Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR

• The Divergent assay is a single-tube real-time PCR reliable for SNV genotyping.
• The assay relies on a primer extension method and intercalating dyes.
• Two completely different allele-specific amplicons are generated during PCR.
• The amplicons could be distinguished as different melt peaks in post-PCR melting analysis.

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR.In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5 °C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157 pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations.