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Received: February 24, 2026

Revised: March 16, 2026

Published: March 30, 2026

 

The article presents the results of a study on model-based optimizing primer design for detecting polymorphism in exon 7 of the cattle STAT5A gene using in silico methods. The analysis was performed with online bioinformatics tools (nucleotide BLAST, Pick Primers) and the NCBI (National Center for Biotechnology Information) nucleotide sequence databases. GenBank accessions AJ237937.1 (Bos taurus partial stat5A, exons 5–19) and KM052370.1 (Bos taurus × Bos indicus isolate J95 STAT5A, exon and partial CDS) were used as reference sequences.

In order to determine the effectiveness of using in silico analysis to optimize the primer system, an adapted, modeled reverse primer with a single‑nucleotide deletion was used. Based on the results of in silico analysis using online bioinformatics tools, mismatch nucleotides were identified in the 3′ region of the reverse primer (five nucleotides), which, as part of the overall primer system, flanks the seventh exon of the STAT5A gene in cattle. These mismatches (mismatch nucleotides) complicate the accurate localization of the amplified fragment in the target DNA and can lead to a decrease in the overall efficiency of amplification, the formation of non-specific fragments, and, ultimately, errors in the genotyping of individuals. Based on the reference sequences AJ237937.1 and KM052370.1 from the GenBank database, the reverse primer was modified; while determining the hybridization parameters, expected amplicon size, and primer melting temperatures: forward: Tm = 60.4 °C, GC = 60%, length = 20 bp; reverse: Tm = 60.5 °C, GC = 52.4%, length = 21 bp; Product length: 215 bp. In silico analysis showed that the modified primer pair is characterized by high specificity and predicted hybridization efficiency, which providing a theoretical basis for its further use in the study of STAT5A polymorphism (exon 7). Analysis of sequence KM052370.1 revealed the need to modify the forward primer – specifically, the position of a thymine in the oligonucleotide sequence. This change does not affect the melting temperature or length, but it underscores the need to verify and correct each sequence individually. Experimental validation under laboratory conditions is planned for future studies.

 

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