• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.669-675
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• 1998

Taq DNA polymerase exhibits a sizable drawback compared to the other thermophilic DNA polymerases in that it demonstrates lower proof-reading activity due to the deficiency of 3'-5'exonuclease activity. A study was undertaken to improve the 3'-5' exonuclease activity in the PCR of Taq DNA polymerase. The three-dimensional structural alignment of the polymerase and 3'-5' exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase has just a background level of 3'-5'exonuclease activity. A comparison indicated that the two polymerase domains are very similar in primary and tertiary conformations, even though Taq DNA polymerase carries a much shorter 3'-5'exonuclease domain than that of E. coli DNA polymerase I. Those two polymerase domains were interchanged between Taq DNA polymerase and E. coli DNA polymerase I. The 3'-5' exonuclease domain from E. coli DNA polymerase I was separated and pasted into the polymerase domain of Taq DNA polymerase I, which resulted in a functional fusion-Stoffel fragment. The 3'-5'exonuclease activity of the fusion-Stoffel fragment increased up to 48% of the value of the Klenow fragment, while that of Taq DNA polymerase remained at 6.0% of the Klenow fragment.

• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• Journal of Microbiology and Biotechnology
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• v.8 no.5
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• pp.471-477
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• 1998

Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

• BMB Reports
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• v.29 no.1
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• pp.38-44
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• 1996

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method, which is being used for amplifying DNA. Not only does Taq DNA polymerase have high commercial value commercial value for the polymerase chain reaction application, but it is also important in studying DNA replication, because it is apparently an homologue to E. coli DNA polymerase I, which has long been used for DNA replication study (Lawyer et ai., 1993). The crystal structure determination of Taq DNA polymerase was initiated. An X-ray diffraction pattern breaks down a crystal structure into discrete sine waves in a Fourier series. The original shape of a crystal object in terms of electron density may be represented as the sum of those sine waves with varying amplitudes and phases in three dimensions. The molecular replacement method was initially employed to provide phase information for the structure of Taq DNA polymerase. The rotation search using the program MERLOT resulted in a solution peak with 5.4 r.m.s. PC-refinement of the X-PLOR program verified the result and also optimized the orientation angles. Next, the translation search using the X-PLOR program resulted in a unique solution peak with 7.35 r.m.s. In addition, the translation search indicated $P3_121$ to be the true space group out of two possible ones. The phase information from the molecular replacement was useful in the MIR phasing experiment.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.381-385
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• 1999

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in a polymerase chain reaction. Taq DNA polymerase has a domain at the amino terminus (residues 1 to 290) that has 5'-3' exonuclease activity and a domain at the C-terminus that catalyzes the polymerase reaction. Taq DNA polymerase is classified into the Pol I family, which is represented by E. coli DNA polymerase I. The alignment of amino acid sequences for the 5'-3' exonuclease domains of the Pol I family DNA polymerases shows ten highly conserved carboxylic amino acids. Crystallographic studies suggested that six of the carboxylic amino acids are clustered within a 7 $\AA$ radius by chelating three metal ions in the active site. Those six carboxylic residues are mutagenized to alanines in order to better understand their function. All six carboxylic residues, Asp l8, Glu1l7, Asp1l9, Asp120, Asp142, and Aspl44, are crucial for catalysis of 5'-3' exonuclease.

• Journal of Microbiology
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• v.44 no.1
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• pp.126-128
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• 2006

This study confirms that Taq DNA polymerase could be contaminated with the $blaTEM-1_a$ gene. It also proposes two different methods that could be used to overcome DNA contamination: (i) DNase I treatment prior to PCR amplification; and (ii) the use of a highly purified Taq DNA polymerase which was devoid of detectable contamination.

• BMB Reports
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• v.35 no.2
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• pp.248-250
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• 2002

Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.158-162
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• 2010

The gene encoding N-terminally truncated Tod polymerase ($\Delta$Tod polymerase) from Thermus thermophilus HJ6 was expressed in Escherichia coli under the control of the lambda pR and pL tandem promoters on the expression vector pJLA503. The N-terminal domain (250 amino acids) of Tod polymerase was removed without significant effect on enzyme activity and stability, while no 5'$\rightarrow$3' exonuclease activity was detected. The $\Delta$Tod polymerase was verified to possess very efficient reverse transcriptase (RT) activity in the presence of $MgCl_2$. The cDNA can also be amplified in the polymerase chain reaction (PCR) with this mutant enzyme. The $\Delta$Tod polymerase was exhibited higher activity than the Taq polymerase in a one-step RT-PCR.

• KSBB Journal
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• v.10 no.3
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• pp.264-270
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• 1995

Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

• Korean Journal of Microbiology
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• v.34 no.1_2
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• pp.51-57
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• 1998

A study was carried out to understand some parameters affecting on RAPD analysis with Lactobacillus species. From the results, we found that appearance of specific DNA bands were very influenced by the concentration of $MgCl_2$ but it was overcome by applying enough amount of Taq DNA polymerase. Other parameters such as concentrations of template DNA, random primers and Taq DNA polymerase have enhanced the production of specific DNA bands by increasing their concentration applied. However, we noticed that G/C contents of random primers did not show any correlations with number of specific RAPD bands generated but the RAPD results were heavily influenced by the characteristics of the random primers, that is, the sequences of the oli.