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Abstract

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.

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Acknowledgements

We thank A. Wada for support and encouragement, T. Ishikawa (Tokyo Institute of Technology) for his constructive discussion about the application of this technology and future prospects, M. Matsunaga, J. Nakashima, M. Matsushita and S. Uno for technical assistance. We acknowledge K. Nakano (NTT Software Corporation) for assistance in web support and software design. We also thank H. Daub and M. Nishikawa (RIKEN) for their editorial and coordination efforts. This study was mainly supported by the research grant for the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science and Technology of the Japan (MEXT) to Y. Hayashizaki, and RIKEN “Research Collaborations with Industry” Program to K. Shibata. S. Kuramitsu is supported by the Research Grant for National Project on Protein Structure and Functional Analysis from MEXT.

Author information

Affiliations

  1. Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center (GSC), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.

    • Yasumasa Mitani
    • , Alexander Lezhava
    • , Yuki Kawai
    • , Takeshi Kikuchi
    • , Atsuko Oguchi-Katayama
    • , Yasushi Kogo
    • , Hideki Takakura
    • , Kanako Hoshi
    • , Takahiro Arakawa
    • , Paul E Cizdziel
    •  & Yoshihide Hayashizaki
  2. K.K. Dnaform, 75-1, Ono-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0046, Japan.

    • Yasumasa Mitani
    • , Yuki Kawai
    • , Takeshi Kikuchi
    •  & Chiaki Kato
  3. Genome Science Laboratory, Discovery Research Institute, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

    • Masayoshi Itoh
    • , Kazuhiro Shibata
    •  & Yoshihide Hayashizaki
  4. Wakunaga Pharmaceutical Co. Ltd., 1624 Shimokotachi, Koda-cho, Akitakata-shi, Hiroshima, 739-1195, Japan.

    • Toru Miyagi
    •  & Takanori Oka
  5. Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, 3-9, Fukuura Kanazawa-ku, Yokohama, 236-0004, Japan.

    • Hideki Takakura
    • , Kanako Hoshi
    •  & Hiroshi Shimada
  6. Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.

    • Kenji Fukui
    • , Ryoji Masui
    •  & Seiki Kuramitsu
  7. RIKEN SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kohto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.

    • Kenji Fukui
    • , Ryoji Masui
    •  & Seiki Kuramitsu
  8. Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.

    • Kazuma Kiyotani
    •  & Tetsuya Kamataki
  9. Center for Molecular Medicine, Karolinska Institutet, 171 76 Stockholm, Sweden.

    • Alistair Chalk
  10. Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi-shi, Gunma, 371-8511, Japan.

    • Katsuhiko Tsunekawa
    •  & Masami Murakami

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Competing interests

Some of the authors of this manuscript are employed by or affiliated with the corporations K.K. DNAFORM and Wakunaga Pharmaceutical Co. Ltd. Y.M., Y.K., T.K. and C.K. are employed by K.K. DNAFORM. T.M. and T.O. are employed by Wakunaga Pharmaceutical Co., Ltd.

It is the intention of K.K. DNAFORM to commercialize the technology described in this manuscript for research and diagnostic purposes.

Corresponding author

Correspondence to Yoshihide Hayashizaki.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Comparison of background amplification pathways of SMAP ver.2 and LAMP.

  2. 2.

    Supplementary Fig. 2

    SMAP ver.2 amplification of the type 2 iodothyronine deiodinase (DIO2) gene.

  3. 3.

    Supplementary Fig. 3

    Amplification time course of DIO2 allele by SMAP and LAMP methods.

  4. 4.

    Supplementary Fig. 4

    Electrophoresis photograph of SMAP and LAMP amplification products.

  5. 5.

    Supplementary Fig. 5

    SMAP ver.2 amplification of the human b3 adrenergic receptor (ADRB3) gene.

  6. 6.

    Supplementary Fig. 6

    Sensitivity determination of SMAP ver.2.

  7. 7.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth1007

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