Applied Biosystems 7900HT User Manual page 349

Basics of SYBR
Green Dye
Chemistry
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
®
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 similar to that of the FAM
passive reference, the SYBR Green 1 Dye can be employed to perform several
SDS-related experiments including quantitative PCR and dissociation cure analysis.
Figure D-2 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
Forward
Primer
5´
3´
5´
During the extension phase, the SYBR Green 1 Dye begins binding to the
PCR product
Polymerization Complete
3´
5´
3´
5´
Polymerization is complete and SYBR Green 1 Dye is completely bound,
resulting in a net increase in fluorescence
Figure D-2 Binding Activity of the SYBR Green 1 Dye during the PCR
™
reporter dye. When used in combination with a
DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Fluorescent-Based Chemistries
5´
3´
5´
3´
5´
3´
5´
Reverse
Primer
5´
3´
5´
3´
D-3