Chapter 12 Mapping Zebrafish Mutations by AFLP

Abstract

Publisher Summary Amplified fragment length polymorphism (AFLP) is a robust and efficient method for selectively polymerase chain reaction (PCR)—amplifying genomic DNA restriction fragment length polymorphisms that exist among strains of organisms. This technology is a powerful way to identify markers that are closely linked to mutant loci and facilitate positional cloning of the affected genes. This chapter describes zebrafish DNA isolation methods. AFLP is used to identify markers close to a mutation of interest after testing the relevant markers that have been previously placed on maps of the zebrafish genome. The goal is to identify a marker within at least 1 cM of a mutation to facilitate the shortest possible walk to the gene using genomic clones. The AFLP technique consists of four basic steps: (1) complete digestion of genomic DNA from the offspring of AB/SJD hybrid females, (2) ligation of adapters, (3) selective amplification of a subset of the thousands of possible bands, and (4) gel analysis of the reaction products to detect bands that segregate with the mutation being studied.