title: Discrimination of SNPs in GC-rich regions using a modified hydrolysis probe chemistry protocol.
abstract: Allelic discrimination using TaqMan 5'-nuclease assay chemistry has been in routine use for many years, and the catalog of Life Technologies' predesigned SNP genotyping assays now exceeds 4 million entries. However, predesigned assays are often not available for genomic regions with a high GC content, nor can an assay necessarily be designed in this type of region using the manufacturer's design pipelines. Additionally, when an assay is available, the performance can be poor when using standard protocols. Here we report a modified allelic discrimination protocol for variants that reside in extremely GC-rich (GC > 75%) regions. The approach resolves fluorescent signal from reference and variant alleles, allowing all samples to be successfully assigned a genotype call. This protocol modification adds an extra step to the standard workflow, but the increased time is a productive compromise to generate high-quality data.
authors.affiliation: The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Alireza
authors.initials: A
authors.lastname: Mowjoodi
authors.affiliation: The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Tara A
authors.initials: TA
authors.lastname: Paton
authors.affiliation: The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada; McLaughlin Centre, University of Toronto, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Scherer
mesh_terms: D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D020862:GC Rich Sequence / Q000235:genetics*; D005838:Genotype; D006868:Hydrolysis; D015335:Molecular Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D015335:Molecular Probes; D004247:DNA
keywords: 5′-nuclease; GC-rich; SNP; SNP genotyping; TaqMan; allelic discrimination