6,000 individual genes are represented in four backgrounds, creating over 20,000 distinct knock out strains
Molecular barcodes allow for parallel analysis of gene function
Analyze individually by sequencing, or using whole genome approaches
Whole gene deletions ensure loss-of-function
Yeast Knockout Strain Genotypes
Strain
Background
Genotype
Mat A
BY4730 MATa leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha
BY4739 MATalpha leu2Δ0 lys2Δ0 ura3Δ0
Mat A
BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
Mat Alpha
BY4742 MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0
Het/Hom Diploid
BY4743 4741/4742
Homozygous diploids are in the BY4743 background unless 4730/4739 is indicated
Creation of the yeast knockout mutants
Cartoon of homologous yeast recombination method used to generate the yeast knockout mutants.
The yeast knock out strains were systematically created using a PCR-based strategy (see Reference 3). By means of two sequential PCR reactions - the first to incorporate the appropriate tags and confer the antibiotic resistance gene and the second to incorporate the mitotic recombination sites - each ORF was replaced with a KanMX cassette using homologous recombination. This method allowed for greater than 95% of the ORFs to be knocked-out. Each cassette contains a unique 20 base pair nucleotide sequence of DNA known as a "molecular barcode" allowing for parallel analysis. Also incorporated is a common set of flanking DNA tag sequences allowing amplification of the unique tags.
By means of two sequential PCR reactions - the first to incorporate the appropriate tags and confer the antibiotic resistance gene and the second to incorporate the mitotic recombination sites - each ORF was replaced with a KanMX cassette using homologous recombination. This method allowed for greater than 95% of the ORFs to be knocked-out.
Start screening sooner with yeast knockouts
Evidence for the functional role of a gene can be obtained by analysis of the phenotypic changes exhibited by each mutant strain under a given condition. While this type of analysis has been performed in the past using traditional genetic screens or random mutagenesis, the yeast knock out strains offer advantages over both of these methods.
One advantage is that the phenotypes of the yeast mutant strains reflect a complete loss of function of the given gene, where this is not assured using classical methods. Furthermore, in contrast to traditional screening, the gene identification is known a priori, thereby removing the time-consuming task of determining the responsible gene.
Also eliminated is the time and tediousness involved in creating a single yeast knock out strain. A single knock out strain is already available for nearly all of the yeast genes in the genome, so all you have to do is request the strain of interest.
Molecular Barcodes Facilitate Whole Genome Approaches
The mutant yeast strains can also be analyzed individually via sequencing or by using a whole genome approach. The molecular barcodes allow for parallel analysis of gene function using oligonucleotide arrays. The fitness of a given strain under a specific condition can be determined by calculating the abundance of that strain's DNA. This is achieved using an oligonucleotide array printed with the complementary tag sequence necessary to detect the presence of a molecular barcode (see References 1 and 2).
A wealth of information - including genetic and physical maps along with DNA sequence information - is available from the Saccharomyces Genome Database (SGD) at Stanford University.
