title: Validation of TaqMan® SNP genotyping specificity for rs12979860 of IL-28B: modeling primer specificity in vitro.
abstract: Members of the type III interferon gene family arose by gene duplication events and have retained a high percent identity both in their coding and non-coding regions. In this study, the specificity of a widely used TaqMan(®) SNP genotyping assay for rs12979860 is validated. The 66 bp template for SNP genotyping has only 3 bp at one 5' end that vary between IL-28B and IL-28A; excluding the rs12979860 SNP itself. Conflicting annealing temperatures were found for the mismatched 19 bp primer to IL-28B and IL-28A with in silico melting temperature algorithms, or with in vitro dissociation curves. In order to prove specificity for IL-28B, an in vitro competition assay was setup with genomic DNA and synthetic oligonucleotides. When genomic DNA, containing equimolar concentrations of rs12979860 and the homologous region of IL-28A are present, no off-target amplification was observed. This SNP genotyping assay is therefore specific for rs12979860 and all previously reported results are valid. Finally, using a completely synthetic in vitro competition assay it was possible to calculate the amount of off-target template that will produce 1/2 the maximum on-target (VIC) fluorescent signal, a value that is between a C/C genotype and a T/T genotype. This value is defined in the manuscript as the half maximum positive value, KHPV, and in the present assay KHPV is 15.75±0.0721, represented as the relative fold increase in the amount of IL-28A over rs12979860. This method will be of interest to those performing genotyping on highly conserved gene families.
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada. Electronic address: brad.s.thomas@gmail.com.
authors.forename: Brad S
authors.initials: BS
authors.lastname: Thomas
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Joyce
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada.
authors.forename: Aviad
authors.initials: A
authors.lastname: Levin
authors.affiliation: Department of Medical Microbiology and Immunology, Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2E1, Canada. Electronic address: lorne.tyrrell@ualberta.ca.
authors.forename: D Lorne J
authors.initials: DL
authors.lastname: Tyrrell
mesh_terms: D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007372:Interferons; D007378:Interleukins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013696:Temperature; D000095022:Interferon Lambda
chemical_list: D007372:Interferons; D007378:Interleukins; C000624353:interferon-lambda, human; D000095022:Interferon Lambda
keywords: HCV; IL-28B/IFNλ3; Off-target amplification; QRT-PCR; SNP genotyping; rs12979860