( Methods Optim...)The Gradient Feature: Use in Optimization of Allelic Discrimination Assays, Rev B

Methods
Optimization of T_a
T_a optimization was performed for the amplification of a fragment of the coagulation factor V gene (van den Bergh et al. 2000). Duplicate PCRs were carried out in a volume of 50 l containing 20 mM Tris, pH 8.4, 50 mM KCl, 200 M each dNTP, 3 mM MgCl₂, 0.2 M each PCR primer (M3= 5'-CTTGAAGGAAATGCCCCATTA, and M5= 5'-TGCCCAGTGCTTAACAAGACCA), a 1:100,000 dilution of SYBR Green I (Molecular Probes, Eugene, OR), 1.25 U Platinum Taq polymerase (Invitrogen), and 10 ng human genomic DNA. Reactions were incubated in the iCycler iQ real-time PCR detection system for 3.5 min at 95C and then subjected to 50 two-step cycles of heating at 95C for 10 sec and annealing for 50 sec at 54, 55.2, 57.1, 59.8, 64, 66.9, 68.9, or 70C (temperature gradient). Fluorescent data were collected during the annealing step. A melt-curve protocol immediately followed the amplification. Reactions were incubated first at 95C for 60 sec and subsequently at 55C for 60 sec, followed by 80 repeats of heating for 10 sec starting at 55C with 0.5C increments.

Optimization of Annealing Temperature for SNP Detection Using a TaqMan Assay
For the optimization of a TaqMan assay for SNP detection, an experiment was designed to amplify and detect the factor V Leiden mutation (Bertina et al. 1994). The sequences of the primers and probes used for this experiment are shown in Table 1. Duplicate PCR reactions were carried out in a 50 l volume containing 20 mM Tris pH 8.4, 50 mM KCl, 200 M each dNTP, 3 mM MgCl₂, 0.2 M each PCR primer, 0.15 M each ASO TaqMan probe, 1.25 U Platinum Taq DNA polymerase, and 10 ng human genomic DNA. PCR conditions
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