Multiplex genotyping of cytokine gene SNPs using fluorescence bead array

• Published in: PloS one 2015-02, Vol.10 (2), p.e0118008-e0118008
• Main Authors: Jang, Jung-Pil, Baek, In-Cheol, Choi, Eun-Jeong, Kim, Tai-Gyu
• Format: Article
• Language: English
• Subjects: Alleles; Analysis; Autoimmune diseases; Bone morphogenetic proteins; Cancer; Cytokines; Cytokines - genetics; Deoxyribonucleic acid; DNA; DNA Primers - genetics; Flow cytometry; Fluorescence; Gene sequencing; Genes; Genetic testing; Genotyping; Genotyping Techniques - instrumentation; Health aspects; Humans; Interleukin 1 receptor antagonist; Interleukin 1 receptors; Interleukin 10; Interleukin 12; Interleukin 2; Interleukin 4; Interleukin 6; Medicine; Microspheres; Multiplexing; Mutation; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Primers; Quality Control; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Spectrometry, Fluorescence; Stem cells; Transforming growth factor-b1; Transforming growth factors; Transplantation; Tumor necrosis factor-TNF; Tumor necrosis factor-α
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0118008

Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1α, IL-1R, IL-1RA, IL-1β, IL-2, IL-4, IL-4Rα, IL-6, IL-10, IL-12, TGF-β1, and TNF-α, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.