ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Basics of SYBR
Green Chemistry
The SYBR® Green 1 Double-Stranded Binding Dye is used for the fluorescent
detection of double-stranded DNA (dsDNA) generated during PCR. The SYBR Green
1 Dye binds non-specifically to dsDNA and generates an excitation-emission profile
similartothatoftheFAM reporter dye. When used in combination with a passive
reference, the SYBR Green 1 Dye can be employed to perform several SDS-related
experiments including quantitative PCR and dissociation cure analysis. The following
figure illustrates the action of the SYBR Green 1 dye during a single cycle of a PCR.
3´
5´
When added to the reaction, the SYBR Green 1 Dye binds non-specifically to
the hybridized dsDNA and fluoresces
Dissociation
3´
5´
Denaturation complete, the SYBR Green 1 Dye dissociates from the strand,
resulting in decreased fluorescence
Polymerization
5´
3´
5´
Polymerization Complete
3´
5´
3´
5´
Forward
Primer
Reverse
Primer
During the extension phase, the SYBR Green 1 Dye begins binding to the
PCR product
Polymerization is complete and SYBR Green 1 Dye is completely bound,
resultinginanetincreaseinfluorescence
5´
3´
5´
3´
5´
3´
5´
5´
3´
5´
3´
Theory of Operation A-3