Patent SG-11201811021Y-A (SG)
Title: Method to screen for a mutant within a population of organisms by applying a pooling and splitting approach
Inventor: WENDT TONI
IPC: C12N15/85
Filed: 2017-06-23
Abstract: 12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 04 January 2018 (04.01.2018) W I PO I PCT IiiimmoimiolollmonoloolosolimmommovoimIE (10) International Publication Number WO 2018/001884 Al (51) International Patent Classification: C12N 15/01 (2006.01) Cl 2N 15/85 (2006.01) C12N 15/10 (2006.01) (21) International Application Number: PCT/EP2017/065516 (22) International Filing Date: 23 June 2017 (23.06.2017) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: PA 2016 70485 01 July 2016 (01.07.2016) DK (71) Applicant: CARLSBERG A/S [DK/DK]; Ny Carlsberg Vej 100, 1799 Copenhagen (DK). (72) Inventors: WENDT, Toni; Sankt Nikolaj Vej 30 st. tv., 1953 Frederiksberg (DK). OLSEN, Ole; Holmblads- gade 102, 2.tv., 2300 Copenhagen S (DK). KNUDSEN, Soren; Bakkesvinget 18, 2760 Malov (DK). THOMSEN, Hanne, Cecilie; Bodil Ipsens Vej 23, 2500 Valby (DK). STRIEBECK, Alexander; Sankt Nikolaj Vej 22, 3th, 1953 Frederiksberg C (DK). SKADHAUGE, Birgitte; Linde- hojvej 31B, 3460 Birkerod (DK). RASMUSSEN, Mag- nus Wohlfahrt; Eskildsgade 66, 3.th, 1657 Copenhagen V (DK). CARCIOFI, Massimiliano; Venedigvej 7, 02 tv., 2300 Copenhagen (DK). (74) Agent: HOIBERG P/S; Adelgade 12, 1304 Copenhagen K (DK). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (54) Title: METHOD TO SCREEN FOR A MUTANT WITHIN A POPULATION OF ORGANISMS BY APPLYING A POOLING AND SPLITTING APPROACH (57) : In traditional plant breeding approaches, chemical mutagen- esis may be utilized to introduce nucleotide substitutions at random in the genome of a plant, i.e. without possibilities to control the sites of nucleotide changes. Because of genome complexities, the statistical probability is ex- tremely little when it comes to fmding a predetermined nucleotide substitu- tion. The present invention, however, demonstrates how a novel, alternative use of digital polymerase chain reaction (dPCR), preferably droplet dPCR (ddPCR), is developed to exploit finding of specific nucleotide substitutions in mutated genes. The entire platform comprises a screening method with a library of mutagenized organisms, digital PCR -based systems and a set-up to propagate and analyze identified, mutated organisms. Organism library pool Fraction of organism library pool Secondary sub-pools of = organisms of library pool Sub pool total [D] i t M 1 2 / 3 I 4 Secondary sub-samples —.. from secondary sub-pools of organisms W O 20 18/00 188 4 Al Reproductio 000 0000 ( Mutant organism FIG. 1C Aliquots of secondary sub-samples gDNA analysis (Example of mutant signal in well) 9 [Continued on next page] WO 2018/001884 Al 1111111111111101110111111111111111111111011111111111110111111111111M11E110111111 Declarations under Rule 4.17: — of inventorship (Rule 4.17(iv)) Published: with international search report (Art. 21(3)) with sequence listing part of description (Rule 5.2(a))