Exciton Primer-mediated SNP detection in SmartAmp2 reactions.
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.
Lezhava
Alexander
A
Technology Development Unit, Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan.
Ishidao
Takefumi
T
Ishizu
Yuri
Y
Naito
Kana
K
Hanami
Takeshi
T
Katayama
Atsuko
A
Kogo
Yasushi
Y
Soma
Takahiro
T
Ikeda
Shuji
S
Murakami
Kayoko
K
Nogawa
Chihiro
C
Itoh
Masayoshi
M
Mitani
Yasumasa
Y
Harbers
Matthias
M
Okamoto
Akimitsu
A
Hayashizaki
Yoshihide
Y
D052160:Benzothiazoles; D017931:DNA Primers / Q000378:metabolism*; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011804:Quinolines; D064417:Vitamin K Epoxide Reductases
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D006899:Mixed Function Oxygenases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases