One-Step ARMS-PCR for the Detection of SNPs—Using the Example of the PADI4 Gene

Abstract

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer’s disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.

Analysis of Polymorphisms rs7093069-IL-2RA, rs7138803-FAIM2, and rs1748033-PADI4 in the Group of Adolescents With Autoimmune Thyroid Diseases

“…It catalyzes the citrilution/deimination of arginine residues of proteins such as histones, thus playing a key role in the histone code and regulation of stem cell maintenance. It is found in the synovial membrane of people with rheumatoid arthritis (20).…”

Genetic polymorphisms associated with polycystic ovary syndrome among Iranian women

“…The main advantage of HRM–PCR is its minimal manual handling, which significantly reduces the chance of error; however, the results of HRM–PCR are not as easy to interpret as those of other methods. Overall, it has been suggested that the most economic and least time‐consuming approach is either ARMS‐PCR or PCR–RFLP because, although these methods involve more handling than HRM‐PCR, their results are far easier to interpret and analyze 130 …”

Single Nucleotide Polymorphisms: A Modern Tool to Screen Plants for Desirable Traits

“…A study by Ehnert et al [45] using tetra ARMS allele specific PCR method described three common single nucleotide polymorphisms in the PADI4 gene involved in diverse post-translational modifications of proteins in eukaryotes. The SNPs which are thought to affect PAD4 expression activity are rs874881, rs11203366 and rs11203367.…”