Primer design and restriction site analysis targeting POU domain class 1 transcription factor 1 gene of Friesian Holstein in silico

Abstract

This study was conducted to design primer set and to map restriction site targeting synonymous mutation c.297A>G of the POU domain class 1 transcription factor 1 gene (POU1F1) of Friesian Holstein in silico. Primer set was designed to target exon 3 of the POU1F1 based on nucleotide sequence at GenBank database with accession number NC_037328.1 using primer3. Simulations of amplification targeting POU1F1 fragment and restriction fragment length polymorphism (RFLP) were conducted in silico using serial cloner version 2.6 and NEBcutter version 3.0, respectively. This study used a primer pair (5′-CCT TTT AGA ACT GAG ACT GGC TG-3′ and 5′-CCC ACA GCT GTT AAC AAG CA-3′) that produce 356 bp of estimated product size. Moreover, a synonymous mutation, c.297A>G of the POU1F1, could be detected using BsII restriction enzyme in silico. The BsII did not have restriction site for AA genotype. On the other hand, it could cut the PCR product size into two fragments (167 and 189 bp) for GG genotype. It can be concluded that in silico analysis successfully amplified target region using primer designed in this study and RFLP simulation using BsII could detect synonymous mutation c.297A>G of the POU1F1. Further in vitro study should be conducted to identify c.297A>G in Friesian Holstein population.