title: Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.
abstract: 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 157pg 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.
authors.affiliation: Department of Veterinary Medical Sciences, Alma Mater Studiorum University of Bologna, via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy. Electronic address: fabio.gentilini@unibo.it.
authors.forename: Fabio
authors.initials: F
authors.lastname: Gentilini
authors.affiliation: Genefast srl, Via Castelfranco 17/d, 40053 Bazzano, Bologna, Italy. Electronic address: me.turba@libero.it.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Turba
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D002138:Calibration; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards; D003959:Diamines; D004283:Dog Diseases / Q000235:genetics; D004285:Dogs; D034284:Dynamin I / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006359:Heat Exhaustion / Q000235:genetics / Q000662:veterinary; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D013696:Temperature
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D034284:Dynamin I
keywords: Divergent assay; Dogs; Exercise-induced collapse; Genotyping; SNV; Single-tube real-time PCR