( consisted of a 3.5 min incubatio...)The Gradient Feature: Use in Optimization of Allelic Discrimination Assays, Rev B

Results and Discussion
The iCycler iQ gradient feature was used to optimize the T_a of PCR experiments for the amplification of a 220 bp fragment of the coagulation factor V gene. The iCycler iQ system was programmed to perform a two-step (denaturation-annealing) PCR protocol to test a range of annealing temperatures between 54 and 70C. To identify the number of amplified PCR products by the incorporation of SYBR Green I, a melt-curve protocol followed the amplification reactions. Melt-curve analysis, which is used to determine the melt curve of doublestranded DNA fragments, is a useful tool to check for the specificity of PCR amplifications. The presence of primerdimers as well as nonspecific PCR fragments is clearly identified by the presence of peaks with melting temperatures (T_m) that differ from that of the expected PCR fragment. Results from the T_a optimization experiments are shown in Table 2 and Figure 1. Data analysis showed the presence of the expected PCR products, as confirmed by a 1.5% agarose gel (Figure 2), within the temperature range of 5468.9C. As a result of reduced oligonucleotide priming efficiency, no amplification products were observed when using 70C as T_a. Maximal yield of PCR products, measured by relative fluorescence and threshold cycle (C_T), was obtained within the range of 5459.8C. Additional information regarding PCR specificity is derived from the analysis of the melt curves (Figure 1 and Table 2). Melt-curve analysis showed
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