getpdf  NLM-PubMed-Logo  doi: 10.17113/ftb.54.03.16.4230 

Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

 

Remigiusz Arnak, Burcin Altun, Valentina Tosato and Carlo V. Bruschi*
 

Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy





Article history:
Received   April 23, 2015
Accepted   February 26, 2016
cc
 

Key words:
DNA manipulation, multiple-antibiotic resistance, multi-marker vectors, gene knock-out, functional analysis, yeast



Summary:
We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the fi rst vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.




 


*Corresponding author:  email3  chabravo@gmail.com
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