Confronting two biomolecular techniques to detect NRF2 gene polymorphism biomarkers. (11th December 2018) - Chiarella, Pieranna
Aim: Gene polymorphism biomarkers identify individual susceptibility to environmental and occupational hazards. The conventional approach considers polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP), a reliable but expensive and time-consuming two-step procedure. Therefore we evaluated the simpler method confronting two-pair primers (CTPP)–PCR for its robustness and applicability to epidemiologic studies.Materials & methods: We compared CTPP–PCR and PCR–RFLP techniques to detect two NRF2 polymorphisms in a set of biological samples.Results: CTPP–PCR produced contradictory results and required the orthogonal technique for confirming the data.Conclusion: In contrast to PCR-RFLP, CTPP–PCR of NRF2 polymorphisms resulted in ambiguous genotyping which strongly jeopardized heterozygosis classification. The necessity of long optimization and control procedures nullified the potential advantages of CTPP–PCR in terms of costs and time. Lay abstract: Confronting two-pair primers-polymerase chain reaction (CTPP–PCR) is potentially a fast, simple and cost-efficient method for the detection of gene polymorphism biomarkers. This technique was used to genotype two SNPs of NRF2 gene in a set of biological samples but resulted in ambiguous results. Therefore, it was necessary to validate the CTPP–PCR genotyping data with the orthogonal technique PCR-RFLP. Since reproducibility is a critical issue in both biomedical research and diagnostics, we advise pairing CTPP–PCR with control methods and evaluating whether the overall package is still convenient for massive genetic screening.