A Robust Closed-Tube Method for Resolving tp53^M214K Genotypes

The tp53^M214K zebrafish mutant is a versatile platform with which to model a diverse spectrum of human diseases. However, currently available genotyping methods for this mutant require lengthy hands-on processes such as restriction digests and outsourced Sanger sequencing. To address this deficiency, we leveraged high-resolution melting analysis technology in conjunction with a parallel, in-tandem wild-type spike-in approach to develop a robust genotyping protocol capable of discriminating tp53^M214K zygosity. In this study, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the tp53^M214K zebrafish mutant by offering reliable results with a shorter turnaround time, lower personnel involvement, and higher throughput than traditional methods, thereby decreasing the burden of genotyping and maximizing research efficiency.