Original scientific paper
https://doi.org/10.17113/ftb.54.03.16.4230
Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning
Remigiusz Arnak
; Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy
Burcin Altun
; Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy
Valentina Tosato
; Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy
Carlo V. Bruschi
; Yeast Molecular Genetics Laboratory, ICGEB, AREA Science Park, Padriciano 99, IT-34149 Trieste, Italy
Abstract
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 first 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.
Keywords
DNA manipulation; multiple-antibiotic resistance; multi-marker vectors; gene knock-out; functional analysis; yeast
Hrčak ID:
165222
URI
Publication date:
8.9.2016.
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