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

Novel Herculase Hotstart DNA polymerase results in greater specificity and sensitivity

Michael Borns Brad Scott Holly Hogrefe

Stratagene

Stratagenes Herculase DNA polymerase *^, is the best enzyme for difficult PCR applications, particularly those involving long, complex, low-copy number, or GC-rich templates. We have expanded our hot-start PCR enzyme product line to include Herculase Hotstart DNA polymerase.*^, This polymerase provides high specificity and detection sensitivity, while facilitating high-throughput robotic PCR applications.

Stratagene is dedicated to developing high-performance PCR enzymes that excel in both routine and demanding PCR applications. We designed the novel Herculase Enhanced DNA polymerase with the goal of realizing higher product yields over an extremely broad range of target sizes and complexities.^1,2 To achieve hot-start capability, Herculase Hotstart DNA polymerase is formulated with a combination of antibodies that effectively neutralize DNA polymerase and 3'-5' (proofreading) exonuclease activities. Full enzyme activity is regained once the antibody is denatured during ramp-up to initial denaturation temperatures.

Enhanced Amplification with Hot-Start Enzymes

Amplification systems that are most likely to benefit from hot start are those designed to detect low-copy-number targets in complex DNA backgrounds, are prone to primer-dimer formation, or are assembled robotically (high-throughput PCR procedures). 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 from limiting amounts of template DNA in a complex genomic DNA background. The target was readily amplified from 10, 100, and 1000 copies of lambda DNA using Herculase Hotstart DNA polymerase, while 1000 copies of target were required for successful amplification with non-hot-start enzymes ( Figure 2).

Conclusions

Use Stratagenes Herculase Hotstart PCR polymerase to achieve greater sensitivity and specificity in detecting DNA, as well as superior product yield in complex PCR systems. This enzyme exhibits the same performance characteristics as the original enzyme formulation, but with the enhanced sensitivity and specificity of a hot-start format.

REFERENCES

1. Borns, M. and Hogrefe, H. (2000) Strategies 13: 1-3.

2. Borns, M. and Hogrefe, H. (2000) Strategies 13: 76-79.

* U.S. Patent Nos. 6,183,997, 5,545,552 and 5,556,772 and 5,866,395 and 5,948,663 and patents pending.