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2022, № 95

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Development of a genetic test system: Obtaining of cattle beta-casein gene fragments containing nucleotide polymorphisms characteristic for various allelic protein forms

Improving the average standard of living of citizens and increasing the availability of food poses new challenges for agricultural science. Not only the quantity of food comes to the fore, but also ensuring its best possible quality becomes important. Modern methods of biological science make it possible to approach the tasks at the junction of agriculture, food industry and medicine, making food a tool for disease prevention and treatment. A vivid example is the so-called A2-milk, the interest in which, both in terms of preventing allergic and inflammatory emergence and in terms of commercial benefits for producers of milk and dairy products, is growing in the world and in Russia. The biological point of the issue is that upon digestion of cow's milk proteins in the human body, several bioactive peptides evolve that affect the nutritional properties of dairy products. Thus, when beta-casein protein (β-CN) is cleaved, among others, β-casomorphine-7 (BCM7) (amino acid residues 60-66 of β-CN) is produced, a peptide that is generated from beta-casein variants carrying His67 (type A1) instead Pro67 (type A2). The two most abundant variants of β-CN protein are A2 (original) and A1, which differs from A2 in one amino acid substitution (Pro67-His67). Thus, variants with Pro67 are preferred, wherein the C nucleotide is in the second position of the triplet (CCT [Pro]) rather than A as in CAT [His]. Currently, A2-milk is produced in a number of countries, given its potential benefits in the area of human health. Numerous studies in different countries have shown that the BCM7 peptide generated by a cleavage of A1 beta-casein in the gastrointestinal tract may be one of the factors in the development of childhood autism, leading to a delay in psychomotor development and impaired muscle tone. In the proposed project, the object is to develop a set of modified fluorescently labeled primers and create on their basis a kit for qPCR identification of animals carrying the beta-casein gene variant (β-CN), which provides A2 milk production. At present, a relatively cheap, but time- and labor-consuming method for an identification of the mutation of interest based on RFLP analysis is widely used in Russia. This method incudes obtaining a PCR fragment carrying the mutation, its isolation and purification (or exchanging the buffer in which the DNA fragment is resides), processing the resulting fragment with a specific restriction enzyme, and electrophoretic analysis of the cleavage products. In addition to the upscaled requirements for staff qualification, the complexity and duration of this outdated approach at all stages, the test sample can easily be contaminated with DNA of other animals. The issues of the aforementioned method provide reasonable doubts on the results of this type of analysis in a high throughput screening of a large number of cattles. The advantages of proposed real-time PCR approach are accuracy, speed, lack of contamination, reliable identification of homo- and heterozygotes, as well sa the possibility of early postnatal identification of animals bearing the desired genotype. This article is the first part of the publication, which describes the production of beta-casein gene fragments, each of which carries its own variant of the nucleotide polymorphism characteristic of certain protein isoforms. The obtained DNA fragments are necessary for the development of the optimal design of gene-specific primers and allele-specific probes. Using the DNA fragments described in this article, the performance of the developed genetic test system will be tested and the analysis conditions will be optimized.
Keywords: Mutation, polypeptide, hypersensitivity, A2-milk, restriction fragment length polymorphism (RFLP), real-time polymerase chain reaction, herd allelic frequencies.
DOI: 10.21515/1999-1703-95-157-164

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Authors:

1. Knyazeva Valeriya Vladimirovna, postgraduate student, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
2. Garkovenko Alexey Vyacheslavovich, master of biological sciences, LLC "DNA Expertise"; Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
3. Radchenko Vitaliy Vladislavovich, PhD in Biology, Leading Researcher, LLC "DNA Expertise".
4. Koshchaev Andrei Georgievich, DSc in Biology, professor, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.