US 11,884,972 B2
Method to screen for a mutant within a population of organisms by applying a pooling and splitting approach
Toni Wendt, Frederiksberg (DK); Ole Olsen, Copenhagen S (DK); Søren Knudsen, Malov (DK); Hanne Cecille Thomsen, Valby (DK); Birgitte Skadhauge, Birkerod (DK); Magnus Wohlfahrt Rasmussen, Copenhagen V (DK); Massimiliano Carciofi, Copenhagen (DK); and Alexander Striebeck, Frederiksberg C (DK)
Assigned to Carlsberg A/S, Copenhagen V (DK)
Appl. No. 16/311,450
Filed by Carlsberg A/S, Copenhagen (DK)
PCT Filed Jun. 23, 2017, PCT No. PCT/EP2017/065516
§ 371(c)(1), (2) Date Dec. 19, 2018,
PCT Pub. No. WO2018/001884, PCT Pub. Date Jan. 4, 2018.
Claims priority of application No. PA201670485 (DK), filed on Jul. 1, 2016.
Prior Publication US 2019/0194723 A1, Jun. 27, 2019
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/686 (2018.01); C12N 15/01 (2006.01); C12N 15/10 (2006.01); A01H 6/46 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6876 (2018.01); C40B 30/00 (2006.01); C40B 40/02 (2006.01)
CPC C12Q 1/686 (2013.01) [A01H 6/4624 (2018.05); C12N 15/01 (2013.01); C12N 15/1024 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6876 (2013.01); C40B 30/00 (2013.01); C40B 40/02 (2013.01)] 22 Claims
 
1. A method of identifying an organism of a predefined species carrying one or more predetermined mutations in one or more nucleotides of interest (NOIs) 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, representing a plurality of genotypes;
b) Dividing said pool into one or more sub-pools of organisms or reproductive parts thereof;
c) Preparing genomic DNA (gDNA) samples, each sample comprising gDNA from each genotype within a sub-pool, while maintaining the potential for multiplication of organisms of each genotype within said sub-pool;
d) Performing a plurality of PCR amplifications, each comprising (i) the gDNA sample from one sub-pool, (ii) one or more sets of primers, each set flanking one or more target sequences in the gDNA sample, and (iii) PCR reagents; thereby amplifying the one or more target sequences, wherein each PCR amplification comprises a plurality of compartmentalized PCR amplifications, each compartmentalized PCR amplification comprising part of said gDNA sample;
e) Detecting one or more PCR amplification products from (d), the one or more amplification products comprising one or more target sequences comprising the one or more predetermined mutations in the one or more NOIs, thereby identifying one or more sub-pools that comprise said one or more predetermined mutations;
f) Dividing the organisms or reproductive parts thereof of the one or more identified sub-pools of (e) into secondary sub-pools;
g) 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;
h) Performing a plurality of PCR amplifications, each comprising (i) the gDNA sample from one secondary sub-pool, (ii) one or more sets of primers, each set flanking one or more target sequences in the gDNA sample, and (iii) PCR reagents, thereby amplifying the one or more target sequences;
i) Detecting PCR amplification products from (h) that comprise one or more target sequences comprising the one or more predetermined mutations in the one or more NOIs, thereby identifying one or more secondary sub-pools that comprise said one or more mutations; and
j) Identifying an organism or reproductive part thereof within one or more secondary sub-pools that carries said one or more predetermined 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.