NATURE METHODS abstract

Yasumasa Mitani^1,2, Alexander Lezhava¹, Yuki Kawai^1,2, Takeshi Kikuchi^1,2, Atsuko Oguchi-Katayama¹, Yasushi Kogo¹, Masayoshi Itoh³, Toru Miyagi⁴, Hideki Takakura^1,5, Kanako Hoshi^1,5, Chiaki Kato², Takahiro Arakawa¹, Kazuhiro Shibata³, Kenji Fukui^6,7, Ryoji Masui^6,7, Seiki Kuramitsu^6,7, Kazuma Kiyotani⁸, Alistair Chalk⁹, Katsuhiko Tsunekawa¹⁰, Masami Murakami¹⁰, Tetsuya Kamataki⁸, Takanori Oka⁴, Hiroshi Shimada⁵, Paul E. Cizdziel¹, Yoshihide Hayashizaki^1,3,*

We have developed a novel and rapid SNP (single nucleotide polymorphism) detection system named SMAP version 2 (SMAP 2). SMAP 2 (SMart Amplification Process) is isothermal and achieved high levels of amplification within 30 minutes when performed with a new DNA polymerase cloned from Alicyclobacillus acidocaldarius (Aac). In SMAP 2, DNA amplification only occurred with a perfect primer match; hence, amplification alone was sufficient to identify the target allele. In establishing SMAP 2, two unique approaches were employed for suppression of exponential background DNA amplification. A mismatch binding protein Thermus aquaticus MutS (Taq MutS), was added to the reaction solution to inhibit background DNA from entering the amplification cycle by specifically binding to mis-primed amplification products. And, an asymmetrical primer design using a combination of turn-back and folding primers was employed to minimize alternative mis-amplification pathways. Furthermore, to assist the scientific community in configuring SMAP assays, we developed a software specific for SMAP primer design. With these new tools, a high precision and rapid DNA amplification technology becomes available for applications in pharmacogenomics and molecular diagnostics.

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
2. K.K. Dnaform, 75-1, Ono-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0046, JAPAN
3. Genome Science Laboratory, Discovery Research Institute, RIKEN Wako Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
4. Wakunaga Pharmaceutical Co. Ltd, 1624 Shimokotachi, Koda-cho, Akitakata-shi, Hiroshima, 739-1195, Japan
5. Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, 3-9, Fukuura Kanazawa-ku, Yokohama, 236-0004, Japan
6. Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
7. RIKEN SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kohto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
8. Faculty of Pharmacy, Takasaki University of Health and Welfare 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan
9. Center for Molecular Medicine, Karolinska Institutet, 171 76 Stockholm Sweden
10. Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi-shi, Gunma, 371-8511, Japan

        * Corresponding author: Yoshihide Hayashizaki [yosihide@gsc.riken.go.jp]

Nature Methods (2007 March), Vol.4, No.3, 257-262.