Sequence-Specific Primer Polymerase Chain Reaction Genotyping of the Human IL33 Polymorphism rs1929992 (T > C) with Sanger Sequencing Validation.

Single-nucleotide polymorphisms (SNPs) may influence gene expression and contribute to interindividual variability in immune responses and disease susceptibility. Interleukin-33 (IL33), a cytokine involved in inflammation, immune regulation, epithelial barrier homeostasis, and tissue remodeling, has been implicated in allergic, autoimmune, inflammatory, and neoplastic disorders. The IL33 rs1929992 (T > C) polymorphism, located in intron 3, has been associated with altered IL33 expression, with the T allele linked to increased cytokine production compared with the C allele. This article describes a standardized sequence-specific primer polymerase chain reaction (PCR-SSP) protocol for genotyping the human IL33 rs1929992 polymorphism, with validation by Sanger sequencing. The protocol includes optimization of annealing temperature, MgCl2 concentration, primer balance, deoxynucleotide triphosphates (dNTPs), and DNA input to ensure amplification specificity and reproducibility. Allele-specific amplification is achieved using forward primers specific for the T and C alleles in independent reactions and analyzed by agarose gel electrophoresis, with the human growth hormone (HGH) gene used as an internal control. This protocol provides a simple, reproducible, and accessible method for rs1929992 genotyping, suitable for population-based and genetic association studies involving immune-mediated and inflammatory diseases. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: PCR-SSP genotyping of IL33 rs1929992 Support Protocol 1: Extraction of genomic DNA by salting-out.