Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    Article  CAS  Google Scholar 

  2. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    CAS  Google Scholar 

  3. Miller, R.D. et al. High-density single-nucleotide polymorphism maps of the human genome. Genomics 86, 117–126 (2005).

    Article  CAS  Google Scholar 

  4. Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 (2004).

  5. Sachidanandam, R. et al. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928–933 (2001).

    Article  CAS  Google Scholar 

  6. Hardenbol, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 21, 673–678 (2003).

    Article  CAS  Google Scholar 

  7. Kennedy, G.C. et al. Large-scale genotyping of complex DNA. Nat. Biotechnol. 21, 1233–1237 (2003).

    Article  CAS  Google Scholar 

  8. Lyamichev, V. et al. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nat. Biotechnol. 17, 292–296 (1999).

    Article  CAS  Google Scholar 

  9. Shen, R. et al. High-throughput SNP genotyping on universal bead arrays. Mutat. Res. 573, 70–82 (2005).

    Article  CAS  Google Scholar 

  10. Biswas, I. & Hsieh, P. Identification and characterization of a thermostable MutS homolog from Thermus aquaticus. J. Biol. Chem. 271, 5040–5048 (1996).

    Article  CAS  Google Scholar 

  11. Iwasaki, M. & Yonokawa, T. Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole blood. Genome Lett. 2, 119–126 (2003).

    Article  CAS  Google Scholar 

  12. Mentucci, D. et al. Association between a novel variant of the human type 2 deiodinase gene Thr92Ala and insulin resistance. Diabetes 51, 880–883 (2002).

    Article  Google Scholar 

  13. Harada, S., Okubo, T., Tsutsumi, M., Takase, S. & Muramatsu, T. Investigation of genetic risk factors associated with alcoholism. Alcohol. Clin. Exp. Res. 20, 293A–296A (1996).

    Article  CAS  Google Scholar 

  14. Harada, S. & Zhang, S. New strategy for detection of ALDH2 mutant. Alcohol Alcohol. (Suppl.) 1A, 11–13 (1993).

    CAS  Google Scholar 

  15. de Morais, S.M. et al. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J. Biol. Chem. 269, 15419–15422 (1994).

    CAS  PubMed  Google Scholar 

  16. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000).

    CAS  Google Scholar 

  17. Walston, J. et al. Time of onset of non-insulin-dependent diabetes mellitus and genetic variation in the beta 3-adrenergic-receptor gene. N. Engl. J. Med. 333, 343–347 (1995).

    Article  CAS  Google Scholar 

  18. Berard, C., Cazalis, M.A., Leissner, P. & Mougin, B. DNA nucleic acid sequence-based amplification-based genotyping for polymorphism analysis. Biotechniques 37, 680–686 (2004).

    Article  CAS  Google Scholar 

  19. Pickering, J. et al. Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms. Nucleic Acids Res. 30, e60 (2002).

    Article  Google Scholar 

  20. Wang, S.S., Thornton, K., Kuhn, A.M., Nadeau, J.G. & Hellyer, T.J. Homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the BD ProbeTec ET system. Clin. Chem. 49, 1599–1607 (2003).

    Article  CAS  Google Scholar 

Download references

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

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yoshihide Hayashizaki.

Ethics declarations

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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). https://doi.org/10.1038/nmeth1007

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1007

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing