Direct DNA hybridization screening of primary yeast transformants in the construction of targeted yeast artificial chromosome (YAC) libraries.
A simple and inexpensive method for direct, large-scale DNA hybridization screening of primary yeast colonies following introduction of foreign DNA into Saccharomyces cerevisiae by spheroplast transformation is presented. An optimally thin layer of standard regeneration agar containing the transformed spheroplasts is spread on selective plates using carefully controlled temperature conditions; the colonies regenerate initially within the thin layer of hardened regeneration agar, but as they grow, greater than 90% of them break the surface and become accessible to direct lifting and screening methods. The technique avoids the manual or mechanized picking of transformants from within the regeneration top agar, does not require specialized (and often expensive) regeneration matrices such as alginate or low-melting-point agarose, and yields transformation efficiencies and screening results identical to those obtained using the standard spheroplast transformation protocol. We demonstrate the utility of this method for the construction of a targeted human-YAC library from a hamster hybrid cell line that contains chromosome 13 as its only human DNA.
Macina
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Wistar Institute, Philadelphia, Pennsylvania 19104.
Riethman
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D015139:Blotting, Southern; D015825:Chromosomes, Fungal; D002882:Chromosomes, Human, Pair 13; D003001:Cloning, Molecular; D015698:Genomic Library; D006801:Humans; D006822:Hybrid Cells; D009693:Nucleic Acid Hybridization; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013104:Spheroplasts / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic