Figure 1

Selection of points. (A) Fluorescence difference (dF) as a function of cycle number with data from a hydrolysis probe assay run on a LightCycler™. Filled circles were used for fitting with function EAvPeak; fitting results are displayed as line graph. Circles marked by an asterisk indicate the 5 point peak. (B) As part A, but at higher magnification on the y axis. Points for definition of background fluorescence are selected as follows: each position i, going down cycle by cycle from the 5 point peak, is checked until the dF values of at least 3 points in the interval i-8 to i-1 surpass the reference level, which is the average dF of points i, i+1, and i+2. The upper limit of the background definition interval, denoted by a filled arrow, corresponds to i; the lower limit, denoted by an open arrow, is at i-8. Finally, slope and offset of the background line is determined by linear regression on the raw fluorescence data at i-8 to i. (C) Corresponding raw fluorescence data. Fitting results from function EAv are displayed as line graph. Open circles represent points that were excluded from fitting. Asterisks and arrows as above.