Technique of PCR analysis of MT2A genes in different animal species

Abstract

Metallothioneins are a family of low-molecular-weight cysteine-rich proteins that participate in the metabolism of essential metals and protect against heavy metal toxicity. Metallothioneins are represented by different isoforms in many eukaryotic and prokaryotic species. The promoter regions of metallothionein genes contain sequences of elements that regulate expression in response to metals (MRE), which makes it an urgent task to investigate polymorphisms in the promoter regions of metallothionein genes. Objective. To develop and optimize the technique of PCR analysis of orthologous metallothionein genes in various biological species using the example of the MT2A isoform of rats and pigs. Methods. Analysis of the primary structure of MT2A isoform genes was performed using the NCBI and Ensembl databases. The design ofprimers for PCR analysis was carried out using the Primer3 program. Biomaterial samples were used for the study: livers of Wistar rats and blood of large white pigs of intrabreed type ULW-1. DNA was isolatedfrom rat liver using the NeoPrep DNA Magnet plant DNA kit, and from pig blood using Chelex 100 reagent. Amplification of DNA fragments was carried out using PCR followed by electrophoretic separation of the amplicons in a 2 % agarose gel. Results. In the course of the study, the primary structure of the MT2A genes of rats and pigs was analyzed. Primers were designed to cover the promoter regions of the MT2A genes of these two animal species. The expected sizes of the amplicons are 583 bp and 512 bp for rats andpigs, respectively. The conditions for the synthesis of PCR amplicons were selected, the optimality of which is confirmed by the presence of the corresponding PCR amplicons on the electrophoregram. Conclusions. The developed PCR analysis system for the MT2A genes of rats and pigs allows to study polymorphisms in the promoter regions that contain MRE sites. Considering the importance of MRE sequences for the regulation of metallothioneins’ expression under the influence of metals, as well as the participation of metallothioneins in various physiological and pathological processes, it can be expected that polymorphisms in the studied areas may have associations with adaptation potential, resistance to heavy metals, economic qualities of organisms, etc. In the future, research involving the MT2A gene as a candidate for the development of molecular genetic markers and their use in the practice of marker-associated selection is promising. Keywords: metallothioneins, polymorphism, Rattus norvegicus, Sus scrofa domesticus, polymerase chain reaction, promoter.