Method to screen for a mutant within a population of organisms by applying a pooling and splitting approach

A method of identifying a unicellular organism of a predefined species carrying one or more predetermined mutation(s) in nucleotide(s) of interest [NOI(s)], in a target sequence, said method comprising the steps of:

• a) Providing a pool of unicellular organisms of said species representing a plurality of genotypes;
• b) Dividing said pool into one or more sub-pools of organisms, and subjecting each sub-pool to a step of reproduction, so that each sub-pool comprises more than one individual organism of each genotype;
• and
• c) randomly dividing each sub-pool into parts in a manner such that each part in theory comprises organisms representing each genotype of the sub-pool;
• d) Preparing genomic DNA (gDNA) samples from one of said parts of each sub-pool, while storing another part of each sub-pool under conditions maintaining the potential for multiplication of organisms of each genotype within said sub-pool;
• e) Performing a plurality of PCR amplifications, each comprising the gDNA sample from one sub-pool, wherein each PCR amplification is a digital PCR comprising a plurality of compartmentalised PCR amplifications, each compartmentalised PCR amplification comprising part of said gDNA sample, one or more set(s) of primers each set flanking a target sequence and PCR reagents, thereby amplifying the target sequence(s);
• f) Detecting PCR amplification product(s) comprising one or more target sequence(s) comprising the mutation(s) in the NOI(s), thereby identifying sub-pool(s) comprising said mutation(s);
• g) Dividing the organisms of said identified sub-pool into secondary sub-pools;
• h) Preparing gDNA samples, each comprising gDNA from each genotype within a secondary sub-pool, while maintaining the potential for multiplication of organisms of each genotype within said secondary sub-pool;
• i) Performing a plurality of PCR amplifications, each comprising the gDNA sample from one secondary sub-pool, one or more set(s) of primers each set flanking a target sequence and PCR reagents, thereby amplifying the target sequence(s);
• j) Detecting PCR amplification products comprising one or more target sequence(s) comprising the predetermined mutation(s) in the NOI(s), thereby identifying secondary sub-pools under step j) comprising said mutation(s);
• k) Identifying an organism within said secondary sub-pool carrying said mutation(s).