Abstract
The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.
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References
Nisson, P. E., Rashtchian, A., and Watkins, P. C. (1991) Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. Genome Res. 1, 120–123.
Rashtchian, A. (1995) Novel methods for cloning and engineering genes using the polymerase chain reaction. Curr. Opin. Biotech. 6, 30–36.
Watson, D. E., and Bennet, G. N. (1997) Cloning and assembly of PCR products using modified primers and DNA repair enzymes. Biotechniques 23, 858–862.
New England Biolabs. (2004) USER Friendly Cloning Kit (http://www.neb.com/nebecomm/ManualFiles/manualE5500.pdf) (access in August 31st, 2009).
Fogg, M. J., Pearl, L. H., and Connolly, B. A. (2002) Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat. Struct. Biol. 9, 922–927.
Stratagene. (2004) PfuTurbo® Cx Hotstart DNA polymerase (http://www.stratagene.com/manuals/600410.pdf) (access in August 31st, 2009).
Nour-Eldin, H. H., Hansen, B. G., Norholm, M. H. H., Jensen, J. K., and Halkier, B. A. (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res. 34, e122.
Nour-Eldin, H. H., Norholm, M.H., and Halkier, B. A. (2006) Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes. Plant Methods 2, 17.
Geu-Flores, F., Olsen, C., and Halkier, B. (2009) Towards engineering glucosinolates into non-cruciferous plants. Planta 229, 261–270.
Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T., Halkier, B. A. (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 35, e55.
Ossowski, S., Schwab, R., and Weigel, D. (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J. 53, 674–690.
Hamann, T., and Moller, B. L. (2007) Improved cloning and expression of cytochrome P450s and cytochrome P450 reductase in yeast. Protein Expr. Purif. 56, 121–127.
Frandsen, R. J., Andersson, J. A., Kristensen, M. B., and Giese, H. (2008) Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Mol. Biol. 9, 70.
Bitinaite, J., Rubino, M., Varma, K. H., Schildkraut, I., Vaisvila, R., and Vaiskunaite, R. (2007) USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35, 1992–2002.
Bang, D., and Church, G. M. (2008) Gene synthesis by circular assembly amplification. Nature Methods 5, 37–39.
Smith, C., Day, P. J., Walker, M. R. (1993) Generation of cohesive ends on PCR products by UDG-mediated excision of dU, and application for cloning into restriction digest-linearized vectors. Genome Res. 2, 328–332.
Jiang, D., Hatahet, Z., Melamede, R. J., Kow, Y. W., and Wallace, S. S. (1997) Characterization of Escherichia coli Endonuclease VIII. J. Biol. Chem. 272, 32230–32239.
Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide, H., and Wallace, S. S. (1994) Isolation and Characterization of Endonuclease VIII from Escherichia coli. Biochemistry 33, 1255–1264.
Norholm, M. (2010) A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology 10, 21.
Acknowledgements
The development of the methods described herein was financially supported by the Danish Council for Independent Research, through a Technology and Production Sciences (FTP) research grant (J.nr. 274-05-0451). Lis B. Møller is thanked for excellent technical assistance, and both Bjarne G. Hansen and Morten H. H. Nørholm are thanked for constant fruitful collaborations in the development of USER cloning.
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Nour-Eldin, H.H., Geu-Flores, F., Halkier, B.A. (2010). USER Cloning and USER Fusion: The Ideal Cloning Techniques for Small and Big Laboratories. In: Fett-Neto, A. (eds) Plant Secondary Metabolism Engineering. Methods in Molecular Biology, vol 643. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-723-5_13
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DOI: https://doi.org/10.1007/978-1-60761-723-5_13
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