Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Fluorescent-Based ChemistriesBasics of SYBRGreen DyeChemistryThe SYBR ® Green 1 Double-Stranded Binding Dye is used for the fluorescentdetection of double-stranded DNA (dsDNA) generated during PCR. The SYBRGreen 1 Dye binds non-specifically to dsDNA and generates an excitation-emissionprofile similar to that of the FAM reporter dye. When used in combination with apassive reference, the SYBR Green 1 Dye can be employed to perform severalSDS-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 aPCR.3´5´DissociationWhen added to the reaction, the SYBR Green 1 Dye binds non-specifically tothe hybridized dsDNA and fluoresces5´3´3´5´5´3´PolymerizationDenaturation complete, the SYBR Green 1 Dye dissociates from the strand,resulting in decreased fluorescence5´3´5´Polymerization Complete3´5´3´5´ForwardPrimerReversePrimerDuring the extension phase, the SYBR Green 1 Dye begins binding to thePCR productPolymerization is complete and SYBR Green 1 Dye is completely bound,resulting in a net increase in fluorescence5´3´5´5´3´5´3´Figure D-2Binding Activity of the SYBR Green 1 Dye during the PCRApplied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide D-3DRAFTSeptember 1, 2004 11:39 am, App_Theory.fm