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

Chapter 6Analyzing Real-Time DataOverviewAboutDissociationCurve AnalysisEmploying theSYBR Green 1DyeMathematicalTransformationsThe Applied Biosystems 7900HT Fast Real-Time PCR System supports dissociationcurve analysis of nucleic acids using SYBR ® Green 1 double-stranded DNA bindingdye chemistry. The objective of dissociation curve analysis is to accurately determinethe melting temperature (T m ) of a single target nucleic acid sequence within anunknown PCR sample. Typical uses of dissociation curves include detection ofnon-specific products and primer concentration optimization.Dissociation curve analysis on the 7900HT instrument is made possible through theuse of the fluorogenic SYBR Green 1 double-stranded DNA binding dye chemistry(see page D-3). Dissociation curves are commonly performed following the PCRstage of a SYBR Green dye experiment to screen for non-specific products. Togenerate the data needed to create a curve, the 7900HT instrument performs aprogrammed temperature ‘ramp’ in which it slowly elevates the temperature of theplate over several minutes. The specific binding characteristic of the SYBR Green 1Dye permits the 7900HT instrument to monitor the hybridization activity of thenucleic acids present in the sample. During the run, the instrument records thedecrease in SYBR Green fluorescence resulting from the dissociation of dsDNA.After the run, the SDS software processes the raw fluorescence data from the SYBRGreen 1 Dye to generate a more meaningful representation of the relationshipbetween spectral change and temperature for the dissociation curve run.Multicomponenting and NormalizationThe first mathematical transformation involves the conversion of the raw data,expressed in terms of Fluorescent Signal vs. Wavelength, to pure dye componentsusing the extracted pure dye standards. At the same time, the software determines thecontribution of each dye in the raw data using the multicomponent algorithm.Afterwards, the software normalizes the data using the component of the passivereference dye as shown below.R n=R---------------------------------------------( SYBR)R ( PassiveReference)Derivation of Dissociation Curve DataThe SDS software then computes the first derivative of the normalized data (R n ) foreach reading taken by the 7900HT instrument during the temperature ramp. Theresulting derivative data (R n´) is the rate of change in fluorescence as a function oftemperature (see below).dRR n′ = --------- ndTThe software plots the negative of the resulting derivative data on graph of -R n´versus temperature (T) that visualizes the change in fluorescence at each temperatureinterval. The T m for the target nucleic acid can be determined from the graph byidentifying the maximum for the rate of change (displayed as a peak) for theappropriate amplification curve.6-38 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Real-Time.fm