Exciton Primer-mediated SNP detection in SmartAmp2 reactions

Abstract

Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so‐called Exciton Primers can overcome this limitation by functioning as “sequence‐specific dyes.” After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence‐specific signals for real‐time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (−1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real‐time monitoring detect wild‐type and mutant alleles in a one‐tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point‐of‐care testing. Hum Mutat 31:208–217, 2010. © 2010 Wiley‐Liss, Inc.

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

“…We have reported fluorescent oligonucleotide probes labeled with a TO dimer unit, which we call ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes, for the detection of single-stranded DNA and RNA. [21][22][23][24][25][26][27][28][29][30][31][32] ECHO probes emitting low background fluorescence were expected to be also applicable to detect double-stranded DNA as TO molecules emit intense fluorescence in the presence of DNA triplexes. 15,17 However, DNA triplexes formed by ECHO probes have not been reported and the photophysical properties are unknown.…”

Direct and Rapid Genotyping of SLCO1B1 388A>G and 521T>C in Human Blood Specimens Using the SmartAmp-2 Method

“…Another advantage of the SmartAmp-2 assay is that the reaction proceeds in an isothermal condition and only simple equipment is needed to maintain the appropriate temperature. The method can be improved further with fluorescence-labeled oligonucleotides called "exciton primers," where visible fluorescent signals are emitted using UV or regular halogen lamps (14). These inventions will allow medical personnel to determine these genotypes even at the bedside in the near future.…”

Pharmacogenomics: Current State-of-the-Art

“…POC Genotype technologies continue to advance and molecular biology methodologies, such as SmartAmp 2 [37] are reducing the time to obtain a genotype further. It is already possible to determine genotypes for pharmacogenomics-related variants such as those associated with warfarin dose requirement ( VKORC1 and CYP2C9 ) in 30–40 min [37,38].…”