Characterization of Cloned Mitochondrial DNA from the TeleostFundulus heteroclitusand its Usefulness as an Interspecies Hybridization Probe

Abstract

Analysis of mitochondrial DNA endonuclease restriction patterns is a powerful tool for studying related species and variation within species. The ethidium bromide staining technique has limited the number of digestions of mitochondrial DNA per individual. Because32P-end-labeling also imposes severe limitations, we have resorted to cloning the fish (Fundulus heteroclitus) mitochondrial genome in the lambda replacement vector EMBL-3. The clone was used as a radioactive probe via Southern blotting to detect mitochondrial DNA restriction fragments obtained by multiple restriction endonuclease digestions from small amounts of tissue. This technique offers much greater sensitivity than ethidium bromide staining. Moreover, it eliminates the expense and time to obtain highly purified mitochondrial DNA for the32P-end-labeling procedure. It is also useful when the mtDNA is prepared from frozen tissue which has been a problem with the32P-end-labeling technique. Because the cloned mitochondrial DNA has a high degree of cross-hybridization with the mitochondrial DNA of certain other fishes, it can be used to probe the mitochondrial DNA restriction patterns of a variety of fish species. However, its usefulness is restricted by the degree of relatedness to the species being cloned.