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.jpg): Failed to open stream: No such file or directory in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 117 J+Biol+Eng
2015 ; 9
(ä): 15
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English Wikipedia
QuickStep-Cloning: a sequence-independent, ligation-free method for rapid
construction of recombinant plasmids
#MMPMID26388935
Jajesniak P
; Wong TS
J Biol Eng
2015[]; 9
(ä): 15
PMID26388935
show ga
BACKGROUND: Molecular cloning is an essential step in biological engineering.
Methods involving megaprimer-based PCR of a whole plasmid are promising
alternatives to the traditional restriction-ligation-based molecular cloning.
Their widespread use, however, is hampered by some of their inherent
characteristics, e.g., linear amplification, use of self-annealing megaprimers
and difficulty with performing point insertion of DNA. These limitations result
in low product yield and reduced flexibility in the design of a genetic
construct. RESULT: Here, we present a novel technique of directional cloning,
which overcomes these problems yet retaining the simplicity of whole-plasmid
amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer
pair with 3'-overhangs, and hence, facilitates the subsequent exponential
whole-plasmid amplification. QuickStep-Cloning generates nicked-circular
plasmids, thereby permitting direct bacterial transformation without DNA
ligation. It allows DNA fragment integration into any plasmid at any position, in
an efficient, time- and cost-effective manner, without tedious intermediate DNA
gel purification, modified oligonucleotides, specialty enzymes and
ultra-competent cells. The method is compatible with competent E. coli cells
prepared using the conventional calcium chloride method. CONCLUSION:
QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an
excellent addition to the cloning toolbox, for the benefit of protein engineers,
metabolic engineers and synthetic biologists.