Herculase® is a DNA Polymerase designed to deal with difficult to amplify targets such as long or GC-rich DNA templates. Herculase® comes in two formats, Herculase® Enhanced and Herculase® Hotstart DNA Polymerases. The two formats of Herculase® are optimized combinations of Stratagene’s high fidelity DNA polymerase
Pfu,
Taq DNA polymerase, and ArchaeMaxx® polymerase-enhancing factor. This combination amplifies a broad range of DNA lengths (0.1 – 48 kb) with reportedly greater yield and higher fidelity than
Taq DNA polymerase alone. Stratagene states that the ArchaeMaxx® polymerase-enhancing factor has been demonstrated to improve PCR yield by reducing dUTP-poisoning which is caused by a build up of dUTP during cycling via dCTP deamination. The Herculase® Hotstart DNA Polymerase utilizes a monoclonal antibody bound to the polymerase inhibiting its 3'-5' exonuclease activity. Heat-denaturation of the antibody when cycling begins releases the antibody, thus activating the polymerase. The Herculase® DNA Polymerase is also provided with DMSO which is known to be a useful addition to PCR reactions to assist with amplification of extra-long or GC-rich DNA targets.
As with all PCR amplifications, optimization of conditions is necessary in order to obtain acceptable yield and specificity; Stratagene provides a table within the Herculase® manual with useful guidelines for PCR optimization. Parameters of particular importance for Herculase® reactions include DNA template quality and concentration, DMSO concentration, and cycling parameters. As with all DNA polymerases, DNA target purity is important; we typically use Qiagen purification kits which have provided us with adequate DNA purity. DMSO is a useful additive to a PCR reaction where the DNA target has GC rich regions. DMSO has been shown to facilitate DNA strand separation in these regions by disrupting base pairing and thus, improves PCR efficiency. However, optimization is required as too high a concentration of DMSO can lead to an increased error rate of the polymerase. Of particular importance with cycling parameters is the denaturation temperature. As high denaturation temperatures can result in DNA target damage, a denaturation temperature of 92ºC is recommended; however, a higher denaturation temperature of 98ºC is recommended for GC rich targets.
We have used Stratagene’s Herculase® Enhanced DNA Polymerases to amplify a number of long DNA targets (8-10 kb) with great success. After optimization following Stratagene’s recommendations, we were able to obtain a good yield of the targets of interest. As these amplifications were for cloning purposes, we were also satisfied with the low rate of PCR-induced mutations. However, the product proved most useful when amplifying through GC-rich areas of our DNA targets. To date, Herculase® DNA Polymerases are the most successful reagents we have used for amplifying GC-rich areas. We have also employed Herculase® when introducing site-directed mutations into GC-rich DNA targets. Due to difficulties amplifying through GC-rich areas of our DNA target, we substituted the Pfu provided with Stratagene’s Quikchange II Site Directed Mutagenesis Kit with Herculase® DNA Polymerases, buffer and DMSO and obtained comparable results to when using the standard kit with a non-GC rich DNA target.
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals