Modeling of PCR-RFLP genotyping of cattle by polymorphic markers of iNOS gene

Abstract

Relevance. The study of the Bos taurus iNOS gene polymorphism and its association with resistance to bovine leukemia, as well as with breeding value in terms of milk productivity, is a topical subject of genetic selection research.The purpose of this study was to identify and map polymorphic restriction sites in 4 SNP markers (AH13-1, AH13-2, AH13-3, AH13-4) of the Bos taurus iNOS gene, followed by PCR-RFLP profiling of the encountered genotypes and modeling of the method of gene testing of cattle by the listed polymorphic markers of the analyzed locus.Methods. An effective tool for visualizing the result of computer simulation of the experiment, which contributed to predicting the applicability of the developed method of gene testing, was in silico modeling of the generated PCR-RFLP profiles of the encountered genotypes of polymorphic markers of the Bos taurus iNOS gene. At the same time, the very result of mapping the identified polymorphic restriction sites and subsequent profiling of the encountered genotypes indicated the technical feasibility of detecting polymorphic positions of 4 SNP markers of the Bos taurus iNOS gene by PCR-RFLP analysis. This, in turn, initiated the modeling of the method of gene testing of cattle by the listed polymorphic markers with the provision of successive stages of DNA isolation from whole preserved blood of cattle, PCR with oligonucleotide primers iNOS-F and iNOS-R, RFLP with selected restriction endonucleases (HinfI, Sau96I (AspS9I isoschizomer), HpyAV and MluCI (Sse9I isoschizomer), as well as performing gel electrophoresis detection with visualization of the obtained electrophoregrams in a UV transilluminator.