Methods of conducting real-time PCR for genotyping cattle by analyzed SNP markers of the iNOS gene

Modern development of genetic and selection technologies requires in-depth study of molecular genetic mechanisms determining cattle resistance to leukemia. In this context, the study of the iNOS gene polymorphism in Bos taurus is of particular scientific and practical importance, since its results can be used to improve breeding programs aimed at increasing both animal productivity and their resistance to chronic infectious diseases. The aim of this study was to develop methods for real-time PCR in the format of hybridization-fluorescence detection of single nucleotide polymorphisms for genotyping cattle by SNP markers AH13-1 and AH13-6 of the iNOS gene. The design of modified and unmodified oligonucleotides forming for a certain polymorphic marker their own set of 5′-fluorescently labeled allele-specific primers, an anti-primer labeled with a fluorescence quencher from the 3′-end of the oligonucleotide, and a common primer was performed in the OligoAnalyzer 1.2 program. The developed methods tested in this work belong to a variety of anti-primer-mediated quantitative real-time PCR, the hybridization-fluorescence detection format of which ensures correct interpretation of the fluorescence signal growth curve data. Their reliability is supported by PCR-RFLP analysis with selected primers and restriction endonucleases, which are also capable of identifying the genotypes of the sought SNP markers. Moreover, the proposed methods for conducting real-time PCR are more expressive compared to PCR-RFLP analysis, since they do not require time-consuming procedures of endonuclease cleavage and subsequent electrophoretic separation of the generated fragments.