Taq can be isolated either from its original source or from its cloned gene expressed in E. While many similarities in sequence and structure exist between E.
This caveat is explained later. The probe is hydrolyzed concomitant with strand replication so that the accumulating fluorescent signal correlates with amplification.
The error rate has been reported to be as low as 10 -5 for base substitution errors and 10 -6 for frameshift errors. Monovalent and divalent cation dependencies: Salt concentrations required for optimum performance are considered to be 40 mM NaCl and 60 mM KCl. Concentrations greater than mM for these monovalent cations are prohibitive to catalytic activity.
This is in contrast to the salt-insensitivity of the E. In addition, Taq may depend — like other polymerases — upon the presence of divalent cations, namely MgCl 2 or MnCl 2 , with optimum concentrations depending on the experimental design. Higher concentrations of manganese can lead to an increased error rate of nucleoside incorporation. Activity with other divalent ions may be significantly decreased or absent, as is the case with greater than 0.
Taq requires the presence of all four species of deoxyribonucleoside triphosphates and DNA for optimum catalytic activity. In a 25 mM Tris-hydrochloride buffer for example, the alkalinity optimum is 7. On occasion, cloned Taq polymerase has been shown to have contaminating bacterial DNA that is possibly carried over from the expression vector system or other sources used during polymerase manufacture.
This residual contamination may limit the use of cloned Taq in the detection of dilute bacterial DNA in certain samples. Trace contamination may be impossible to completely remove, and indeed certain estimates of contamination counts in commercially available Taq have claimed as many as genome equivalents of bacterial DNA per unit of enzyme. Several methods for removing bacterial DNA from Taq polymerase have been tested and appear in the literature.
Methods such as exposure to ultraviolet light below nm UVB or UVC has the effect of making DNA resistant to amplification; however it also affects the integrity of the Taq polymerase, reducing the efficiency of nucleoside incorporation. The crystal structure of the large fragment of Taq polymerase Klentaq -DNA complex revealed that Glu directly interacts with the template DNA in the closed conformation, but not in the open conformation Li et al. As shown in the right panel of Figure 4 , the residues Glu and Ala magenta are located in the finger subdomain and face to the template DNA blue.
The basic amino acid cluster in the chimeric Taq polymerases is supposed to interact with the template DNA. We focused on this finding, and made a series of mutant polymerases by substitutions at positions and in WT Taq polymerase to change the affinity of the enzymes with DNA. Fourteen mutant recombinant enzymes were purified to homogeneity from E. Thermal stabilities of the mutant Taq polymerases were similar to WT enzyme data not shown.
Figure 3. A multiple alignment of the amino acid sequences of the substituted regions in the chimeric Taq polymerases with higher extension rates. The conserved motifs are shown on the top Loh and Loeb, The distinctive region observed in this alignment is indicated by a red line with two arrowheads. The basic cluster is indicated by a blue line.
Figure 4. The mutational sites of Taq DNA polymerase. The polymerase domain is composed of a right hand with finger green , palm cyan , and thumb red subdomains Eom et al. The thumb and finger subdomains hold DNA backbones are colored orange.
The residues E and A for the mutations are shown in magenta. The site, K and S, where the interesting 9 amino acids were inserted was colored brown. The residues V and V corresponding to the junctions of the substitutions were shown in gray.
The in vitro primer extension rates were compared for these mutant Taq polymerases, as well as WT Taq polymerase. As shown in Figure 5 , all of the mutant Taq polymerases exhibited faster extension reactions compared with that by the WT.
The results of these experiments were quantified. The increased extension rate is generally related to the number of positive charges at this site Table 4. The basic residues gave the varied effects. The positive charge of His appeared to have lower effect than those of Arg and Lys.
The relative degree of positive charge of His is estimated to be low. The increased number of positive charge at the positions and appeared to provide higher binding efficiency of Taq polymerase. Although there is no difference in the apparent K d among these mutants, the second-shifted bands appeared in the gel images of EMSA in the case of the mutants, which possess Arg or Lys at the positions and The positive charge of Arg or Lys at the positions and might cause a nonspecific binding, in addition to the functional binding, of the enzyme to DNA.
Figure 5. Primer extension activities of WT and mutant Taq polymerases. M13 ssDNA annealed with a 32 P-labeled deoxyoligonucleotide 55mer was used as the substrate. For each DNA polymerase, 0. The names of the proteins were indicated on the top. Figure 6. DNA binding ability of mutant Taq polymerases. The names of the mutant proteins were indicated on the top of each panel. Lane 1 was 32 P-labeled 27mer ssDNA. Lane 2 had no protein with primed DNA.
The main goal of this study was to create PCR enzymes with superior performance, as compared to that of WT Taq polymerase. A representative example of the PCR experiments is shown in Figure 7. The other mutant enzymes prepared in this study did not work well in the same conditions. These experiments showed inconsistency with the results of primer extension experiment. These results indicated that the positions of and in Taq polymerase are important for DNA strand synthesis, and the electrostatic environment of this site severely affects its PCR performance.
Figure 7. Lambda DNA was used as the template. Therefore, numerous structural and functional investigations of DNA polymerase have been reported to date. In this study, we developed PCR enzymes that provide a superior extension reaction as compared to Taq polymerase, the standard enzyme for PCR. As compared to the PCR performance of Taq polymerase, these enzymes achieved the amplification of either the same length of DNA in a shorter time or a longer DNA in the same reaction time.
Metagenomic analysis is a revolutionary technique for microbiological ecology. The amplification of target genes from metagenomic DNA is a very powerful method to investigate many different DNA polymerases from uncultivated microbes. In this study, we focused on thermophilic bacteria as useful genetic resources for new thermostable family A DNA polymerases.
We obtained many new sequences encoding a region of a family A DNA polymerase from the hot spring soil samples. These results suggested that our strategy to amplify a specific region of the family A DNA polymerase genes is actually applicable to the analysis of microbial populations in any habitat. We employed the same strategy to search for new family B DNA polymerases, and some of this work was published previously Matsukawa et al. We constructed chimeric enzymes between Taq polymerase and the products of the various pol genes amplified from the metagenomic DNAs, and their primer extension abilities were compared.
Many chimeric polymerases possessing excellent extension ability were obtained by this experiment. However, none of the chimeric enzymes were sufficiently thermostable for PCR use. The microbial sources of the gene fragments used for the construction of the chimeric genes are not necessarily extreme thermophiles, and some moderate thermophiles and mesophiles may be included among the amplified genes. The chimeric Taq polymerases showing faster extension ability than WT Taq polymerase would have gene fragments from the organisms, which are not extreme thermophiles.
However, the amino acid sequence comparison of the chimeric Taq polymerases provided an important clue to design a mutant Taq polymerase with superior speed for the primer extension reaction, by site-specific mutagenesis. We focused on positions and in this study.
The positions and are located in the finger subdomain and affected the interaction with DNA. The conversion of the electrostatic environment at this position, from a negative charge to a positive charge, will affect the stabilization of the DNA binding near the active site of Taq polymerase.
It is important to check whether the mutations in this site affect the fidelity of Taq polymerase. Our preliminary data revealed that the fidelities of these enzymes are not different from that of WT Taq polymerase data not shown. We will confirm this with more experiments and provide statistical data in the future. In addition to positions and , we found one more remarkable feature in the sequences of the chimeric enzymes. These enzymes have an insertion of either 9 or 3 amino acids between positions and of Taq polymerase.
It will be interesting to investigate the effects of these insertions in the finger subdomain on the PCR performance of Taq polymerase. Characterizations of the mutant Taq polymerases with the different inserted sequences are now underway.
In conclusion, we designed a method for engineering Taq polymerase to improve its primer extension rate, by using information obtained from the metagenomic analysis of soil samples from various hot-spring areas. The created enzymes showed robust PCR performances that were better than that of Taq polymerase. The enzymes created in this study basically retain the properties of Taq polymerase, and therefore, they are applicable to many uses that have already been optimized with Taq polymerases.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Drs. Masaaki Takahashi and Yukiko Miyashita for valuable discussions and encouragement. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan [grant numbers , , and to Yoshizumi Ishino].
Baar, C. The heat stability of the enzymes is directly related to the temperature, at which the organism thrives. Generally, hyperthermophiles have the potential to provide more heat-stable enzymes than normal thermophiles.
Hyperthermophilic archaea became popular not only as sources of useful enzymes for application, but also as interesting model organisms for molecular biology. In the early s, the metabolic phenomena in archaeal cells were just barely understood, and therefore, the molecular biology of Archaea, the third domain of life, became a novel and exciting field. Heat resistance of the DNA polymerases. Residual DNA polymerase activities after incubation at the indicated temperature for 30 min were plotted.
DNA polymerases from Pyrococcus furiosus open circles , Thermus aquaticus closed circles , and Bacillus caldotenax open squares were used as representatives from hyperthermophiles, extreme thermophiles, and moderate extremophiles, respectively. When choosing thermostable DNA polymerases as reagents for genetic engineering, research scientists generally do not consider the biology of the source organisms. The properties of the obtained enzyme are important, regardless of the source.
To obtain a thermostable DNA polymerase, the growth temperature of the thermophile attracts the most attention. One report described no significant differences in the fidelities of the ULTIMA and Taq polymerases, when using optimal buffer conditions for each enzyme, for sequencing purposes Diaz and Sabino, We cloned the pol gene from P.
We thought ours would be the first report of the full-length sequence of an archaeal family B DNA polymerase, which had been predicted earlier because of the aphidicolin-sensitive phenotype of a halophile and a methanogen Forterre et al.
However, two papers showing the deduced total amino acid sequences of DNA polymerases from the hyperthermophilic archaea, Sulfolobus solfataricus Pisani et al. It is also interesting that the T.
Thereafter, many cases of DNA polymerases containing various pattern of inteins, inserted in motifs A, B, and C, were discovered Perler, The fidelity of DNA synthesis in vitro is markedly affected by the reaction condition.
However, the archaeal family B enzymes generally perform more accurate DNA synthesis as compared with Taq polymerase Cariello et al. To date, the enzymes utilized for genetic engineering have been only from families A and B among them. Taq polymerase from family A has strong extension ability and performs efficient amplification of the target DNA.
However, their fidelity is low. On the other hand, the Pfu polymerase from family B performs highly accurate PCR amplification, but their extension rate is slow and a long extension time is required for each cycle of PCR. One simple idea that researchers considered trying was to combine one enzyme each from family A and family B in a single PCR reaction mixture. However, the actual PCR performance was not so simple, and persevering trials were necessary to find suitable conditions to develop a long and accurate LA PCR system.
Distribution of DNA polymerases in the three domains of life. The names of DNA polymerases vary, depending on the domains. Only DNA polymerases with in vitro activity, if applicable, are shown.
This enzyme has the typical amino acid sequence of the archaeal family B enzymes, but it showed a high extension rate while maintaining high fidelity, and therefore, the commercial product, KOD DNA polymerase KOD Pol , was developed and became popular as a PCR enzyme. The underlying reason why this family B enzyme shows high extension speed is interesting.
Comparisons of the crystallographic structures and amino acid sequences of KOD Pol with other archaeal family B enzymes revealed the logical explanation for the efficient extension ability of this enzyme. Many basic residues are located around the active site in the finger domain of KOD Pol. In addition, many Arg residues are located at the forked point, which is the predicted as the junction of the template binding region and the editing cleft. Research on DNA polymerases in hyperthermophilic archaea is motivated by not only industrial applications, but also basic molecular biology, to elucidate the molecular mechanisms of genetic information processing systems at extremely hot temperatures.
To identify all of the DNA polymerases in the archaeal cell, we tried to separate the DNA polymerase activities in the total cell extract of P. Three major fractions showed nucleotide incorporation activity after anion exchange column chromatography Resource Q column, GE Healthcare; Imamura et al. In addition to the further purification of each fraction, the screening of the DNA polymerase activity from the heat-stable protein library, made from E.
This was the first report of a eukaryotic-like initiator protein for DNA replication in Archaea. After the discovery of this DNA polymerase, the total genome sequence of Methanococcus jannaschii was published as the first complete archaeal genome Bult et al. The two genes were not present in tandem, but were located separately on the genome. We cloned and expressed them in E.
Three more total genome sequences were subsequently reported, and the genes for DP1 and DP2 were found in all them. Due to the lack of sequence homology to other DNA polymerases, we proposed a new family, family D, for this enzyme Cann and Ishino, Physical map of the P.
In parallel to the identification of DNA polymerase activities in the cell extract of P. By using a set of mixed primers based on the conserved sequences of motifs A and C in the family B DNA polymerase, a single band was amplified. However, two different fragments were found after the cloning and sequencing of the PCR product. The full-length sequences of both pol -like genes were cloned from the P. Both of the gene products exhibited the heat stable DNA polymerase activity Uemori et al.
Unfortunately, the performance of these two enzymes in PCR was not better than Pfu polymerase, and we discontinued further research on them. However, this was the first report that an archaeal cell has two different family B DNA polymerases.
In the early stages of the total genome sequences, all sequences were from Euryarchaeota Archaeoglobus fulgidus, Methanothermobacter thermautotrophicus, Pyrococcus horikoshii and the determination of the genome sequence of a crenarchaeal organism was delayed until that of A. Taken together with the new knowledge at that time, it was predicted that euryarchaeal organisms have one DNA polymerase each from family B and family D, respectively, and crenarchaeal organisms have at least two family B enzymes in the cell.
DNA polymerases in Archaea. The evolutionary relationships of six phyla in the domain Archaea are schematically shown with the DNA polymerases encoded in their genomes. The family B DNA polymerases from extrachromosomal elements were excluded. All of the original biochemical data for P. However, PolD has not been commercially developed.
At the early stage, hot start PCR was one of the big improvements for the specific amplification. This hot start PCR method is generally effective to prevent non-specific amplification. For this purpose, another idea was tested.
A chemical modification of Taq polymerase inactivated its enzymatic activity at low temperatures, but the modification can be released by high temperature resulting in activation of Taq polymerase to start PCR.
This temperature-dependent reversible modification of the Taq protein led to the commercial product, AmpliTaq Gold, as the hot start PCR enzyme. Taq polymerase is a family A enzyme, and is applicable to practical dideoxy sequencing. However, the output of the sequencing data was not ideal as compared with that from T7 DNA polymerase known commercially as Sequenase; see below.
An ingenious protein engineering strategy produced a mutant Taq polymerase that is more suitable for dideoxy sequencing than the wild type Taq polymerase. For this property, the strength of each signal is not uniform, but is distinctly unbalanced. However, T7 DNA polymerase equally incorporates deoxynucleotides and dideoxynucleotides, and therefore, it is easy to adjust the reaction conditions to provide very clear signals Tabor and Richardson, A detailed comparison of E.
This work was applied to Taq polymerase and a modified Taq with FY, which endows Taq with T7-type substrate recognition, was created Tabor and Richardson,
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