNA polymerases have sequences similar to the eukaryotic replicative DNA polymerases, Pol α, δ, and ε (family B). It is also interesting that the T. litoralis pol has intens that must be spliced ​​out after translation (Perler et al., 1992). Subsequently, many instances of DNA polymerases with different patterns of introns, inserted into A, B, and C motifs, were discovered (Perler, 2002). The reaction condition greatly affects the fidelity of DNA synthesis in vitro. However, the archaeal B enzymes generally perform more accurate DNA synthesis compared to Taq polymerase (Cariello et al., 1991; Ling et al., 1991; Lundberg et al., 1991; Mattila et al., 1991), suggesting that the strong 3′–5′ exonuclease activities of the hyperthermophilic family B polymerase in vitro affect PCR fidelity.

Pdf) Development Of A Recombinant Taq Dna Polymerase Enzyme Expressed Using A Synthetic Gene And Its Comparison With A Commercial Enzyme

DNA polymers are classified into seven families based on amino acid sequence similarity (Figure 2). So far, the enzymes used for genetic engineering have only been from the A and B families. Taq polymerase from the A family has strong elongation ability and efficiently amplifies the target DNA. However, their fidelity is low. On the other hand, the Pfu polymerase from the B family performs highly accurate PCR amplification, but its extension rate is slow and a long extension time is required for each cycle of PCR. Therefore, a method for accurate PCR amplification of long DNA regions was needed. One simple idea that researchers have considered trying is to combine one enzyme from family A and one from family B in a single PCR reaction mixture. However, the actual PCR performance was not so simple, and persistent trials were needed to find suitable conditions for long and long development.

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