Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays.
To analyse the reliability and accuracy of genotype analysis with high-density oligonucleotide microarrays, this method and other experimental approaches were used to analyse genomic DNA of two popular Saccharomyces cerevisiae laboratory strains. S288C was used for systematic sequencing of 'the' S. cerevisiae genome; CEN.PK113-7D is a popular strain for physiological studies and functional genomics. Random amplified polymorphic DNA, electrophoretic karyotyping and microarray analysis all indicated a high level of sequence similarity between the two strains. In the microarray analysis, as few as 288 (4.5%) of the ca. 6300 represented yeast genes were identified that yielded significantly different hybridisation intensities between the two strains. These could be classified as amplified, absent, or with sequence polymorphism in CEN.PK113-7D compared to S288C. A detailed analysis focused on the subset of 25 genes called absent in CEN.PK113-7D. Among these absent genes, 17 were clustered together on five chromosomes, mainly in subtelomeric regions. Thorough analysis of these regions by polymerase chain reaction (PCR) and restriction fragment length polymorphism confirmed the absence of these genes in CEN.PK113-7D. Surprisingly, three of these regions were not smaller in CEN.PK113-7D chromosomes, indicating that they may harbour unidentified and potentially new sequences. In addition, eight genes called absent by the microarrays were scattered over the chromosomes. Using diagnostic PCR most of these genes were actually found to be present in CEN.PK113-7D, but after sequencing were found to differ significantly at the DNA level from S288C, explaining the poor hybridisation to the arrays. Our results indicate that DNA microarrays are a powerful tool for determining genotypic similarity between different yeast strains. However, to obtain meaningful information at the individual gene level, this method should be backed up by additional techniques.
Daran-Lapujade
Pascale
P
Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands. p.lapujade@tnw.tudelft.nl
Daran
Jean Marc
JM
Kötter
Peter
P
Petit
Thomas
T
Piper
Matthew D W
MD
Pronk
Jack T
JT
D015342:DNA Probes; D004271:DNA, Fungal / Q000032:analysis; D005800:Genes, Fungal; D005838:Genotype; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016366:Open Reading Frames; D012150:Polymorphism, Restriction Fragment Length; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics* / Q000254:growth & development
D015342:DNA Probes; D004271:DNA, Fungal