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Transglutaminase-Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

Authors

  • Dr. Momoko Kitaoka,

    1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
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  • Yukito Tsuruda,

    1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
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  • Yukari Tanaka,

    1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
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  • Prof. Masahiro Goto,

    1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
    2. Center for Future Chemistry, Kyushu University (Japan)
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  • Dr. Masayuki Mitsumori,

    1. Aloka Co., Ltd., 3-7-19 Imai, Ome-shi, Tokyo (Japan)
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  • Kounosuke Hayashi,

    1. Aloka Co., Ltd., 3-7-19 Imai, Ome-shi, Tokyo (Japan)
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  • Dr. Yoshiyuki Hiraishi,

    1. Aloka Co., Ltd., 3-7-19 Imai, Ome-shi, Tokyo (Japan)
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  • Dr. Katsuyuki Miyawaki,

    1. Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2–1 Minamijosanjima-cho, Tokushima (Japan)
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  • Prof. Sumihare Noji,

    1. Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2–1 Minamijosanjima-cho, Tokushima (Japan)
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  • Prof. Noriho Kamiya

    Corresponding author
    1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
    2. Center for Future Chemistry, Kyushu University (Japan)
    • Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan), Fax: (+81) 92-2810
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Abstract

A new synthetic strategy for DNA–enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA–(enzyme)n conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA–(enzyme)n probe could clearly detect 104 copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten–antibody reaction-based DNA-detection system.

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