Development of a system for identifying alleles of the BoLA-BRD3.2 locus in cattle samples containing degraded DNA

Purpose: Development a system for identifying alleles of the BoLA-DRB3.2 locus aimed at amplifying short target fragments with subsequent Sanger sequencing, suitable for studying samples containing damaged DNA.
Materials and methods. For the first time, a test system for sequencing the polymorphic region of the BoLA-DRB3 gene according to Sanger was developed, suitable for analyzing degraded DNA. The system was tested on 10 modern and 4 ancient cattle samples. Sequence alignment and allele typing were performed using the UGENE v1.32.0 program and the BLAST online resource.
Results. A test system was developed that allows amplifying a fragment of the BoLA-DRB3.2 locus suitable for further analysis using Sanger sequencing and allele typing. Four DNA samples obtained from ancient samples provided by the Institute of Archaeology of the Russian Academy of Sciences were analyzed. A total of 13 samples of modern cattle of different breeds were used as a comparison group. For all studied samples, it was possible to obtain sequences of a highly polymorphic fragment of the second exon of the BoLA-DRB3.2 locus, 114 bp long, located at BTA23:25730221-25730334 (ARS-UCD2.0 assembly, GenBank: GCF_002263795.3). However, some nucleotides were identified ambiguously during sequencing for some samples. It was possible to confidently type alleles for 10 out of 13 samples: the identity of the analyzed sequences with the reference allele sequences was more than 99 %. Three modern samples were identified with a lower identity level (95,536, 96,429 and 98,214 %, respectively, for MOD1, MOD9 and MOD8), which may be due to inaccurate reading of the nucleotide during sequencing.

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