Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR.
BACKGROUND
Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression. The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli. In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated.


RESULTS
The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF). Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing.


CONCLUSIONS
In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective.
Antolovic
Dalibor
D
Department of Surgery, University of Heidelberg, Germany. dalibor.antolovic@med.uni-heidelberg.de <dalibor.antolovic@med.uni-heidelberg.de>
Koch
Moritz
M
Bohlmann
Inga
I
Kienle
Peter
P
Büchler
Markus
M
Weitz
Jürgen
J
D000256:Adenoviridae / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D043525:Cytosine Deaminase / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D016158:Genes, p53; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011995:Recombination, Genetic; D019076:Transgenes
D017931:DNA Primers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D043525:Cytosine Deaminase