Chemical structure of thymine labeled with fluorescent dyes. A : Thiazole pink-labeled thymine. B : Thiazole orange-labeled thymine. doi:10.1371/journal.pone.0060151.g002 

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... primer set and an enzyme, Aac polymerase [18], which has strand displacing activity and a high resistance to cellular contaminants. More recently, we have developed exciton-controlled hybridization-sensitive fluorescent primers, named ‘‘Exciton Primers’’ or ‘‘Eprimers,’’ which function as sequence-specific dyes that significantly enhance the signal/noise ratio [26–28]. After hybridization to complementary sequences, the Eprimer provides a sequence-specific fluorescent signal for real-time monitoring of amplification reactions. Eprimers show high signal strength with low background leading to a superior specificity and sensitivity compared with the commonly used SYBR H Green I [28]. To further advance our technology, in the present study, we introduced novel primers as well as two Eprimers to detect both 309T and 309G alleles simultaneously in one tube. We examined the specificity and reliability of the new method by comparison with the conventional PCR-based method. We here present validation data obtained with the new method named ‘‘Duplex SmartAmp’’ and demonstrate its genotyping data with clinical samples of lung cancer patients. Figure 1A depicts a schematic illustration of the strategy for detecting SNP (c.309G . T) in the MDM2 gene by the Duplex SmartAmp method. To achieve high fidelity of SNP typing, we introduced two novel primers, namely, the outer Boost Primer (oBP) and the neutral Boost Primer (nBP), in addition to the standard SmartAmp primers ( i.e ., TP, FP, BP, and OP). The reason for adopting such a SNP typing strategy is that GC- rich nucleotide sequences were found in both 5 9 - and 3 9 -regions adjacent to the SNP (c.309T . G), as shown in Figure 1B. In other words, the melting temperature of the GC-rich regions was so high that dissociation of the DNA strands became slower during the isothermal DNA amplification reaction. Two oBPs, i.e ., oBP (WT) and oBP (SNP), were designed to discriminate T and G, respectively, at nucleotide c.309 between the wild type (WT) and SNP alleles (Figure 1B). These two oBPs were labeled with differently colored fluorescence dyes, as described below. The nBP served as a pacemaker for the detection reaction, where it is annealed at a locus proximal to the SNP position (Figure 1B). The Duplex SmartAmp method requires the use of seven different primers: TP, FP, BP, OP, nBP, oBP (WT), and oBP (SNP). These primer candidates were selected on the basis of algorithms for free energy, probability of base-pairing, and product size range [29]. After extensive screening with a variety of synthesized oligo-nucleotides as primer candidates, we have selected one optimal set to use in the Duplex SmartAmp method for detecting SNP 309T . G in the MDM2 gene. Table 1 summarizes the sequences of those primers comprising the optimal set. The genomic sequence located between the annealing sites of TP and FP (Figure 1A) is the target region that is amplified by the isothermal DNA amplification reaction. oBP (WT) and oBP (SNP) were separately labeled with fluorescence dyes named thiazole pink and thiazole orange, respectively. One thymine in each of those oBPs was chemically linked with either thiazole pink or thiazole orange molecules at the position ‘‘Z’’ or ‘‘U’’ in the primer sequence (Table 1). Thyamine bases, except for both 3 9 - and 5 9 -terminals, can be labeled with exciton dyes. The sequence of Eprimers should be computation- ally designed to avoid any possibility of inter-primer dimmer formation and self-folding within a primer. Figure 2 represents the chemical structures of the thiazole pink (A) and thiazole orange (B) molecules. To detect the fluorescence of those dyes intercalated into DNA double strands during the Duplex SmartAmp reaction, thiazole pink and thiazole orange were excited at 585 and 492 nm, respectively, and their fluorescence was detected through the respective ROX (610 nm) and FAM (516 nm) filters in a real-time PCR machine Mx3000P (Agilent Technologies, Santa Clara, CA, USA). These primers selectively recognized the target sequence of SNP c.309T . G of the MDM2 gene to discriminate homozygous 309T/T, heterozygous 309T/G, and homozygous 309G/G. As shown in Figure 3, the fluorescence intensity reached a plateau in 30 minutes over time for both the genomic DNA (upper panels) and blood (lower panels) samples. To verify amplification products, by agarose gel electrophoresis, we analyzed the DNA amplicons formed during the reaction of Duplex SmartAmp assay. We sampled aliquots of ...