A rapid and efficient method of genotyping zebrafish mutants.
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.
Parant
John M
JM
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.
George
Stephen A
SA
Pryor
Rob
R
Wittwer
Carl T
CT
Yost
H Joseph
HJ
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D020732:Cytogenetic Analysis / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods*; D004526:Efficiency; D004625:Embryo, Nonmammalian; D017353:Gene Deletion; D005838:Genotype; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000032:analysis / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D012190:Retroviridae / Q000235:genetics / Q000502:physiology; D013997:Time Factors; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D050505:Mutant Proteins