Realtime Exonuclease-Mediated Allelic Discrimination (Read): A Simple Homogeneous Genotyping Assay for Snps at the ABC Gene Loci

Abstract

Aims: Members of the ATP-binding-cassette transporter family are implicated in the traffic of drugs/xenobiotics. Several SNPs in these ATP-binding-cassette genes were previously identified to show evidence of recent positive selection. These recent positive selection SNPs may confer functional effects and account for variation in drug response. To facilitate association studies between these SNPs and drug response, we report the development of a homogeneous (realtime exonuclease-mediated allelic discrimination) assay to genotype these SNPs. Materials & methods: Realtime exonuclease-mediated allelic discrimination involves real-time PCR using a proof-reading enzyme and simultaneous genotype determination by product presence/absence as detected using SYBR^® Green I stain. Results: A total of 29 recent positive selection SNPs from 17 ATP-binding-cassette transporter genes were evaluated. Of the 777 eealtime exonuclease-mediated allelic discrimination assays, 773 genotypes (∼99.5%) were concordant with the Perlegen data and other genotyping methods. Conclusion: Therefore, this simple, robust, rapid, cost-effective single-step, closed-tube assay with a scalable and automatable platform has potential applications in population genetic screening and association studies.

KEYWORDS::

• ABC transporters
• allelic discrimination
• exonuclease
• homogeneous
• SNP genotyping

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2217/3dp-2022-0019

Financial & competing interests disclosure

This work is supported by grants from the Biomedical Reseach Council (BMRC) of Singapore to Samuel S Chong and Caroline G Lee (BMRC 04/1/21/19/210) as well as grants from the Academic Research Fund – Ministry of Education Tier 2 (MOE T206B3105) and the National Medical Research Council (NMRC) (NMRC/0993/2005) to Caroline G Lee. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This work is supported by grants from the Biomedical Reseach Council (BMRC) of Singapore to Samuel S Chong and Caroline G Lee (BMRC 04/1/21/19/210) as well as grants from the Academic Research Fund – Ministry of Education Tier 2 (MOE T206B3105) and the National Medical Research Council (NMRC) (NMRC/0993/2005) to Caroline G Lee. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.