Applied Biosystems 7900HT - Page 349

DRAFT
September 1, 2004 11:39 am, App_Theory.fm
Fluorescent-Based Chemistries
Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-3
Basics of SYBR
Green Dye
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 similar to that of the FAM
™
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.
Figure D-2 illustrates the action of the SYBR Green 1 dye during a single cycle of a
PCR.
Figure D-2 Binding Activity of the SYBR Green 1 Dye during the PCR
When added to the reaction, the SYBR Green 1 Dye binds non-specifically to
the hybridized dsDNA and fluoresces
5´
3´
5´
3´
5´
3´
5´
3´
5´
3´
5´
3´
Polymerization Complete
Denaturation complete, the SYBR Green 1 Dye dissociates from the strand,
resulting in decreased fluorescence
Polymerization is complete and SYBR Green 1 Dye is completely bound,
resulting in a net increase in fluorescence
5´
3´
5´
3´
Polymerization
Dissociation
5´
3´
5´
3´
5´
5´
During the extension phase, the SYBR Green 1 Dye begins binding to the
PCR product
Forward
Primer
Reverse
Primer