A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering
© Nørholm. 2010
Received: 21 October 2009
Accepted: 16 March 2010
Published: 16 March 2010
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© Nørholm. 2010
Received: 21 October 2009
Accepted: 16 March 2010
Published: 16 March 2010
The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.
Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.
The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.
In 1999, the crystal structure of the DNA polymerase Tgo from the archea Thermococcus gorgonarius was solved , revealing the nature of the uracil-binding pocket, and allowing the design of mutant Tgo- and Pfu-polymerases with reduced stalling at uracil-containing DNA . With mutants like PfuV93Q, high-fidelity PCR became compatible with uracil-excision cloning. This led to development of an improved uracil-excision cloning technology  and to a new way of doing seamless PCR product fusion that may eventually replace overlapping PCR in many applications [8, 9]. The useful application of these technologies was further demonstrated by their use in artificial gene synthesis .
In addition to PCR, and cloning and fusing genes, site-directed mutagenesis is an indispensable tool for molecular biologists. One of the early methods for doing site-directed mutagenesis was the Kunkel method  that uses template DNA isolated from a ung- dut- E. coli strain. This strain lacks dUTPase and uracil deglycosidase and therefore accumulates soluble dUTP and DNA-bound dU nucleotides. In the method, dU-containing DNA is used as template in a linear amplification reaction with a mutagenic primer, as well as the Klenow enzyme, dNTPs and a ligase. Subsequently, the DNA is transformed into ung+ bacteria, where only the newly synthesized, mutant DNA survives and the primer-introduced mutation is isolated. Later, PCR entered the scene and variants, known as inverse PCR or whole plasmid synthesis (WHOPS), largely seems to have replaced the Kunkel method. In the typical PCR-based approach, template carry-over is inhibited by treatment with the restriction enzyme DpnI, that restricts dam methylated plasmid DNA, but leaves unmethylated PCR-derived DNA intact.
Here, uracil-excision-based artificial gene synthesis is used to create a combination of a PfuV93Q mutant  and a highly processive Pfu-SSo7d fusion polymerase . This new DNA-polymerase has several properties that make it uniquely suitable for several of the described applications, including site-directed mutagenesis of large plasmids, and a combination of the modern WHOPS mutagenesis and the classical Kunkel-method to avoid carry-over of template DNA.
In one of the early PCR-based mutagenesis protocols, WHOPS was performed with non-overlapping phosphorylated primers and was followed by a ligation step prior to transformation of the mutant DNA  - the method became available as the Exsite™ PCR-based Site-Directed Mutagenesis Kit (Stratagene) and a similar product is today available as the Phusion™ Site-directed Mutagenesis Kit (Finnzymes). The uracil-excision site-directed mutagenesis approach is very similar. However, compared to the latter technique, ligation is not necessary and the uracil-defined overlaps add an extra quality insurance step since erroneous mismatching extensions on the amplified plasmid will not yield stable recombinant molecules . In the older approach, any phosphorylated DNA species can be ligated to create a circular recombinant molecule.
Today, probably the most widely used technique is WHOPS with a perfectly overlapping primer pair that negates the need for ligation prior to transformation - commercially available from e.g. Stratagene as the QuikChange® Site-Directed Mutagenesis Kit. Due to the exponential nature of WHOPS, and the availability of modern high-fidelity DNA polymerases, such as the highly processive Pfu-Sso7d fusion protein DNA polymerase , this is a highly efficient technology, but the technique is not as versatile as uracil-excision.
In site-directed mutagenesis, the DpnI template removal step adds extra time to the procedure and template carry-over is an error frequently observed in our lab. PfuX7 allows the combined use of DpnI-treatment and dU-containing DNA to avoid carry-over of template. In our experimental setup, both the Kunkel-method and the use of DpnI was more than 90% efficient in the prevention of template recovery, and it was therefore not possible to detect a large effect of the combined use. However, in a linear amplification mutagenesis strategy, the combined use of DpnI and dU-DNA was previously reported to increase the efficiency of a mutagenesis from 38% (DpnI alone) to 70-91% . Therefore, the Kunkel-approach to limit tempate carry-over could be a useful and time-saving alternative or addition to DpnI-treatment, in site-directed mutagenesis.
Pfu-Sso7d fusion polymerases, such as Phusion, are marketed as polymerases for direct PCR on complex samples such as blood. Indeed, we have found that in some cases PfuX7 is the only DNA polymerase that produces a PCR product in combination with DNA isolated from e.g. plant material (Nour-Eldin, H. H., personal communication), human cDNA (Lange, J. B., personal communication) or when doing WHOPS on large plasmid DNA templates (this work). Forensic PCR deals with complex samples of low quantity and quality, and prevention of carry-over contamination from previous PCRs is important to this field. PfuX7 polymerase performs better than other DNA polymerases in virtually all applications in our laboratory. Furthermore, none of the wildtype Pfu-based versions (including Phusion) have ever been able to amplify dU-containing DNA. Hence, PfuX7 polymerase may be useful in forensic PCR, both due to its high performance as well as compatibility with the well-established UNG-method for prevention of carry-over contamination.
The combination of the USER enzyme and the new PfuX7 polymerase described here, constitutes a simple, yet comprehensive molecular toolkit that enables one-tube, ligase-free cloning , easy conversion or design of compatible vectors , seamless PCR fusions [8, 9], artificial gene synthesis , site-directed mutagenesis and prevention of carry-over contamination. No other single molecular cloning technology exhibits a comparable versatility.
Oligonucleotides used in this work
Sso7d was fused to the C-terminus of PfuV93Q by amplifying part of the sequence upstream from the fusion site with the primers Pfu-upstream-F and Pfu-R, and part of the sequence downstream from the fusion site with the primers Pfu-F and Pfu-downstream-F (Table 1 and Figure 3A). Next, PCR products were mixed with varied concentrations of three pairs of complementary oligonucleotides (Sso7d-1F + Sso7d-1R, Sso7d-2F + Sso7d-2R and Sso7d-3F + Sso7d-3R, Table 1) and treated with the USER enzyme (New England Biolabs, NEB). Fragments were ligated using the Quick ligase kit (NEB), gel purified the product using the Qiagen gel purification kit and finally used as template in a standard Phusion PCR with the primers Pfu-upstream-F and Pfu-downstream-R. The resulting PCR products and the pET-Pfu vector was treated with the SacI- and BlpI-Fastdigest restriction enzymes (Fermentas) and subsequently fragments of the expected size were gel purified as described above. Finally, the Pfu-Sso7d fragment was ligated into the pET-Pfu-(V93Q) vector with the Quick ligase kit and transformed into a standard E. coli cloning strain. The sequence of the Pfu-Sso7d fusion (termed PfuX7) was confirmed by standard DNA sequencing.
The pET-PfuV93Q and pET-PfuX7 plasmids were transformed into BL21 cells and transformants were inoculated in LB with ampicillin (50 μg/ml) overnight (ON). An ON culture was back diluted to an optical density of 0.1 at 600 nm and grown to OD 0.3, before protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranosid for three hours. The culture was then centrifuged at 15.000 g for 10 minutes, the supernatant discarded and the pellet stored at -80°C ON. The his- tagged protein was purified under native conditions using the Qiagen Ni-NTA Spin Kit according to the manufacturers prescriptions. The protein was desalted on Vivaspin 20 columns (Sartorius) columns and stored at -20°C in a solution of 50% glycerol, 100 mM Tris/HCl pH 8.0, 0.2 mM EDTA, 2 mM DTT, 0.2% NP40, 0.2% Tween 20. The protein activity was tested in a dilution series under standard Pfu PCR conditions (as described above) to obtain the optimal performance.
Prof. Tatsuo Kakimoto is thanked for providing the pTEF423- CRE1 plasmid and Jens Lykke-Andersen is thanked for providing the pET-Pfu expression construct. Joanna Slusky, Roger Draheim and Gunnar von Heijne are thanked for comments on the manuscript. The work was supported by a grant from the Lundbeck Foundation.
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