title: Alternative method of allelic discrimination.
abstract: 5' nuclease assays for allelic discrimination use both a wild-type and a mutant probe. Here we present a new method for genotyping by 5' nuclease assays that dispenses with the mutant probe, using the wild-type probe together with a probe for a reference gene known to be present in two copies to determine the copy number of the wild type allele relative to the reference gene. The copy number of the wild-type allele then determines the genotype: two copies indicates homozygous wild-type; one copy indicates heterozygous; and zero copies indicates homozygous mutant. We were able to use our method to correctly genotype three alleles of the thiopurine methyl transferase (TPMT) gene: TPMT *2 (c.G238C), *3B (c.G460A) and *3C (c.A719G). Our approach can be used as an alternate allelic discrimination strategy that is cost effective when multiple TaqMan assays are performed on a sample.
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: Rajeswari
authors.initials: R
authors.lastname: Avula
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: Dennis
authors.initials: D
authors.lastname: O'Kane
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
authors.forename: John L
authors.initials: JL
authors.lastname: Black
mesh_terms: D000483:Alleles; D056915:DNA Copy Number Variations / Q000235:genetics; D003851:Deoxyribonucleases / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*
chemical_list: D008780:Methyltransferases; C022745:thiopurine methyltransferase; D003851:Deoxyribonucleases
keywords: TaqMan; copy number variation; thiopurine methyl transferase allelic discrimination