کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6133716 | 1593473 | 2014 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Validation of TaqMan® SNP genotyping specificity for rs12979860 of IL-28B: Modeling primer specificity in vitro
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موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ویروس شناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 5â² 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 203, July 2014, Pages 39-47
Journal: Journal of Virological Methods - Volume 203, July 2014, Pages 39-47
نویسندگان
Brad S. Thomas, Michael A. Joyce, Aviad Levin, D. Lorne J. Tyrrell,