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The Uracil-Specific Excision Reagent
(short USER) FriendlyTM cloning technology is dependent on the
excision of a single uracil base from either end of a PCR generated insert.
The uracil bases are included in the primers as part of a 9 bp long 5’
extension. Following PCR amplification with the primers, excision of the
uracil bases results in the generation of 9 bp long 3’ overhangs on the PCR
product complementary to 3’ overhangs in the vector fragments. The 3’
overhangs in the vector DNA is generated by the combined cutting of PacI
(standard restriction enzyme) and Nt.Bbc.CI (nicking enzyme).
Annealing of the 9 bp long sequences in the PCR insert and the vector DNA is
stable enough to survive chemical transformation into E. coli. Inside
E. coli the DNA fragments are covalently linked by the formation of
phosphodiester bonds between the free DNA ends.
Figure:
Top)
Vector with the two new unique USER cloning sites, that each consists of a
PacI site, two Nt.BbvC IB sites and two times two variable
bases. The variable bases, shown in yellow, green, gray and pink boxes,
ensure directional cloning of the PCR amplified inserts.
Bottom)
The two vector fragments following the combined cutting of PacI and
Nt.BbvC IB and purification. PacI is a standard restriction
enzyme that cuts both strands, while Nt.BbvC IB is a nicking enzyme
that only cuts one strand.
Instruction for use of the USER Friendly
cloning system for targeted gene replacement in fungi
To obtain the Agrobacterium tumefaciens plasmid with the shown
USER Friendly cloning sites contact:
Rasmus Frandsen by
e-mail (ras(at)bio.dtu.dk)
OR
The Fungal Genetics
Stock Center (http://www.fgsc.net/)
Articles dealing with the
USER Friendly cloning technique:
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Rasmus J.N. Frandsen,
Jens A. Andersson, Matilde B. Kristensen and Henriette Giese: "Efficient
four fragment cloning for the construction of vectors for targeted gene
replacement in filamentous fungi", BMC Molecular Biology 2008, 9:70.
OPEN ACCESS |
| Nour-Eldin
HH, Hansen BG, Nørholm 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. Epub 2006 |
| Bitinaite J,
Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R.: "USER
friendly DNA engineering and cloning method by uracil excision", Nucleic
Acids Res. 2007;35(6):1992-2002. |
| Fernando
Geu-Flores, Hussam H. Nour-Eldin, Morten T. Nielsen, and Barbara A. Halkier:
"USER fusion: a rapid and efficient method for simultaneous fusion and
cloning of multiple PCR products", Nucleic Acids Res. 2007;35(7):e55. Epub
2007 |
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