Patent ID: 11884972
Assignee: CARLSBERG A/S
Field: Biotechnology (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 18:
19. A method of identifying a sub-pool of organisms from a predefined species or reproductive parts thereof, wherein the sub-pool comprises an organism with one or more predetermined mutations in one or more nucleotides of interest (NOI) in a target sequence, wherein the predetermined mutation is a deletion or substitution of one or more NOIs, said method comprising the steps of:
a) Subjecting the organisms of said species, or reproductive parts thereof, to random mutagenesis, thereby providing a pool of organisms from said species or reproductive parts thereof, the pool representing a plurality of genotypes;
b) Dividing said pool into one or more sub-pools of organisms or reproductive parts thereof, wherein each sub-pool comprises more than one individual organism or reproductive part thereof of each genotype, wherein all progeny of one organism or reproductive part thereof from said pool are comprised within one sub-pool;
c) Randomly dividing the one or more sub-pools into parts such that each part is intended to comprise organisms or reproductive parts thereof representing each genotype of the sub-pool;
d) Preparing genomic DNA (gDNA) samples from one of said parts and storing the remainder of organisms or reproductive parts thereof under conditions maintaining the reproductive potential;
e) Performing a plurality of PCR amplifications, each amplification comprising the gDNA sample from the parts of one sub-pool, and further comprising one or more sets of primers, wherein each set of primers flanks a target sequence, and PCR reagents, thereby amplifying one or more target sequences; and
f) Detecting one or more PCR amplification products comprising one or more target sequences, said one or more target sequences comprising the one or more mutations in the one or more NOI, thereby identifying one or more sub-pools comprising said one or more mutations,, wherein the pool comprises at least 100,000 organisms or reproductive parts thereof; or, wherein each sub-pool comprises at least 100 organisms or reproductive parts thereof with different genotypes.