Razvoj genetskega testa za določanje genotipov beta kazeina A1A1, A2A2 in A1A2

Kozmos, Martin

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Title:	Razvoj genetskega testa za določanje genotipov beta kazeina A1A1, A2A2 in A1A2
Authors:	ID Kozmos, Martin (Author) ID Langerholc, Tomaž (Mentor) More about this mentor... ID Kozmos, Maša (Comentor)
Files:	MAG_Kozmos_Martin_2020.pdf (1,58 MB) MD5: F6C164D964507200F234AE96FD4E88E4 PID: 20.500.12556/dkum/37af6cc5-0215-4ccd-aa0b-905458eb7061
Language:	Slovenian
Work type:	Master's thesis/paper
Typology:	2.09 - Master's Thesis
Organization:	FKBV - Faculty of Agriculture and Life Sciences
Abstract:	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.
Keywords:	beta kazein, qPCR, A2-mleko, genetski test
Place of publishing:	Maribor
Year of publishing:	2020
PID:	20.500.12556/DKUM-77715
NUK URN:	URN:SI:UM:DK:KSQ1YKEZ
Publication date in DKUM:	25.09.2020
Views:	1332
Downloads:	61
Metadata:
Categories:	FKBV
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Licences

License:	CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Link:	http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:	The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:	11.09.2020

Secondary language

Language:	English
Title:	Genetic Test Development for Determining A1A1, A2A2 and A1A2 Genetic Beta-Casein Variants
Abstract:	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.
Keywords:	beta-casein, qPCR, A2 Milk, genetic test

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