Page 54 - 2022-23 BIOFACT Catalogue

Fluorescence

Detection System

F
F
F F
F : uorescence
F F

F
F
DNA-Binding Dye System
F F
F : uorescence
This method detects the uorescence of dye bound to the ampli ed DNA (or cDNA). SFCgreen® I, SYBR® Green I and EvaGreen™
F
F
dye are typically used for this system. A false signal may occur due to non-speci c ampli cation, so the melting curve should
SYBR® Green I
be analyzed together. F F F F unbound-no
uorescence Forward TaqMan Q
primer probe
R Primer and
Probe-base Detection System (annealing and extension) probe annealing
PCR ampli cation
F
F
In addition to the ampli cation primer, a probe to which a uorescent dye is bound is also added for detection. Target-speci c
F
F
F : uorescence
Fluorescence
Fluorescence
probes do not require melting curve analysis, and multiplex PCR is available as the region combined with Q-R primer can be ampli-
F
SYBR® Green I
F
F
F
unbound-no
F
F
F
ed at the same time. In this case, di erent types of reporter (dye) should be used for detection. (e.g. FAM, HEX, VIC, ROX, Cy5 etc.) Forward TaqMan
F
uorescence
F
F
primer probe Q
F F F F R Primer and
PCR ampli cation probe annealing
Principle of the Intercalating Dye F F F F (annealing and extension)
F : Fluorescence Fluorescence
< SYBR® Green I Dye > < EvaGreen™ Dye > Fluorescence Fluorescence
EvaGreen™ has the same principle of action as SYBR® Green I, but exhibits R
F
F
SYBR® Green I F F R
F F F F unbound-no much higher sensitivity and DNA binding capacity than SYBR® Green I due Q
F
F
F
F
uorescence Forward TaqMan Q Extension cleavage of
to the "release on demand" feature. In addition, EvaGreen™ is character- uorescent label
primer
probe
F
F
R
F
F
Primer and
probe annealing
PCR ampli cation ized by being non-toxic and non-mutagenic, making it safe for users. Fluorescence
: Fluorescence
F
(annealing and extension) R R
Novel “Release-on-Demand” DNA Binding Mechanism
R
Fluorescence Fluorescence R
DNA Q
F F
Extension cleavage of
F F : Taq polymerase
F F F F EvaGreen™ , EvaGreen™ , EvaGreen™ - DNA complex uorescent label
inactive form active form R : Reporter (형광물질)
F F F F R R
Fluorescence
B) 30 min.
F : Fluorescence A) 5 min. Novel “Release-on-Demand” DNA Binding Mechanism Q : Quencher (상쇄물질)
B) 30 min.
incubation, long photographing
incubation incubation exposure time
DNA
R
<SFCgreen® Ⅰ> R : Taq polymerase
SFCgreen® I has the same principle of action as SYBR® green I or SYBR® EvaGreen™ , EvaGreen™ , Q EvaGreen™ - DNA complex R : Reporter (형광물질)
inactive form
active form
Green I Extension cleavage of
EvaGreen™, but has higher DNA binding strength and higher uorescent label
B) 30 min. Q : Quencher (상쇄물질)
sensitivity. As it has lower PCR inhibition than other intercalating A) 5 min. R B) 30 min. incubation, long photographing
incubation R
exposure time
incubation
Novel “Release-on-Demand” DNA Binding Mechanism
dyes, PCR ampli cation e ciency is high even at high concentra-
tions, and it is stable under light, heat, and chemicals, resulting
DNA
in a longer uorescence half-life compared to other uorescent SYBR®
Green I
EvaGreen™ : Taq polymerase
dyes.
EvaGreen™ , EvaGreen™ , EvaGreen™ - DNA complex
inactive form active form Minor groove binder Bis-intercalator R : Reporter (형광물질)
B) 30 min. Q : Quencher (상쇄물질)
A) 5 min. B) 30 min. incubation, long photographing
incubation incubation exposure time
Major groove Minor groove
EvaGreen™
SYBR®
Green I Principle of Probe Reporter-Quencher (Hydrolysis) Fluorescence
R
R
Forward TaqMan Q
primer probe Q
R
: Taq polymerase
EvaGreen™ R
R
R : Reporter (형광물질)
Reporter (Fluorescent substance)
Primer and Extension cleavage of Q Quencher (O setting substance)
: Quencher (상쇄물질)
probe annealing uorescent label
54 BIOFACT CATALOGUE