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MULTIPLEX ANALYSIS OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNP) IN CYTOKINE GENES.
Anne Hutchings PhD , Henry Fortinberry BS , Stacie Jenkins BS and Judith M. Thomas, PhD . Birmingham AL, University of Alabama at Birmingham, 35294, Surgery, Transplant Immunology .

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Individual variation in cytokine protein production has been reported, related to DNA base changes due to single nucleotide polymorphisms (SNP), especially within the promoter regions of TNF-α, IL-10, IL-6, TGF-β and IFN-γ. Also, associations are being uncovered between SNPs and disease incidence. To increase analytical efficiency, we have developed a primer extension assay for the simultaneous analysis of multiple SNPs using the Luminex® platform. SNPs from 3 cytokine loci (TNF-α, IL-6 and IL-10) were investigated. Human DNA samples were amplified in a multiplex format using allele-specific primers previously described for ARMS-PCR protocols. After amplification, a second extension reaction was performed using reagents containing a biotin-labeled dCTP, and allele-specific primers carrying an additional sequence (tag) that was unique to each locus. Finally pre-dyed beads (MiraiBio Inc), containing the complementary sequences to each of the cytokine tag sequences were incubated with the samples, and the biotinylated tags were reacted with streptavidin-phycoerythrin. The fluorescent intensity of the samples was determined using a Luminex-100 instrument. Analysis of approximately 30 samples was compared to results obtained by direct ARMS-PCR and gel electrophoresis with ethidium bromide detection. The results indicate that multiplex analysis of these cytokine SNPs is a practical and efficient alternative method for large-scale analysis.