One-Step ARMS-PCR for the Detection of SNPs—Using the Example of the <i>PADI4</i> 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.

Keywords

• <i>PADI4</i> (protein arginine deiminase 4)
• SNPs (single nucleotide polymorphisms)
• ARMS-PCR (tetra-primer amplification refractory mutation system PCR)
• PCR-RFLP (restriction fragment length polymorphism analysis)
• HRM-PCR (high resolution melting curve analysis)