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Isao Kitajima - One of the best experts on this subject based on the ideXlab platform.

  • eukaryote made Thermostable DNA Polymerase enables rapid pcr based detection of mycoplasma ureaplasma and other bacteria in the amniotic fluid of preterm labor cases
    PLOS ONE, 2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made Thermostable DNA Polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made Thermostable DNA Polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

  • The workflow of the rapid detection method for Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Using this PCR-based method, pathogens can be detected within three hours of amniotic fluid sample collection. To prevent the occurrence of unreliable results in PCR-based assaying of amniotic samples for bacterial pathogens, eukaryote-made Thermostable DNA Polymerase (or Taq Polymerase), which is free from bacterial DNA contamination, is used in combination with bacterial universal primers (along with two specific primers in the second, nested PCR). In contrast, for fungal pathogens, conventional bacterially-made Thermostable DNA Polymerase (or Taq Polymerase), which is usually free from fungal DNA contamination, is used in combination with fungal universal primers.

  • The strategy used for the primer design.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    (A) In an attempt to detect Mycoplasma, Ureaplasma and other bacteria, nested PCR was performed using the primer for the first PCR (Bacterial Universal Primer for 1st PCR) at the start. For the second (nested) PCR, four kinds of primers (Bacterial Universal Primer for 2nd PCR, Mycoplasma Specific Primer, Ureaplasma Specific Primer, and NotMycoUrea Bacterial Universal Primer) were used. *The amplicon sizes are described, and the amplified positons on Escherichia coli 16S ribosomal RNA (Accession No. J01859) are shown. Mycoplasma and Ureaplasma Specific Primers do not bind to E. coli 16S rDNA. (B) The sequence homology between the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR, NotMycoUrea Bacterial Universal Primer) and the target regions of Mycoplasma, Ureaplasma and other bacteria. Seven examples are shown as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The base sequence differences between the primers and the target regions are shown in red. Two of the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR) can detect almost all kinds of bacteria including Mycoplasma and Ureaplasma species. On the other hand, the NotMycoUrea Bacterial Universal Primer can detect almost all kinds of bacteria, but does not detect Mycoplasma or Ureaplasma species because of the primer’s low sequence homology, which is a key point of our method. (C) The PCR amplification products of Mycoplasma, Ureaplasma and other bacteria amplified by each primer set. Six examples are used as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The gels showed no bacterial contamination using eukaryote-made Thermostable DNA Polymerase and also showed the specificity of each primer set. PCR amplification products were detected precisely according to the presence or absence of the targeted bacterial DNA templates.

  • a novel eukaryote made Thermostable DNA Polymerase which is free from bacterial DNA contamination
    Journal of Clinical Microbiology, 2011
    Co-Authors: Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Tomohiro Ueno, Shirou Hayashi, Isao Kitajima
    Abstract:

    To achieve the production of a Thermostable DNA Polymerase free from bacterial DNA contamination, we developed eukaryote-made Thermostable DNA (Taq) Polymerase. The novel eukaryote-made Thermostable DNA Polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made Thermostable DNA Polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made Thermostable DNA Polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.

Hideki Niimi - One of the best experts on this subject based on the ideXlab platform.

  • polymicrobial amniotic fluid infection with mycoplasma ureaplasma and other bacteria induces severe intra amniotic inflammation associated with poor perinatal prognosis in preterm labor
    American Journal of Reproductive Immunology, 2016
    Co-Authors: Noriko Yoneda, Hideki Niimi, Tomohiro Ueno, Shirou Hayashi, Satoshi Yoneda, Mika Ito, Arihiro Shiozaki, Daichi Urushiyama, Kenichiro Hata, Wataru Suda
    Abstract:

    Problem To study the relationship between perinatal prognosis in cases of preterm labor (PTL) and polymicrobial infection in amniotic fluid (AF) and intra-amniotic (IA) inflammation using a highly sensitive and reliable PCR-based method. Method of Study To detect prokaryotes using a nested PCR-based method, eukaryote-made Thermostable DNA Polymerase without bacterial DNA contamination was used in combination with bacterial universal primers. We collected AF aseptically from 118 PTL cases and 50 term subjects. Results The prevalence of microorganisms was 33% (39/118) by PCR and only 7.6% (9/118) by culture. PTL caused by a combination of positive Mycoplasma/Ureaplasma and other bacteria had significantly higher AF IL-8 levels and a significantly shorter amniocentesis-to-delivery interval. Conclusions Our newly established PCR method is useful for detecting IA microorganisms. Polymicrobial infection with Mycoplasma/Ureaplasma and other bacteria induces severe IA inflammation associated with poor perinatal prognosis in PTL.

  • eukaryote made Thermostable DNA Polymerase enables rapid pcr based detection of mycoplasma ureaplasma and other bacteria in the amniotic fluid of preterm labor cases
    PLOS ONE, 2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made Thermostable DNA Polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made Thermostable DNA Polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

  • The workflow of the rapid detection method for Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Using this PCR-based method, pathogens can be detected within three hours of amniotic fluid sample collection. To prevent the occurrence of unreliable results in PCR-based assaying of amniotic samples for bacterial pathogens, eukaryote-made Thermostable DNA Polymerase (or Taq Polymerase), which is free from bacterial DNA contamination, is used in combination with bacterial universal primers (along with two specific primers in the second, nested PCR). In contrast, for fungal pathogens, conventional bacterially-made Thermostable DNA Polymerase (or Taq Polymerase), which is usually free from fungal DNA contamination, is used in combination with fungal universal primers.

  • The strategy used for the primer design.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    (A) In an attempt to detect Mycoplasma, Ureaplasma and other bacteria, nested PCR was performed using the primer for the first PCR (Bacterial Universal Primer for 1st PCR) at the start. For the second (nested) PCR, four kinds of primers (Bacterial Universal Primer for 2nd PCR, Mycoplasma Specific Primer, Ureaplasma Specific Primer, and NotMycoUrea Bacterial Universal Primer) were used. *The amplicon sizes are described, and the amplified positons on Escherichia coli 16S ribosomal RNA (Accession No. J01859) are shown. Mycoplasma and Ureaplasma Specific Primers do not bind to E. coli 16S rDNA. (B) The sequence homology between the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR, NotMycoUrea Bacterial Universal Primer) and the target regions of Mycoplasma, Ureaplasma and other bacteria. Seven examples are shown as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The base sequence differences between the primers and the target regions are shown in red. Two of the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR) can detect almost all kinds of bacteria including Mycoplasma and Ureaplasma species. On the other hand, the NotMycoUrea Bacterial Universal Primer can detect almost all kinds of bacteria, but does not detect Mycoplasma or Ureaplasma species because of the primer’s low sequence homology, which is a key point of our method. (C) The PCR amplification products of Mycoplasma, Ureaplasma and other bacteria amplified by each primer set. Six examples are used as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The gels showed no bacterial contamination using eukaryote-made Thermostable DNA Polymerase and also showed the specificity of each primer set. PCR amplification products were detected precisely according to the presence or absence of the targeted bacterial DNA templates.

  • a novel eukaryote made Thermostable DNA Polymerase which is free from bacterial DNA contamination
    Journal of Clinical Microbiology, 2011
    Co-Authors: Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Tomohiro Ueno, Shirou Hayashi, Isao Kitajima
    Abstract:

    To achieve the production of a Thermostable DNA Polymerase free from bacterial DNA contamination, we developed eukaryote-made Thermostable DNA (Taq) Polymerase. The novel eukaryote-made Thermostable DNA Polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made Thermostable DNA Polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made Thermostable DNA Polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.

Tomohiro Ueno - One of the best experts on this subject based on the ideXlab platform.

  • polymicrobial amniotic fluid infection with mycoplasma ureaplasma and other bacteria induces severe intra amniotic inflammation associated with poor perinatal prognosis in preterm labor
    American Journal of Reproductive Immunology, 2016
    Co-Authors: Noriko Yoneda, Hideki Niimi, Tomohiro Ueno, Shirou Hayashi, Satoshi Yoneda, Mika Ito, Arihiro Shiozaki, Daichi Urushiyama, Kenichiro Hata, Wataru Suda
    Abstract:

    Problem To study the relationship between perinatal prognosis in cases of preterm labor (PTL) and polymicrobial infection in amniotic fluid (AF) and intra-amniotic (IA) inflammation using a highly sensitive and reliable PCR-based method. Method of Study To detect prokaryotes using a nested PCR-based method, eukaryote-made Thermostable DNA Polymerase without bacterial DNA contamination was used in combination with bacterial universal primers. We collected AF aseptically from 118 PTL cases and 50 term subjects. Results The prevalence of microorganisms was 33% (39/118) by PCR and only 7.6% (9/118) by culture. PTL caused by a combination of positive Mycoplasma/Ureaplasma and other bacteria had significantly higher AF IL-8 levels and a significantly shorter amniocentesis-to-delivery interval. Conclusions Our newly established PCR method is useful for detecting IA microorganisms. Polymicrobial infection with Mycoplasma/Ureaplasma and other bacteria induces severe IA inflammation associated with poor perinatal prognosis in PTL.

  • eukaryote made Thermostable DNA Polymerase enables rapid pcr based detection of mycoplasma ureaplasma and other bacteria in the amniotic fluid of preterm labor cases
    PLOS ONE, 2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Background Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. Methods We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made Thermostable DNA Polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made Thermostable DNA Polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. Results Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. Conclusions We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

  • The workflow of the rapid detection method for Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    Using this PCR-based method, pathogens can be detected within three hours of amniotic fluid sample collection. To prevent the occurrence of unreliable results in PCR-based assaying of amniotic samples for bacterial pathogens, eukaryote-made Thermostable DNA Polymerase (or Taq Polymerase), which is free from bacterial DNA contamination, is used in combination with bacterial universal primers (along with two specific primers in the second, nested PCR). In contrast, for fungal pathogens, conventional bacterially-made Thermostable DNA Polymerase (or Taq Polymerase), which is usually free from fungal DNA contamination, is used in combination with fungal universal primers.

  • The strategy used for the primer design.
    2015
    Co-Authors: Tomohiro Ueno, Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Noriko Yoneda, Satoshi Yoneda, Shigeru Saito, Isao Kitajima
    Abstract:

    (A) In an attempt to detect Mycoplasma, Ureaplasma and other bacteria, nested PCR was performed using the primer for the first PCR (Bacterial Universal Primer for 1st PCR) at the start. For the second (nested) PCR, four kinds of primers (Bacterial Universal Primer for 2nd PCR, Mycoplasma Specific Primer, Ureaplasma Specific Primer, and NotMycoUrea Bacterial Universal Primer) were used. *The amplicon sizes are described, and the amplified positons on Escherichia coli 16S ribosomal RNA (Accession No. J01859) are shown. Mycoplasma and Ureaplasma Specific Primers do not bind to E. coli 16S rDNA. (B) The sequence homology between the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR, NotMycoUrea Bacterial Universal Primer) and the target regions of Mycoplasma, Ureaplasma and other bacteria. Seven examples are shown as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The base sequence differences between the primers and the target regions are shown in red. Two of the primers (Bacterial Universal Primer for 1st PCR, Bacterial Universal Primer for 2nd PCR) can detect almost all kinds of bacteria including Mycoplasma and Ureaplasma species. On the other hand, the NotMycoUrea Bacterial Universal Primer can detect almost all kinds of bacteria, but does not detect Mycoplasma or Ureaplasma species because of the primer’s low sequence homology, which is a key point of our method. (C) The PCR amplification products of Mycoplasma, Ureaplasma and other bacteria amplified by each primer set. Six examples are used as representative of Mycoplasma, Ureaplasma and other bacteria, respectively. The gels showed no bacterial contamination using eukaryote-made Thermostable DNA Polymerase and also showed the specificity of each primer set. PCR amplification products were detected precisely according to the presence or absence of the targeted bacterial DNA templates.

  • a novel eukaryote made Thermostable DNA Polymerase which is free from bacterial DNA contamination
    Journal of Clinical Microbiology, 2011
    Co-Authors: Hideki Niimi, Masashi Mori, Homare Tabata, Hiroshi Minami, Tomohiro Ueno, Shirou Hayashi, Isao Kitajima
    Abstract:

    To achieve the production of a Thermostable DNA Polymerase free from bacterial DNA contamination, we developed eukaryote-made Thermostable DNA (Taq) Polymerase. The novel eukaryote-made Thermostable DNA Polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made Thermostable DNA Polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made Thermostable DNA Polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.

Nadejda I Rechkunova - One of the best experts on this subject based on the ideXlab platform.

  • effect of DNA and bivalent metal ions on the interaction of Thermostable DNA Polymerase tte with dntps
    Russian Chemical Bulletin, 2005
    Co-Authors: Ekaterina A Belousova, Nadejda I Rechkunova, Kh S Degtyarev, Vladimir V Koval, Olga S Fedorova, O I Lavrik
    Abstract:

    The interaction of DNA Polymerase Tte from Thermus thermophilus B35 with dUTP analog containing a fluorescein residue bound to C(5) of the base (Flu-dUTP) was studied by fluorescence titration. The dissociation constants of the enzyme—substrate complexes in the absence and in the presence of a DNA duplex containing an extended template and bivalent metal ions and the kinetic parameters of polymerization by DNA Polymerase Tte in the presence of Flu-dUTP were determined.

  • photoreactive dttp analogues as substrates for Thermostable DNA Polymerase from thermus thermophilus b35
    Bioorganicheskaia khimiia, 2004
    Co-Authors: N A Lebedeva, Nadejda I Rechkunova, S V Dezhurov, Kh S Degtyarev, O I Lavrik
    Abstract:

    Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP–NAB-x-dUTP (where x = 2, 4, 7–13 is the number of atoms in the linker), were studied. All the analogues are substrates for Thermostable Thermusthermophilus B35 DNA Polymerase in the elongation reaction of the 5′-32P-labeled primer–template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUМP residues into the 3′ primer end did not impede further elongation of the chain in the presence of natural dNTP.

  • Thermostable DNA Polymerase from thermus thermophilus b35 preparation and study of a modified form of the enzyme with high affinity to ddntp
    Biochemistry, 2003
    Co-Authors: A. G. Akishev, N A Lebedeva, Nadejda I Rechkunova, O I Lavrik, J E Tomilova, Kh S Degtyarev
    Abstract:

    The hybrid protein consisting of Tte DNA Polymerase fragment and mutant Taq DNA Polymerase (F667Y) fragment in the ratio 20 : 1 was constructed. Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme. The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68%. In the Polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA Polymerase.

  • Thermostable DNA Polymerase from thermus thermophilus b35 influence of divalent metal ions on the interaction with deoxynucleoside triphosphates
    Biochemistry, 2000
    Co-Authors: Nadejda I Rechkunova, S K Degtyarev, S S Okhapkina, Rashid O Anarbaev, I A Lokhova, O I Lavrik
    Abstract:

    The interaction of DNA Polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the Polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.

  • Thermostable DNA Polymerase from thermus thermophilus b35 cloning sequence analysis and gene expression
    Biochemistry, 1999
    Co-Authors: A. G. Akishev, N A Lebedeva, Nadejda I Rechkunova, O I Lavrik, S K Degtyarev
    Abstract:

    The nucleotide sequences of three Thermostable DNA Polymerase (Taq, Tth, and Tfl) genes were analyzed and high conserved regions typical for this Polymerase family were identified. Using primers for one of the conserved regions, the genomic DNA fragment of T. thermophilus B35 strain was amplified. The resulting fragment was cloned into a plasmid and used as a hybridization probe with digests of T. thermophilus B35 DNA cleaved by different restriction endonucleases. A restriction DNA fragment carrying the full-length Tte Polymerase gene was found, cloned, and sequenced. The primary structures of the Tte and Tth DNA Polymerase genes were analyzed. The Tte-pol gene was recloned into an expression vector and recombinant protein was purified to homogeneity. The properties of Tte-pol in the Polymerase chain reaction were investigated.

Shinsuke Fujiwara - One of the best experts on this subject based on the ideXlab platform.

  • Development of an efficient one-step real-time reverse transcription Polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection.
    'Public Library of Science (PLoS)', 2021
    Co-Authors: Yukiko Nakura, Yuya Okamoto, Muneyuki Takeuchi, Koichiro Suzuki, Yoshitaka Tamura, Yuichiro Oba, Fumiko Nishiumi, Nobuaki Hatori, Shinsuke Fujiwara
    Abstract:

    The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and Thermostable DNA Polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two Thermostable DNA Polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA Polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the Thermostable DNA Polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method's sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2

  • enhanced detection of rna by mmlv reverse transcriptase coupled with Thermostable DNA Polymerase and DNA rna helicase
    Enzyme and Microbial Technology, 2017
    Co-Authors: Hiroyuki Okano, Shinsuke Fujiwara, Yuta Katano, Misato Baba, Ayako Fujiwara, Ryota Hidese, Itaru Yanagihara, Tsukasa Hayashi, Kenji Kojima, Teisuke Takita
    Abstract:

    Detection of mRNA is a valuable method for monitoring the specific gene expression. In this study, we devised a novel cDNA synthesis method using three enzymes, the genetically engineered Thermostable variant of reverse transcriptase (RT), MM4 (E286R/E302K/L435R/D524A) from Moloney murine leukemia virus (MMLV), the genetically engineered variant of family A DNA Polymerase with RT activity, K4polL329A from thermophilic Thermotoga petrophila K4, and the DNA/RNA helicase Tk-EshA from a hyperthermophilic archaeon Thermococcus kodakarensis. By optimizing assay conditions for three enzymes using Taguchi's method, 100 to 1000-fold higher sensitivity was achieved for cDNA synthesis than conventional assay condition using only RT. Our results suggest that DNA Polymerase with RT activity and DNA/RNA helicase are useful to increase the sensitivity of cDNA synthesis.