20.500.12556/DKUM-77715 Razvoj genetskega testa za določanje genotipov beta kazeina A1A1, A2A2 in A1A2 Genetic Test Development for Determining A1A1, A2A2 and A1A2 Genetic Beta-Casein Variants Kravje mleko predstavlja pomemben vir potrebnih hranil za človeka, vendar pa mnogim ljudem povzroča zdravstvene težave. Alergija na mlečne proteine in preobčutljivost sta med najbolj razširjenimi prehranskimi težavami, za katere je med drugim odgovoren tudi mlečni protein beta kazein oziroma njegova alelna različica A1. Ena od rešitev je selekcija goveda (Bos taurus) na različico beta kazeina A2, pri katerem naj ne bi bilo tovrstnih težav oziroma naj bi se pojavljale v močno zmanjšanem obsegu. V okviru magistrskega dela je bil razvit zanesljiv ter cenovno in časovno ugoden genetski test za ločevanje alelnih različic beta kazeina pri govedu. Pri razvoju testa so bili načrtovani začetni oligonukleotidi, ki so sposobni zanesljivo ločevati ob alelni različici. Skozi optimizacijo je bil razvit protokol za izolacijo dezoksiribonukleinske kisline iz dlake goveda. Z optimizacijo števila ciklov, temperaturnih območij in trajanja posameznih korakov je bil ustvarjen protokol polimerazne verižne reakcije, ki uspešno in zanesljivo pomnoži le tisto obliko beta kazeina, ki je prisotna v izoliranem vzorcu dezoksiribonukleinske kisline. Pregled dobljenih rezultatov in končno določanje oblike beta kazeina (A1A1, A1A2 ali A2A2) v vzorcu dezoksiribonukleinske kisline sta bila izvedena s pomočjo talilnih krivulj. Zanesljivost testa je bila odvisna predvsem od uspešnosti načrtovanja in sprememb začetnih oligonukleotidov, njihove temperature prileganja in števila ciklov polimerazne verižne reakcije. Ta test manjšim kmetom ponuja cenovno ugodno genetsko testiranje govedi na prisotnost A2-beta kazeina in nadaljnjo selekcijo, zbiranje ter trženje mleka in mlečnih izdelkov višje kakovosti. Cow's milk is a source of essential nutrients for humans, but it causes health problems to many people. Milk protein allergies, as well as lactose intolerance are among the most common dietary problems. Among other less known agents, the milk protein beta-casein, and in particular its allelic version A1, are responsible for the observed allergies. One of the solutions to the problem is the selection of cattle (Bos taurus) with an A2-beta-casein genotype, which milk could eliminate or at least mitigate such adverse health effects. In this master's thesis, a reliable and cost-effective genetic test for the determination of allelic variants of beta-casein in cattle was developed. For this purpose, oligonucleotide primers capable of distinguishing allelic variants were designed. A protocol was successfully optimized to isolate DNA from bovine hair. By optimizing the number of cycles, temperature and the duration of individual steps, a PCR protocol was prepared that successfully and reliably amplified specific allelic forms of beta-casein in isolated DNA. An analysis of the results and final determination of beta-casein allelic variant (A1A1, A1A2, or A2A2) in genomic DNA samples was performed using temperature melting curves obtained from qPCR instrument. The reliability of the assay was primarily dependent on the primer design and proper modifications, annealing temperature, and the PCR cycle number. The newly developed test offers smaller farmers a cost-effective genetic testing of cattle for the presence of A2-beta-casein allelic variants and hence further selection, collection, and marketing of higher quality milk and dairy products. beta kazein qPCR A2-mleko genetski test beta-casein qPCR A2 Milk genetic test true true false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-11 08:41:47 2020-09-25 13:19:19 2025-03-26 11:23:36 2020-09-26 03:26:19 2020 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2023-09-25 URN:SI:UM:DK:KSQ1YKEZ MAG_Kozmos_Martin_2020.pdf MAG_Kozmos_Martin_2020.pdf 1 F6C164D964507200F234AE96FD4E88E4 deecabd41ea702d45cd1998fe3e21c0c5bccb0149392d4f8de6e064086ed5e5f e6b7bab1-7c11-11eb-bb7a-00155d0001ca 20.500.12556/dkum/37af6cc5-0215-4ccd-aa0b-905458eb7061 https://dk.um.si/Dokument.php?lang=slv&id=146489 Fakulteta za kmetijstvo in biosistemske vede 0 0 0