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Pure Appl. Chem., 2006, Vol. 78, No. 12, pp. 2305-2312

Highly selective fluorescent nucleobases for designing base-discriminating fluorescent probes

Isao Saito1, Yoshio Saito1, Kazuo Hanawa1, Keigo Hayashi1, Kaori Motegi1, Subhendu Sekhar Bag1, Chikara Dohno2, Tomohisa Ichiba2, Kazuki Tainaka2 and Akimitsu Okamoto2

1 NEWCAT Institute, School of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan
2 Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Katsura, Kyoto 615-8510, Japan

There is increasing interest in single nucleotide polymorphism (SNP) typing since they can be used as markers to identify the genes that underlie complex diseases and to realize the full potential of pharmacogenomics in analyzing variable response to drugs. Among the different methodologies for SNP genotyping, the homogenous assay is more amenable than the heterogeneous one.
In this article, we will describe some of our most recently developed novel base-discriminating fluorescent (BDF) nucleosides useful for homogenous SNP typing. Our novel concept led to the investigation of a new type of pyrene-labeled BDF nucleosides PyU, PyC, 8pyA, and MePydA, which emitted strong fluorescence only when the bases opposite the BDF bases are A, G, T, and C, respectively. The DNA probes containing four different BDF bases enabled us to distinguish single-base alterations by simply mixing with a sample solution of target DNA. An example of SNP typing of c-Ha-ras SNP sequence has also been demonstrated. Detection of base insertion in insertion/deletion (indel) polymorphisms using pyrene excimer fluorescent probe has also been explored.