Abstract
BackgroundInnate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases. This immune response has been reported to be genetically diverse, due to polymorphisms coded by different genes. For this reason, our purpose was to develop a multiplex assay that allows the genotyping of candidate single nucleotide polymorphisms (SNPs) in innate immune genes.FindingsWe developed three multiplex PCR panels coupled with the minisequencing (SNaPshot) technique (multiplex PCR, multiplex primer extension, and capillary electrophoresis). The panels were tested in a sample set composed of 100 anonymous DNAs from healthy blood donors living in São Miguel Island (Azores, Portugal). Sixteen relevant SNPs among nine genes of the innate immune system – IL1α, IL1β, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14 – were genotyped and validated by direct sequencing, with the exception of one that was undetected by minisequencing. We suggest that these panels can be used in future studies for detection of risk gene variants in several populations and/or diseases.ConclusionsIn summary, we propose a multiplex assay that is able to identify the most frequent candidate SNPs in innate immune genes, using a medium scale genotyping platform. The assays can be used to evaluate the risk gene variants in populations of various geographic origins.
Highlights
Innate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases
In summary, we propose a multiplex assay that is able to identify the most frequent candidate single nucleotide polymorphisms (SNPs) in innate immune genes, using a medium scale genotyping platform
In order to apply this technique to candidate genes studies associated with autoimmune and infectious diseases, we developed a multiplex assay designed to genotype 16 single nucleotide polymorphisms (SNPs) among nine genes of the innate immune system: IL1α, IL1β, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14
Summary
We were able to develop combined multiplex PCR amplifications and SNaPshot reactions that genotypes 15 SNPs among nine innate immune response genes. These multiplex panels can be useful to investigate candidate immune response genes, using a medium scale genotyping platform, rather than singleplex PCR-based techniques. Competing interests The authors declare that they have no competing interests. Authors’ contributions LME developed and performed the multiplex experiments and drafted the manuscript. SMB and MJB did the validation assays by Sanger DNA sequencing. LMV provided scientific orientation and revised the manuscript. All authors read and approved the final manuscript
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