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

Appendix DTheory of OperationFluorescence vs.AmplifiedProductWhen using TaqMan fluorogenic probes with the 7900HT instrument, fluorescenceemission increases in direct proportion to the amount of specific amplified product.As Figure D-4 on page D-5 demonstrates, the graph of normalized reporter (R n ) vs.cycle number during PCR appears to have three stages. Initially, R n appears as a flatline because the fluorescent signal is below the detection limit of the 7900HTinstrument. In the second stage, the signal can be detected as it continues to increasein direct proportion to the increase in the products of PCR. As PCR productcontinues to increase, the ratio of AmpliTaq Gold polymerase to PCR productdecreases. When template concentration reaches 10 -8 M, PCR product ceases to growexponentially. This signals the third stage of R n change, which is roughly linear andfinally reaches a plateau at about 10 -7 M (Martens and Naes, 1989).The progressive cleavage of TaqMan fluorescent probes during the PCR makespossible the correlation between initial template concentration and the rise influorescence. As the concentration of amplified product increases in a sample, sodoes the R n value. During the exponential growth stage (the geometric phase), therelationship of amplified PCR product to initial template can be shown in thefollowing equation:N c= N1 ( + E) cwhere N c is the concentration of amplified product at any cycle, N is the initialconcentration of target template, E is the efficiency of the system, and c is the cyclenumber.CalculatingThreshold CyclesThe Applied Biosystems 7900HT Fast Real-Time PCR System creates quantifiablerelationships between test samples based on the number of cycles elapsed beforeachieving detectable levels of fluorescence. Test samples containing a greater initialtemplate number cross the detection threshold at a lower cycle than samplescontaining lower initial template. The SDS software uses a Threshold setting todefine the level of detectable fluorescence.The threshold cycle (C T ) for a given amplification curve occurs at the point that thefluorescent signal grows beyond the value of the threshold setting. The C T representsa detection threshold for the 7900HT instrument and is dependent on two factors:• Starting template copy number• Efficiency of DNA amplification the PCR systemHow the SDS Software Determines C T sTo determine the C T for an Amplification plot, the SDS software uses data collecteddata from a predefined range of PCR cycles called the ‘baseline’ (the defaultbaseline occurs between cycles 3 and 15). First, the software calculates amathematical trend based on the baseline cycles’ R n values to generate a baselinesubtracted Amplification plot of ∆R n versus cycle number. Next, an algorithmsearches for the point on the Amplification plot at which the ∆R n value crosses thethreshold setting (the default threshold setting is 0.2). The fractional cycle at whichthe intersection occurs is defined as the threshold cycle (C T ) for the plot.Note: It may be necessary to adjust the baseline and threshold settings to obtainaccurate and precise data. For further information on resetting the baseline andthreshold settings, see “Setting the Baseline and Threshold Values” on page 6-46.D-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, App_Theory.fm