Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe.
A typical TaqMan™ real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
Dmitry Y
DY
Ryazantsev
Dmitry A
DA
Tsybulsky
Igor A
IA
Prokhorenko
Maksim V
MV
Kvach
Yury V
YV
Martynenko
Pavel M
PM
Philipchenko
Vadim V
VV
Shmanai
Vladimir A
VA
Korshun
Sergey K
SK
Zavriev
D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D019914:Taq Polymerase / Q000737:chemistry*
D015342:DNA Probes; D005456:Fluorescent Dyes; D019914:Taq Polymerase