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Unique DNA Polymerase Formulation Excels in a Broad Range of PCR,,,Applications


Herculase enhanced DNA polymerase delivers high fidelity and great performance

Michael Borns Holly Hogrefe
Stratagene

Stratagene has recently released Herculaseenhanced DNA polymerase,*, a novel enzyme formulation that provides superior performance in both routine and demanding PCR applications. Comparisons demonstrate that Herculase enhanced DNA polymerase is even more robust than commercial DNA polymerase blends, producing superior product yields in amplifications of both short (<1 kb) and extra-long (>20 kb) genomic targets. In addition to high product yields over a broad range of target sizes, the Herculase enhanced DNA polymerase exhibits greater fidelity than Taq DNA polymerase, several proofreading DNA polymerases, and all commercial DNA polymerase blends tested.

Over the past few years, a number of DNA polymerases and polymerase blends have been developed to fulfill a variety of PCR application requirements. Taq DNA polymerase is generally used in routine amplifications of short targets (<2 kb), while Stratagenes Pfu and PfuTurbo DNA polymerases*, are the best choices for cloning applications requiring the highest fidelity PCR enzyme available. DNA polymerase blends are used to successfully amplify long targets (>5 kb), and additionally may provide higher product yields, greater sensitivity, increased accuracy, and faster cycling times than are achieved with Taq DNA polymerase alone. Polymerase vendors may offer as many as 2 to 5 different DNA polymerase blends, developed to satisfy particular fidelity, target-length, and DNA template (%GC content, cDNA, genomic DNA) requirements.

Until now, no PCR enzyme possesses all of the features necessary to fulfill a broad range of PCR application needs. Single-enzyme formulations exhibit limited target-length capability compared to DNA polymerase blends. Although DNA polymerase blends can be formulated to favor increased fidelity (higher proportion of proofreading enzyme) or enhanced amplification of longer targets (higher proportion of nonproofreading enzyme), variations in polymerase ratios and buffer composition to achieve increased fidelity generally lead to reduced product yield and poor amplification of long targets. Moreover, DNA polymerase blends and PCR buffers optimized for amplification of long targets generally exhibit poor fidelity and inefficient amplication of short targets.

Stratagenes Herculase enhanced DNA polymerase was developed to provide customers with a single PCR reagent to fulfill all PCR application needs. The Pfu-based Herculase formulation exhibits several unique features, including high fidelity, broad target-length capability, and superior amplification of extra-long targets which make it ideal for a full range of routine and demanding PCR applications (Table 1).

Comparisons to Taq(= x)a

Herculase Enhanced DNA Polymerase

PfuTurbo DNA Polymerase

TaqPlus Precision PCR System

TaqPlus Long PCR System

Performance
(yield & length)

XXXXX

XX

XXX

XXXX

Fidelity

XXXX

XXXXX

XXX

XX

a The results of numerous amplification reactions have been used to rank the PCR performance of Stratagenes PCR enzymes from highest performance

(XXXXX: Greatest yield/successful amplification of >20 kb targets) to lowest performance (X: Product yield and target-length capability (<5 kb) comparable to Taq DNA polymerase). Relative fidelity, determined from error rate measurements, is indicated from most accurate (XXXXX) to least accurate (X: Comparable to Taq, 8 x 10-6 MF/bp/d1,3).

Herculase Enhanced DNA Polymerase

The Herculase enzyme formulation has been extensively tested using primer-template systems of varying lengths, complexities, GC content, and DNA template sources. It has been used successfully to amplify genomic targets 0.1 kb to 37 kb in length and lambda DNA targets up to 48 kb in length. The Herculase enhanced DNA polymerase is provided with an optimized PCR reaction buffer and DMSO. As discussed below, adding DMSO can increase product yield and extend target-length capability while having minimal effects on fidelity. For genomic targets greater than 23 kb and lambda targets greater than 30 kb, we recommend adding DMSO to a final concentration of 3% to 7%, depending upon template source, template GC content, and amplicon length. Since the optimal DMSO concentration varies with each primer-template system, providing DMSO as a supplement rather than including it in the PCR reaction buffer ensures that maximal product yields are achieved.

Yield and Target-Length Comparisons

Fig.3

The PCR performance of Herculase enhanced DNA polymerase was tested relative to Platinum Taq DNA polymerase High Fidelity (Life Technologies, Inc.), a blend developed specifically for high-fidelity PCR applications, and the Expand 20 kbPlus PCR system (Roche Molecular Biochemicals), the only commercial blend specifically formulated for amplifications of extra-long (>20 kb) genomic targets. The range of amplicon lengths synthesized by each formulation was evaluated by amplifying a panel of short genomic DNA targets, ranging in length from 0.1 kb to 0.5 kb (Figure 1) and a panel of extra-long targets, ranging in length from 17 kb to 48 kb (Figure 2). Amplifications were performed under identical conditions (Methods) with the following exceptions: as per the manufacturers recommendations, PCRs were carried out using 2.5 U of Platinum Taq DNA polymerase High Fidelity, 2.6 U of Expand 20 kbPlus PCR system blend, and 2.5 U (<1 kb) or 5 U (>17 kb) of Herculase enhanced DNA polymerase. The 30-kb and 48-kb targets were amplified with the Herculase formulation in the presence of 3% and 7% DMSO, respectively.

Fig.2

The Platinum Taq DNA polymerase High Fidelity exhibited limited target-length capability compared to Herculase enhanced DNA polymerase, which successfully amplified all targets up to 48 kb in length (Figure 1 and Figure 2). Even in amplifications of short targets, the Herculase formulation produced higher product yields than the Platinum Taq blend (Figure 1). Although the Expand 20 kbPlus PCR system successfully amplified the longer 17-kb and 30-kb genomic targets, product yields were highest with the Herculase formulation. Moreover, amplification of the longest 48-kb lambda target was unsuccessful using the Expand 20 kbPlus PCR system (Figure 2). In PCRs employing short targets (<1 kb), the Expand 20 kbPlus PCR system did not produce as much product as the Herculase formulation (Figure 1). Therefore, Herculase enhanced DNA polymerase exhibits superior performance over a full range of amplicon sizes (0.1 kb to 48 kb), and its use eliminates the need for multiple PCR enzymes and specialized long and extra-long PCR blends.

PCR Fidelity Comparisons

Current Assay Results

Published lacI Assay Results

DNA Polymerase

#PCRs

Mean E.R.
(x10-6) ( Std. dev.)

#PCRs

Mean E.R.
(x10-6) ( Std. dev.)

PfuTurbo

4

1.3 0.1

14

1.3 0.21,3,4

Herculase

4

2.8 0.5

Deep Vent

4

2.7 0.21

Vent

6

2.8 0.91

Pfx

4

3.5 1.04

TaqPlus Precision

2

4.6 0.3

11

3.0 1.33

TaqPlus Long

10

7.6 1.23

Taq

4

10.4 4.4

11
4

8.0 3.91,3
11.7 0.84

The fidelity of Herculase enhanced DNA polymerase was measured using the previously described PCR mutation assay.1 The Herculase formulation exhibits an average error rate of 2.8 x 10-6 mutation frequency per base pair per duplication (Table 2). In side-by-side comparisons, the error rate of the Herculase formulation was found to be 2.1-fold higher than the error rate of PfuTurbo DNA polymerase and 1.6-fold and 3.7-fold lower than the error rate of the TaqPlus Precision blend and Taq DNA polymerase, respectively. The Pfu-based composition of the Herculase formulation presumably contributes to its higher fidelity, compared to Stratagenes TaqPlus Precision and TaqPlus Long DNA polymerase blends.

Comparisons to previous data indicate that Herculase enhanced DNA polymerase exhibits an error rate that is comparable (Vent, Deep Vent) or superior [Platinum Pfx (KOD)] to the error rates of certain proofreading single-enzyme preparations (Table 2). Moreover, side-by-side comparisons show that the Herculase formulation exhibits significantly greater accuracy than DNA polymerase blends (Figure 3), including blends formulated specifically for high-fidelity (Platinum Taq DNA polymerase High Fidelity, Advantage HF PCR kit) and long (LA Taq, Expand 20 kbPlus PCR system) PCR applications (Figure 3). The results of a more comprehensive study, comparing the fidelity of TaqPlus Precision DNA polymerase to other vendors DNA polymerase blends, indicate that the Herculase formulation exhibits greater accuracy than at least seven additional blends.2

Fig.3

The effects of DMSO on the error rates of Taq, Pfu, and DNA polymerase blends have not been previously described despite DMSOs widespread use as a PCR adjunct. The utility of DMSO in amplifications of extra-long targets prompted us to test its effects on the fidelity of Herculase enhanced DNA polymerase. Preliminary results have shown that DMSO may increase the error rate of the Herculase formulation, albeit slightly. The average error rate of the Herculase formulation was 32% higher in the presence of 3% DMSO, compared to reactions carried out in the absence of DMSO (data not shown). However, the mean error rate of the Herculase formulation in the presence of 3% DMSO is still lower than the mean error rates of commercial Taq-based blends (Table 2, Figure 3). Therefore, using Herculase enhanced DNA polymerase eliminates the need for specialized high-fidelity DNA polymerase blends.

Conclusions

Stratagenes Herculase enhanced DNA polymerase provides accurate and robust amplification over a broad range of target lengths and complexities. The broad utility and superior performance of the Herculase formulation eliminates the need for multiple specialized PCR enzymes and DNA polymerase blends.

Methods

The PCR fidelity assay was carried out as described,1,3 using PfuTurbo DNA polymerase (Stratagene), Herculase enhanced DNA polymerase (Stratagene), TaqPlus Precision PCR system (Stratagene), Taq2000 DNA polymerase (Stratagene), Platinum Taq DNA polymerase High Fidelity (Life Technologies, Inc.), Advantage HF (ClonTech), LA Taq (Takara), and Expand 20 kbPlus PCR system (Roche Molecular Biochemicals). For fidelity assays, PCR amplifications were carried out in each enzymes recommended PCR buffer using 2.5 U of each DNA polymerase.

For PCR comparisons, amplifications (50 l) employed 200 M (<1 kb) or 500 M (>17 kb) each dNTP and 100 ng (<1 kb) or 200 ng (>17 kb) of each primer. The following amounts of DNA template were used: 100 ng (<1-kb targets) or 240 ng (17-kb and 30-kb targets) of genomic DNA or 15 ng (48 kb) of lambda DNA. As recommended by each enzyme manufacturer, amplifications were carried out with 2.5 U (<1 kb) or 5 U (>17 kb) of the Herculase formulation, 2.5 U of Platinum Taq DNA polymerase High Fidelity, or 2.6 U of the Expand 20 kbPlus blend. The following amounts of DMSO were added to Herculase PCRs: 0% (0.1-kb to 17-kb targets), 3% (30-kb target), or 7% (48-kb target).

Reactions were cycled in 200-l thin-walled PCR tubes using a RoboCycler gradient 96 temperature cycler (Stratagene)(<1-kb targets) or a single-block thermocycler (>17-kb targets) fitted with a Hot Top Assembly. For enzyme comparisons, identical temperature cycling parameters were employed that consisted of (<1-kb targets) 1 cycle at 94C for 1 minutes, followed by 30 cycles at 94C for 40 seconds (denaturation), 58C for 40 seconds (annealing) and 68C for 1 minute, or (>17-kb targets) 1 cycle at 92C for 2 minutes, followed by 10 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target, followed by 20 cycles at 92C for 10 seconds (denaturation), 60 to 65C for 30 seconds (annealing) and 68C for 1 minute per kb of target plus an additional 10 seconds/cycle. After 30 cycles, one final extension cycle of 68C for 10 minutes was carried out.

PCR products less than 1 kb in length were electrophoresed on 4% agarose/1XTBE gels, while the 17-kb to 48-kb products were resolved on 1% agarose/1XTBE gels using a CHEF DRIII system (Bio-Rad). PCR products were visualized by staining with ethidium bromide, and the gels were imaged using the Eagle Eye II still video system.

REFERENCES

  1. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: 3546-3551.

  2. Guide to Pfu DNA Polymerase, Stratagene, 1996.

  3. Nielson, K.B., et al. (1997) Strategies 10: 29-32.

  4. Borns, M., Cline, J., and Hogrefe, H. (1999) Strategies 12: 40-42.

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


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