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.

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

“…SNP genotypes were determined as previously published [29] , [54] , [55] . Briefly, the probe set to discriminate the rs12979860 discriminates the C and T alleles, where C is the major, and T is the minor-allele [55] . For the rs8099917 SNP, the probe set discriminates the T and G alleles, where T predicts the major, and G is the minor-allele.…”

Hepatitis C and the Impact of Host Genetics

“…2e4 For IL28B, the rs12979860 and rs8099917 SNPs were found to best correlate with the clinical phenotype, so many clinical and research studies were conducted using either of those two PCR assays. The commercially available allele-specific real-time PCR assay targeting rs12979860 has been validated for clinical testing 23,24 and a similar validation of an rs12979860 allele-specific real-time PCR method with an allele-specific primer 25 has been published. Several other methods to perform SNP typing in the IL28B region have been described and compared with each other, including high-resolution melting (HRM), amplification refractory mutation system, PCR/restriction fragment length polymorphism (RFLP), Invader (Third Wave Technologies, Madison, WI), ZNA (Zip Nucleic Acids, New York, NY), SNaPShot PCR (Applied Biosystems, Carlsbad, CA), and direct sequencing.…”