Improve reliability of PCR reactions with Herculase enhanced DNA polymerase
Michael Borns Holly Hogrefe
Stratagene recently released Herculase enhanced DNA polymerase,*, a novel enzyme formulation that provides superior performance in both routine and demanding PCR applications. Previous comparisons showed that Herculase DNA polymerase exhibits greater fidelity than commercial DNA polymerase blends, in addition to producing higher product yields over an extremely broad range of target sizes (0.1 to 48 kb). In this follow-up article, we compared Herculase DNA polymerase to single-enzyme formulations and found it to be more reliable than Taq DNA polymerase in PCR applications; it provided higher product yield, greater sensitivity, and improved synthesis of difficult (long, multiplex, GC-rich) targets. Herculase DNA polymerase was also the superior performer when compared to proofreading PCR enzymes. Moreover, its accuracy was equal to or greater than the accuracy of all proofreading DNA polymerases except Stratagenes Pfu and PfuTurbo DNA polymerases. *,
Herculase enhanced DNA polymerase was developed to meet the need for a single PCR reagent for a broad range of PCR applications.1 Because of its unique Pfu-based composition, Herculase DNA polymerase has distinct properties that distinguish it from Taq-based DNA polymerase blends (formulated as described2). Previous comparisons to commercial DNA polymerase blends showed that Herculase DNA polymerase exhibited higher fidelity, broader target-length capability (0.1 to 48 kb), and superior amplification of extra-long targets (>20 kb).1
Hence, we tested Herculase DNA polymerase in PCR applications that are routinely carried out using single-enzyme formulations. The Herculase formulation was compared to Taq DNA polymerase and to proofreading PCR enzymes in a variety of procedures, including RT-PCR, multiplex PCR, and amplifications employing GC-rich and complex genomic DNA targets.
Herculase enhanced DNA polymerase was compared to Taq DNA polymerase (Figure 1) and to proofreading DNA polymerases (Figure 2) in amplifications of genomic DNA targets less than 3 kb in length. PCRs were performed under identical conditions, using each enzymes recommended buffer (Methods).
Herculase DNA polymerase consistently produced high yields of all genomic DNA targets, ranging in length from 105 bp to 2.6 kb (Figure 1 and Figure 2). In contrast, Taq DNA polymerase synthesized each amplicon in lower yield, and recoveries decreased with increasing amplicon size for targets greater than 1 kb in length (Figure 1). Comparisons with proofreading DNA polymerases showed that even in PCRs of relatively short targets (105 to 500 bp), Herculase DNA polymerase produced significantly higher product yields than Vent, Deep Vent, and Platinum Pfx DNA polymerases (Figure 2).
The sensitivity of Herculase enhanced DNA polymerase was demonstrated in PCR amplifications that employed limiting concentrations of genomic DNA template. In Figure 3, Herculase DNA polymerase successfully amplified a 2-kb amplicon using as little as 10 ng of genomic DNA. In contrast, successful amplification with Taq DNA polymerase required at least 25 ng of template DNA. The superior sensitivity of Herculase DNA polymerase compared to Taq DNA polymerase was further demonstrated in template titration studies using additional primer sets (data not shown).
In Figure 3, a visible difference is observed in the migration of products synthesized by Taq DNA polymerase compared to Herculase DNA polymerase. Slight differences in band migration are the result of differences in PCR buffer composition and product yield (data not shown).
Herculase enhanced DNA polymerase was compared to Taq DNA polymerase in RT-PCR reactions using a panel of targets ranging in length from 0.6 to 7.6 kb. Reverse transcription of total RNA was carried out with the reagents and protocol provided in the prostar Ultra HF RT-PCR system.3 Equivalent amounts of cDNA were then added to PCR reactions carried out with Herculase or Taq DNA polymerase in each enzymes recommended PCR buffer.
As shown in Figure 4, Herculase DNA polymerase consistently produced high yields of all cDNA targets up to 7.6 kb in length; however, Taq DNA polymerase could only amplify the 0.6-kb target in sufficient yield. In additional RT-PCR comparisons, Herculase DNA polymerase synthesized a higher yield of a 660-bp b-actin target compared to Taq DNA polymerase (data not shown).
Herculase enhanced DNA polymerase is provided with an optimized PCR reaction buffer and DMSO. As discussed previously,1 the addition of DMSO can increase product yield and extend the target-length capability of the Herculase formulation, while having minimal effects on fidelity. Although it is not required for routine PCRs, we recommend adding DMSO (3 to 7% final concentration) to amplifications of genomic targets greater than 23 kb and lambda targets greater than 30 kb to increase product yield.1 DMSO can also be used to facilitate amplification of GC-rich targets and amplicons containing difficult secondary structures that impede DNA polymerase translocation.4 Such targets can be difficult to amplify in high yield.
We tested the utility of Herculase DNA polymerase in amplifying GC-rich targets (Figure 5). A 300-bp amplicon with 82.5% GC content was amplified with or without DMSO using Herculase or Taq DNA polymerase. Successful amplification of this extremely difficult target required modification of the initial denaturation step, whereby the initial denaturation temperature was increased from 95C to 98C, and the incubation time was increased from 1 minute to 3 minutes. Under these modified conditions, Herculase DNA polymerase successfully amplified the target in the presence of 6 to 9% DMSO, with optimal yields achieved using 8 to 9% DMSO. In contrast, Taq DNA polymerase was unable to amplify this target despite using optimal denaturation conditions and DMSO concentrations up to 9% (final concentration) (Figure 5).
Additional studies show that Herculase DNA polymerase plus DMSO permits superior performance compared to commercial PCR systems developed specifically for amplifying GC-rich targets. In Figure 5, higher product yields were achieved using Herculase DNA polymerase plus DMSO compared to the GC-RICH PCR System (Roche Molecular Biochemicals). For the GC-RICH PCR System, amplifications were performed as recommended, using a Taq-based DN A polymerase blend and varying concentrations (0 to 2 M) of GC-RICH resolution solution (betaine). Amplifications were performed with the GC-RICH PCR System using both the RoboCycler 96 temperature cycler (amplification unsuccessful, data not shown) and the single-block cycler recommended by the manufacturer (Figure 5).
Herculase DNA polymerase was also compared to the Advantage-GC 2 PCR kit (CLONTECH). Amplification was carried out as recommended by the kits manufacturer using a hot start Taq DNA polymerase blend, a PCR buffer containing 5% DMSO, and various concentrations of GC-Melt reagent (0 to 1.5 M). Despite repeated attempts, we were unsuccessful in amplifying the 300-bp target using the Advantage-GC 2 PCR kit (data not shown).
Therefore, Herculase DNA polymerase can be used with the provided DMSO to amplify difficult GC-rich targets, obviating the need for specialized DNA polymerase blends and PCR additives.
Herculase enhanced DNA polymerase was also tested in multiplex PCR applications. In Figure 6, Herculase DNA polymerase provided uniform amplification of three genomic DNA targets (300 bp, 500 bp, 900 bp). Using the same reaction conditions, greater variation in amplicon yield was observed with Taq DNA polymerase (data not shown).
Herculase enhanced DNA polymerase exhibits high fidelity due to its novel pfu-based composition. In previous studies,1 Herculase DNA polymerase was found to exhibit an average error rate of 2.8 x 10-6 mutation frequency per base pair per duplication.1 Comparisons show that the fidelity of Herculase DNA polymerase is comparable to (Vent, Deep Vent) or greater than (Platinum Pfx, KOD) the fidelity of all commercial proofreading DNA polymerases except Pfu and PfuTurbo DNA polymerases (Figure 7). Additional comparisons show that Herculase DNA polymerase also exhibits significantly greater accuracy than Taq and Taq-based DNA polymerase blends, including blends formulated specifically for high-fidelity and long PCR applications.1
As demonstrated here and previously,1 Herculase enhanced DNA polymerase excels in a broad range of PCR applications. Herculase DNA polymerase can be readily used in procedures normally carried out with Taq DNA polymerase to improve reliability of PCR amplification reactions. In addition to producing higher product yields, Herculase DNA polymerase is also superior to Taq in difficult PCR applications, such as multiplexing, amplifying GC-rich or longer complex targets, amplifying cDNA, and producing high product yields from limiting amounts of DNA template. Moreover, superior PCR performance is achieved with high fidelity.
Proofreading DNA polymerases and Taq-based DNA polymerase blends are routinely used for high-fidelity PCR amplification. Comparisons show that Herculase DNA polymerase provides higher product yields, compared to proofreading DNA polymerases, and broader target-length capability and better amplification of extra-long targets (>20 kb) compared to DNA polymerase blends.1 Moreover, superior PCR performance is achieved with comparable or greater accuracy. Only the fidelity of Pfu and PfuTurbo DNA polymerases is higher than that of Herculase DNA polymerase.
While PfuTurbo remains the best choice for routine PCR applications demanding the highest fidelity possible, Herculase DNA polymerase excels in all other PCR application requirements. The use of Herculase DNA polymerase overcomes performance limitations of single-enzyme formulations (e.g. Taq, Platinum Pfx), while eliminating the need for multiple specialized DNA polymerase blends.
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 s followed by 10 cycles at 95C for 30 seconds (denaturation), 58C for 30 seconds (annealing), and 72C for 2 minutes; followed by 20 cycles at 95C for 30 seconds, 58C for 30 seconds, and 72C for 2 minutes plus an additional 5 seconds per cycle. The conditions for RT-PCR (Figure 4) were: (RoboCycler 96 temperature cycler) 1 cycle at 95C for 1 minute followed by 30 cycles at 95C for 1 minute (denaturation), 60C for 1 minute (annealing), and 68C for 1 minute (targets <1 kb) or 2 minutes per kb (targets >4 kb). For each cycling regimen, one final extension cycle of 72C for 7 minutes was performed (after 30 cycles).
PCR products were electrophoresed on 1 to 4% agarose/1X TBE gels. PCR products were visualized by ethidium bromide staining, and the gels were imaged using the Eagle Eye II still video system.
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* U.S. Patent Nos. 5,545,552, 5,556,772, 5,866,395 and 5,948,663 and patents pending.