Synteny analysis provides a route to design genus-specific PCR primers for rapid identification of all Saccharomyces species.
The genus Saccharomyces comprises seven single-genome species (S. arboricola, S. cerevisiae, S. eubayanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum) and two hybrid species - S. pastorianus (S. cerevisiae plus S. eubayanus) and S. bayanus (mostly S. uvarum plus S. eubayanus). Species-specific primers have already been developed for the identification of each of the single-genome species, and these primers can usually detect both genomes in hybrids. It would be advantageous if a single reaction could detect any member of the clade. We have investigated three potentially generic approaches to design genus-specific primers. Two methods that both use sequence alignment differences for primer design were only partly successful. A third method used synteny data to identify 136 target genes that are potentially present only in all species of the Saccharomyces clade. HSP30 (YCR021C) was fully successful; different primer pairs were developed with high G+C content for use at 63 °C. In < 3 h, using a robust colony-PCR followed by gel electrophoresis, the method can reliably detect any member of the genus. This novel approach still uses conventional sequence alignment mismatches but relies principally on the presence of the target gene only within the genus Saccharomyces.
Department of Biology and Biochemistry, University of Bath, Bath, UK.
Ben
B
Sharpe
Michelle
M
Hulin
James
J
Thorne-Wallis
Alan
A
Wheals
D001482:Base Composition; D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D004586:Electrophoresis; D005656:Fungal Proteins / Q000235:genetics; D050890:HSP30 Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012440:Saccharomyces / Q000145:classification* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D026801:Synteny; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D050890:HSP30 Heat-Shock Proteins
colony-PCR; species-specific