Use of allele specific pcr to investigate the presence of β-casein polymorphism in holstein-friesian cows

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Marko Ristanic
Aleksandar Niksic
Mia Niketic
Stefan Jelisic
Milan Rajkovic
Uros Glavinic
Zoran Stanimirovic


Following the “one health” principle, we have conducted optimization of a protocol for β-casein genotyping in cattle in order to select cows with exclusively the A2A2 genotype. Gastrointestinal proteolysis of A1 β-casein in humans releases beta-casomorphin 7, which is believed to cause a number of diseases/conditions (diabetes mellitus type 1, ischemic heart disease, atherosclerosis, sudden infant death syndrome, autism, schizophrenia, gastrointestinal discomfort, and prolonged gastrointestinal passage time). On the contrary, A2 β-casein does not cause similar effects on human health, due to its different metabolism. DNA extraction was conducted from blood samples belonging to the laboratory archive of the Department of Biology, Faculty of Veterinary Medicine, University of Belgrade. Determination of genotypes was performed using the Allele Specific Polymerase Chain Reaction (AS-PCR) method. The amplification was preceded by determination of proper primer annealing temperature (65.50 °C), in order to ensure optimal genotyping results. The results obtained indicated a higher frequency of the A2 allele (0.56) compared to the A1 allele (0.44). Furthermore, in 7 out of 35 tested samples, the A1A1 genotype (20.00%) was found, in 17 samples, the A1A2 genotype (48.60%) was found, and in 11 samples, the A2A2 genotype (31.40%) was found. The molecular methods used ensured reliable β-casein genotyping that would enable selection of cows with the A2A2 β-casein genotype, implying production of milk free of the undesirable A1 β-casein protein with all its potential negative impacts on human health.


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Ristanic, M., Niksic, A. ., Niketic, M. ., Jelisic, S., Rajkovic, M., Glavinic, U., & Stanimirovic, Z. (2022). Use of allele specific pcr to investigate the presence of β-casein polymorphism in holstein-friesian cows. Veterinarski Glasnik, 76(1).
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