A Robust Closed-Tube Method for Resolving tp53 M214K Genotypes

• Published in: Zebrafish Vol. 21; no. 3; p. 250
• Main Authors: Silvius, Katherine M, Kendall, Genevieve C
• Format: Journal Article
• Language: English
• Published: United States 01.06.2024
• Subjects: Animals; Genotype; Genotyping Techniques - methods; Mutation; Tumor Suppressor Protein p53 - genetics; Zebrafish - genetics; Zebrafish Proteins - genetics; tp53; wild-type spike-in; tp53M214K zebrafish mutant; genotyping; HRMA
• DOI: 10.1089/zeb.2023.0030

The 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 zygosity. In this study, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the 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.