A rapid and efficient method of genotyping zebrafish mutants.

ABSTRACT

In order to facilitate high throughput genotyping of zebrafish, we have developed a novel technique that uses High Resolution Melting Analysis (HRMA) to distinguish wild-type, heterozygous mutants and homogyzous mutants. This one hour technique removes the need for restriction enzymes and agarose gels. The generated melting curve profiles are sensitive enough to detect non-specific PCR products. We have been able to reliably genotype three classes of mutations in zebrafish, including point mutants, apc(hu745) (apc(mcr)), and p53(zy7) (p53(I166T)), a small deletion mutant (bap28(y75)) and a retroviral insertion mutant (wdr43(hi821a)). This technique can genotype individual zebrafish embryos and adults (by tail-clip) and is applicable to other model organisms.