Optimization of apa gene replication in Mycobacterium avium subsp paratuberculosis the usage of dimethyl sulfoxide (DMSO)

Mycobacterium avium subsp. paratuberculosis is a remarkably slow-growing bacterium identified as the causative agent of Johne’s disease. This disease causes economic losses especially in dairy cattle breeding, all over the world. Considering the similar clinical and pathological symptoms of Crohn's disease and Johne's disease, it is probable that this bacterium plays a role in the occurrence and progression of Crohn's disease. Hence the potential transmission of MAP through milk has raised significant concerns. Examination of the apa gene sequence in the NCBI database indicates a substantial prevalence of cytosine and guanine nucleotides, signifying elevated GC content. This leads to the disruption of gene amplification throughout the PCR cycle. The abundance of GC-rich regions has caused various problems due to the formation of secondary structures, such as needle-like or ring-shaped topologies in DNA pattern. The aim of this study was the feasibility of enhancing a portion of the apa gene through the modification of dimethyl sulfoxide (DMSO) concentration. A bioinformatics analysis utilizing CpG Plot and Mfold was performed to evaluate the GC content and probable secondary structures of the targeted region of the apa gene. According to the PCR design, the apa gene was amplified using 3μl of DMSO at an annealing temperature of 55 °C, without any unspecific bands. The results of this study demonstrated that apa gene amplification was unachievable without DMSO, and the application of this substance facilitated the gene's amplification.