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Methods

PCR amplifications (50 l) employed 200 M each dNTP (or 300 M each dNTP for Platinum Pfx), 100 ng of each primer, and 100 ng of genomic DNA. As recommended by the enzyme manufacturers, amplifications were carried out in each enzymes recommended buffer using 2.5U of Herculase, AmpliTaq (Applied Biosystems), Vent (NEB), or Deep Vent (NEB) DNA polymerase or 1.25U of Platinum Pfx DNA polymerase. For GC-rich PCR targets, amplifications were carried out using 2.5U of Herculase DNA polymerase, 5U to 6U of AdvantTaq Plus (CLONTECH), or 2U of GC-RICH PCR System DNA polymerase and each enzymes recommended buffer and PCR additive solution.

Reactions were cycled in 200-l thin-walled PCR tubes, using a RoboCycler Gradient 96 Temperature Cycler (Stratagene) or a single-block thermocycler, fitted with a Hot Top device. Enzyme comparisons employed identical temperature cycling parameters. For PCRs of genomic DNA targets (Figure 1, Figure 2, Figure 3 and Figure 6), the following conditions were used: (single-block cycler) 1 cycle at 95C for 1 minute followed by 30 cycles at 95C for 30 seconds (denaturation), 58C for 30 seconds (annealing), and 72C for 40 seconds (<900 bp) or 1 minute (900 bp) or 2 minutes (>1 kb). The conditions used to amplify the GC-rich amplicon (Figure 5) were as follows: (RoboCycler 96 temperature cycler) 1 cycle at 98C for 3 minutes followed by 30 cycles at 95C for 1 minute (denaturation), 58C for 40 seconds (annealing), and 72C for 2 minutes; (single-block cycler) 1 cycle at 95C for 3 minute
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