DNA technologies for identification of alleles and genotypes of polymorphic markers of the analyzed locus of the Bos taurus iNOS gene

The current area of research in genetic and selection focus, which is necessary in the long term for the reproduction of a highly productive herd of cattle with genetic resistance to leukemia, is the study of the polymorphism of the Bos taurus iNOS gene of in association with the breeding value of animals and their resistance to this chronic infectious disease. The aim of.this study was to develop DNA technologies for identification of alleles and genotypes of SNP markers of the Bos taurus iNOS gene by detection and interpretation of diagnostically significant single nucleotide polymorphisms based on simulated methods of nested PCR with dCAPS primers and SNP genotyping by analysis of direct sequencing data of the PCR product. Construction of dCAPS primers iNOS-F1 and iNOS-R1 was performed using dCAPS Finder 2.0 and OligoAnalyzer 1.2 software. Mapping of polymorphic restriction sites and calculation of PCR-RFLP profiles of possible genotypes of SNP markers were performed using the NEBcutter V2.0 online program. The constructed dCAPS primers were successfully tested in nested PCR as internal oligonucleotide pairs followed by endonuclease digestion of the 123 bp PCR amplification product of the second round with selected restriction enzymes that were significant for identification of a particular polymorphic marker. The 258 bp PCR amplification product of the first round was subjected to capillary DNA sequencing according to Sanger for detection and interpretation of diagnostically significant single nucleotide polymorphisms in the context of identification of alleles and genotypes of SNP markers. The DNA technologies used in this work turned out to be effective approaches to correct genotyping of cattle by SNP markers of the iNOS gene.

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