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Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology


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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Figure 1: The mechanism of allele discrimination as mediated by Taq MutS.
Figure 2: SMAP 2 primer design.
Figure 3: SMAP amplification process.
Figure 4: SNP typing of ALDH2 allele.
Figure 5: SNP typing of CYP2C19*2.
Figure 6: SMAP primer design software version 1.


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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.

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Correspondence to Yoshihide Hayashizaki.

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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.

Supplementary information

Supplementary Fig. 1

Comparison of background amplification pathways of SMAP ver.2 and LAMP. (PDF 829 kb)

Supplementary Fig. 2

SMAP ver.2 amplification of the type 2 iodothyronine deiodinase (DIO2) gene. (PDF 1397 kb)

Supplementary Fig. 3

Amplification time course of DIO2 allele by SMAP and LAMP methods. (PDF 1245 kb)

Supplementary Fig. 4

Electrophoresis photograph of SMAP and LAMP amplification products. (PDF 988 kb)

Supplementary Fig. 5

SMAP ver.2 amplification of the human b3 adrenergic receptor (ADRB3) gene. (PDF 1126 kb)

Supplementary Fig. 6

Sensitivity determination of SMAP ver.2. (PDF 1369 kb)

Supplementary Methods (PDF 210 kb)

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Mitani, Y., Lezhava, A., Kawai, Y. et al. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nat Methods 4, 257–262 (2007).

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