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The Best Enzyme for the Toughest PCR Challenges Improved with New Hot-Start Feature

res). Detecting low-copy-number templates is especially problematic in a complex genomic DNA background (multiple false priming sites) due to frequent mispriming events at ambient temperature. In systems prone to primer-dimer formation, the synthesis of primer oligomers leads to reduced yield and obscured detection of specific product. In high-throughput applications, PCR reactions are robotically assembled in multiple 96-well dishes and then stored for varying periods of time prior to temperature cycling. Nonspecific primer extension can occur frequently during prolonged storage, leading to high background. In each of these difficult PCR systems, hot- start methods reduce background and improve detection sensitivity by preventing priming until stringent primer annealing temperatures are reached.

Extreme Specificity and Sensitivity

To demonstrate the high specificity and sensitivity of Herculase Hotstart DNA polymerase, amplicons were synthesized from limiting amounts of target DNA in the presence of denatured genomic DNA. The addition of exogenous DNA creates extremely challenging test systems for evaluating PCR enzyme specificity and sensitivity. Herculase Hotstart DNA polymerase successfully amplified a 115-bp fragment of the gag gene from as few as 5 copies of DNA template, and no extraneous bands were evident (Figure 1). In contrast, amplification with non-hot-start enzymes produces high background and low detection sensitivity.

The enhanced sensitivity of Herculase Hotstart DNA polymerase is also demonstrated in Figure 2, where a 1.7-kb fragment was amplified fr
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