title: PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae.
abstract: BACKGROUND
  Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies.
  
  
  RESULTS
  In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications.
  
  
  CONCLUSIONS
  The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications.
authors.affiliation: christine.bonnet@glycode.fr
authors.forename: Christine
authors.initials: C
authors.lastname: Bonnet
authors.forename: Céline
authors.initials: C
authors.lastname: Rigaud
authors.forename: Emilie
authors.initials: E
authors.lastname: Chanteclaire
authors.forename: Claire
authors.initials: C
authors.lastname: Blandais
authors.forename: Emilie
authors.initials: E
authors.lastname: Tassy-Freches
authors.forename: Christelle
authors.initials: C
authors.lastname: Arico
authors.forename: Christophe
authors.initials: C
authors.lastname: Javaud
mesh_terms: D005800:Genes, Fungal; D005818:Genetic Engineering; D006031:Glycosylation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism