Alternative Method of Allelic Discrimination
Year: 2014
Type: article
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.
Source: BioTechniques
DOI: 10.2144/000114199
Authors: Rajeswari Avula, Dennis O’Kane, John L. Black
Institution: Mayo Clinic
Language: English
Subfield: Pharmacology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
OpenAlex ID: W2408604627