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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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.
Comparison of background amplification pathways of SMAP ver.2 and LAMP.
SMAP ver.2 amplification of the type 2 iodothyronine deiodinase (DIO2) gene.
Amplification time course of DIO2 allele by SMAP and LAMP methods.
Electrophoresis photograph of SMAP and LAMP amplification products.
SMAP ver.2 amplification of the human b3 adrenergic receptor (ADRB3) gene.
Sensitivity determination of SMAP ver.2.
About this article
A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP)
Analytical and Bioanalytical Chemistry (2016)