BioTechniques (Aug 2014)

Alternative method of allelic discrimination

  • Rajeswari Avula,
  • Dennis O'Kane,
  • John L. Black

DOI
https://doi.org/10.2144/000114199
Journal volume & issue
Vol. 57, no. 2
pp. 88 – 90

Abstract

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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.

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