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, MgCl 2 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.