Achilles cleavage of fungal chromosomes

ID: 115

Group:

Fungal Molecular Biology

Prepared by:

Mark L Farman, Univ of Wisconsin (mlf@plantpath.wisc.edu)

Detail:

Achilles’ cleavage (Koob et al. 1993) is the only physical mapping method that enables determination of the actual distance between two markers in a single step. After a little practice, it is a simple, efficient and routinely applicable method for physically mapping and cloning regions of fungal chromosomes. The principle of the RecA- assisted technique is that when double stranded DNA is incubated with RecA protein, an oligonucleotide and a non-hydrolyzable analog of ATP, a DNA triplex is formed between the oligonucleotide and homologous double stranded DNA. The triplex DNA is insensititive to restriction enzymes and methylases. If the oligonucleotide overlaps a region of DNA containing a restriction site and the triplex DNA is treated with a corresponding methylase, the enzyme methylates all the sites that are not protected. Therefore, when the triplex is inactivated, only one cleavable restriction site remains in the entire genome.

Year prepared: 1993

Date uploaded: 2010-09-23 15:47:34

url:

Laboratory Protocols