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Pfu Turbo DNA polymerase was ..

(exo-) DNA polymerase, Pfu Turbo DNA ..

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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.

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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.

Pfu Turbo DNA polymerase, Taq ..

employing the high-fidelity Pfu Turbo DNA polymerase ..

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.

PfuX7 accepts dUTP in place of dTTP in PCR. Agarose gel electrophoresis of PCRs performed with the Phusion (S7) or the PfuX7 (×7) DNA polymerases and either standard dNTPs or with a dUTP/dNTP mix where dUTP replaces dTTP. The molecular marker (M) is kb+ (Invitrogen).

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A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering SpringerLink

To overcome that problem, Thilly used thermolabile polymers already known to have lower error rates to create high-fidelity PCR. When a high-fidelity thermostable polymerase, Pfu, became available, his lab adapted it to measurement of mutations at levels as low as one in a million copies. This mixes the value of Pfu, which has the best fidelity of any thermostable polymerase, with its use in combination with Taq in creating long copies, the approach initially performed by Barnes.

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.

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Role of trehalose in heat and desiccation tolerance in …

Principle of whole plasmid synthesis (WHOPS) and uracil-excision based site-directed mutagenesis (U-SM). (A) In WHOPS, two usually perfectly overlapping oligonucleotides are used to amplify an entire plasmid. WHOPS is used for site-directed mutagenesis by placing mutations (illustrated with an X) in the middle of the two primers. (B) Oligonucleotides for U-SM need only to overlap in the complementary region between the selected A- and T-nucleotides and only one primer needs to carry the mutation. (C) U-SM is compatible with making insertions (illustrated with a loop) and (D) deletions (missing sequence illustrated with a dashed line). (E) Larger insertions, such as in e.g. whole gene fusions, are made by a simple combination of the U-SM principle and uracil-excision based cloning - or even multiple fragments (not shown) as in the uracil-excision based PCR fusion principle.

Role of trehalose in heat and desiccation tolerance in the soil ..

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.

Putative genes for trehalose synthesis (otsAB ..

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.

Stress tolerant transgenic plants over-expressing ascorbic ..

A new Pfu-sso7d fusion DNA polymerase that is compatible with uracil-excision cloning. (A) Illustration of the modular structure of the Pfu-sso7d DNA polymerase and the oligonucleotides used to fuse the Sso7d gene to Pfu. (B) Agarose gel electrophoresis of whole plasmid synthesis PCRs performed with five different Pfu-based DNA polymerases and either standard (normal) oligonucleotides or dU-containing primers. The five different DNA polymerases are PfuTurbo (T), Phusion (S7), PfuTurboCX (TX), Pfu-(V93Q) (X) or PfuX7 (×7) and the molecular marker (M) is kb+ (Invitrogen).

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