A robust closed-tube method for resolving tp53M214K genotypes

Abstract

The tp53M214K zebrafish mutant developed by Berghmans et al 20051 is a versatile platform with which to model a diverse spectrum of human diseases. However, currently available genotyping methods for this mutant require lengthy processes such as restriction digests and outsourced Sanger sequencing. To address this deficiency, we leveraged high-resolution melting analysis (HRMA) technology in conjunction with a parallel, in-tandem wildtype spike-in approach to develop a robust genotyping protocol capable of discriminating tp53M214K zygosity. Here, we describe our method in detail. We anticipate that our genotyping protocol will benefit researchers utilizing the tp53M214K zebrafish mutant by offering reliable results with a faster turnaround time than conventional approaches.