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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Barnett M. P., McNabb W. C., Roy N. C., Woodford K. B., Clarke A. J. 2014. Dietary A1 β-casein affects gastrointestinal transit time, dipeptidyl peptidase-4 activity, and inflammatory status relative to A2 β-casein in Wistar rats. International Journal of Food Science, 65:720-727. 260.

Cade R., Privette M., Fregly M., Rowland N., Sun Z., Zele V. 2000. Autism and schizophrenia: Intestinal disorders. Nutritional Neuroscience, 3:57–72.

Farrell H. M., Jimenez-Flores R., Bleck G. T., Brown E. M., Butler J. E., Creamer L. K., Hicks C. L., Hollar C. M., Ng-Kwai-Hang K. F., Swaisgood H. E. 2004. Nomenclature of the proteins of cows’ milk—sixth revision. Journal of Dairy Science, 87:1641-1674.

Gallinat J. L., Qanbari S., Drögemüller C., Pimentel E. C. G., Thaller G., Tetens J. 2013. DNA-based identification of novel bovine casein gene variants. Journal of Dairy Science, 96(1): 699-709.

Ganguly I., Kumar S., Gaur G. K., Singh U., Kumar A., Kumar S., Mann S., Sharma A. 2013. Status of β-casein (CSN2) Polymorphism in Frieswal (HF X Sahiwal Crossbred) Cattle. International Journal of Biotechnology and Bioengineering Research, 4(3): 249-256

Gustavsson F., Buitenhuis A. J., Johansson M., Bertelsen H. P., Glantz M., Poulsen N. A., Månsson H. L., Stålhammar H., Larsen L. B., Bendixen C., Paulsson M. 2014. Effects of breed and casein genetic variants on protein profile in milk from Swedish Red, Danish Holstein, and Danish Jersey cows. Journal of Dairy Science, 97(6): 3866-3877.

Haq M. R., Kapila R., Shandilya U. K., Kapila S. 2014. Impact of milk derived β-casomorphins on physiological functions and trends in research: a review. International Journal of Food Properties, 17(8): 1726-1741.

Jianqin S., Leiming X., Lu X., Yelland G. W., Ni J., Clarke A. J. 2015. Effects of milk containing only A2 beta casein versus milk containing both A1 and A2 beta casein proteins on gastrointestinal physiology, symptoms of discomfort, and cognitive behavior of people with self-reported intolerance to traditional cows’ milk. Nutrition Journal, 15(1): 35. doi: 10.1186/s12937-016-0147-z

Kamiński S., Ruść A., Cieślińska A. 2006. A note on frequency of A1 and A2 variants of bovine beta-casein locus in Polish Holstein bulls. Journal of Animal and Feed Sciences, 15:195-98. doi:

Kostyra E., Sienkiewicz-Szłapka E., Jarmołowska B., Krawczuk S., Kostyra H. 2004. Opioid peptides derived from milk proteins. Polish Journal of Food and Nutrition Sciences, 13:25-35.

Laugesen M, Elliott R. 2003. Ischaemic heart disease, Type 1 diabetes, and cow milk A1 beta-casein. The New Zealand Medical Journal, 116: 1168.

Malarmathi M., Senthil K. T., Parthiban M., Muthuramalingam T., Palanisammi A. 2014. Analysis of ß-casein gene for A1 and A2 genotype using allele specific PCR in Kangeyam and Holstein-Friesian crossbred cattle in Tamil Nadu. The Indian Journal of Animal Sciences, 43: 310-315. doi: 10.2478/acve-2020-0037

Massella, E., Piva S., Giacometti F., Liuzzo G., Zambrini A. V., Serraino A. 2017. Evaluation of bovine beta casein polymorphism in two dairy farms located in northern Italy. Italian journal of food safety, 6(3): 6904. doi: 10.4081/ijfs.2017.6904

Molee A., Boonek L., Rungsakinnin N. 2011. The effect of beta and kappa casein genes on milk yield and milk composition in different percentages of Holstein in crossbred dairy cattle. Animal Science Journal, 82: 512–516.

Pal S., Woodford K., Kukuljan S., Ho S. 2015. Milk intolerance, beta-casein and lactose. Nutrients, 7(9): 7285-7297.

Ristanic M., Glavinić U., Vejnović B., Maletić M., Kirovski D., Teodorović V., Stanimirović Z. 2020. Beta-casein gene polymorphism in Serbian Holstein-Friesian cows and its relationship with milk production traits. Acta Veterinaria, 70:497-510. doi: 10.2478/acve-2020-0037

Roginski H., Fuquay J. W., Fox P. F. 2003. Encyclopedia of dairy sciences, Volumes 1-4 pp. 2799

Schopen G. C. B., Heck J. M., Bovenhuis H., Visker M. H. P. W., van Valenberg H. J. F., van Arendonk J. A. M. 2009. Genetic parameters for major milk proteins in Dutch Holstein-Friesians. Journal of Dairy Science, 92: 1182-1191.

Singh L.V., Jayakumar S., Sharma A., Gupta S. K., Dixit S. P., Gupta N., Gupta S. C. 2015. Comparative screening of single nucleotide polymorphisms in β-casein and κ-casein gene in different livestock breeds of India. Meta gene, 4: 85-91.

Sun Z., Zhang Z., Wang X., Cade R., Elmir Z., Fregly M. 2003.: Relation of β-casomorphin to apnea in sudden infant death syndrome. Peptides, 24: 937-943.

Tailford K. A., Berry C. L., Thomas A. C., Campbell J. H. 2003. A casein variant in cow’s milk is atherogenic. Atherosclerosis, 170:13-19. S0021-9150(03)00131-X

Visker M. H. P. W., Dibbits B. W., Kinders S. M., van Valenberg H. J. F., van Arendonk J. A. M., Bovenhuis H. 2011. Association of bovine β‐casein protein variant I with milk production and milk protein composition. Animal Genetics, 42:212-218.

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