VALIDATION OF THE RESULTS OF GENOTYPING OF PIGS USING MARKERS OF MITOCHONDRIAL DNA
Abstract
The article provides a method of confirming the transfection of foreign DNA, which leads to a mixed DNA profile of the object under study. Establishing a DNA profile is the process in which a corresponding DNA sample is obtained from a pig (tissue, blood, bristles, etc.) and is amenable to research to establish the origin of the pro-maternal breeds of the studied pigs. Despite the fact that Sus scrofa is a unique biological object, most of the DNA is actually identical to the DNA of other representatives of Sus scrofa. However, it is the specific regions of the polysite system that indicate the difference between the breeds of representatives of the subspecies of wild pig and domesticated, indicating interbreed polymorphism of the mitochondrial genome. Each representative of Sus scrofa inherits a unique combination of polymorphisms from parents. With this in mind, the purpose of the study was to validate the results of genotyping of pigs using polymorphism of mitochondrial DNA markers, to analyze the obtained data to identify the DNA profile of hybrid pigs (Large White × Landrace) × Maxgro. DNA typing to identify the mitochondrial genome of hybrid pigs was performed by examining bristle samples (n=9) and epithelial tissue (n=28) from pig ears. The detected trace prints provide objective evidence that allows us to characterize the prints of cadaveric DNA from other biological objects, discovered at the “scene of the crime” during the selection of samples during the slaughter of pigs at the Globyno meat processing plant. Isolation of DNA from bristles was carried out using Chelex-100 ion exchange resin. However, it was not possible to confirm the results of genotyping, due to the fact that prints of foreign «cadaveric DNA» were found in the studied samples during slaughter at the meat processing plant. This is evidenced by the highly sensitive method of PCR analysis and hydrolysis by TasI endoeuclease of the studied variable site of the D-loop of mtDNA of hybrid pigs, the resulting false-positive result of the eloktrophoregram showed mixed DNA profiles. Before DNA was isolated from the epithelial tissue of the pig’s ear, the samples under study were treated with dry alcohol fire. DNA isolation from epithelial tissue was carried out by the sorbent method using a set of reagents «DNA-Sorb-B». The following haplotypes have been identified: 9 pigs with haplotype C – wild pig subspecies, Landrace, Hampshire, Wales (Ukraine, Poland, France); 5 pigs are representative of the subspecies of wild pigs, breeds of Large White, Wales (Italy) with haplotype G; 5 pigs are carriers of haplotype O – Landrace, wild pig (Sweden) grouped into a European cluster of haplogroup E (E1 and E2); 11 pigs with haplotype N are representatives of a Large White breed, and an Asian-type wild pig, belong to the Asian cluster A(D). Therefore, an important factor determining the validation of the results of genotyping of pigs using mitochondrial DNA markers there is not so much a method of DNA extraction, but a pure sample of the host under study to establish a clear examination of the mitochondrial genome.
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