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. 2010 Mar 16;10:21.
doi: 10.1186/1472-6750-10-21.

A Mutant Pfu DNA Polymerase Designed for Advanced Uracil-Excision DNA Engineering

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Free PMC article

A Mutant Pfu DNA Polymerase Designed for Advanced Uracil-Excision DNA Engineering

Morten H H Nørholm. BMC Biotechnol. .
Free PMC article

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
Principle of uracil-excision based DNA fusion. In uracil-excision based DNA fusions, dU nucleotides replace selected thymidine nucleotides in DNA and is subsequently removed by e.g. the USER™ enzyme, leaving the upstream nucleotide sequence unstable. When the resulting, compatible single stranded overhangs are combined they can be transformed directly into bacteria without prior ligation to yield a stable recombinant molecule.
Figure 2
Figure 2
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.
Figure 3
Figure 3
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).
Figure 4
Figure 4
PfuX7 polymerase is compatible with the Kunkel method for template elimination in site-directed mutagenesis. (A) Screen designed to calculate the efficiency of site-directed mutagenesis. By introducing a stop codon mutation in a plasmid responsible for constitutive expression of the green fluorescent protein in E. coli cells, the efficiency of the mutagenesis is easily calculated as the ratio of non-fluorescent cells to total amount of cells. Shown, is a typical example of an experiment using template DNA isolated from either DH5α or CJ236. (B) Agarose gel electrophoresis of PCRs performed with the PfuTurbo- (T) or the PfuX7 (×7) DNA polymerases using template plasmid DNA isolated from the ung+ E. coli strain DH5α or the ung- strain CJ236. The molecular marker (M) is kb+ (Invitrogen). (C) Quantification of the efficiency of site-directed mutagenesis using the PfuTurbo- or the PfuX7 DNA polymerases using template plasmid DNA isolated from the ung+ E. coli strain DH5α or the ung- strain CJ236, with or without DpnI treatment. Data represents the average of three independent experiments with standard deviations.
Figure 5
Figure 5
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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References

    1. Nisson PE, Rashtchian A, Watkins PC. Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Methods Appl. 1991;1(2):120–123. - PubMed
    1. Smith C, Day PJ, Walker MR. Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. PCR Methods Appl. 1993;2(4):328–332. - PubMed
    1. Nour-Eldin HH, Hansen BG, Norholm MH, Jensen JK, Halkier BA. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 2006;34(18):e122. doi: 10.1093/nar/gkl635. - DOI - PMC - PubMed
    1. Bitinaite J, Nichols NM. DNA cloning and engineering by uracil excision. Curr Protoc Mol Biol. 2009;Chapter 3(Unit 3):21. - PubMed
    1. Watson DE, Bennett GN. Cloning and assembly of PCR products using modified primers and DNA repair enzymes. Biotechniques. 1997;23(5):858–862. - PubMed

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