E4 - Restriction Endonuclease Analysis

Abstract

This chapter focuses on restriction endonuclease analysis (REA). REA has become a most useful taxonomic tool, facilitating the identification and classification of mycoplasmal isolates as well as providing means for evaluating the degree of genotypic heterogeneity of strains within established species. REA provides valuable information on the type and number of specific nucleotide sequences in the genome, serving as a basis for construction of physical genomic maps. The great advantage of chromosomal REA is that it is universally applicable and sensitive because the entire genome is evaluated for restriction fragment length polymorphism (RFLP) and, in addition, the test is relatively easy to perform. The method involves comparison of the number and size of fragments produced by digestion of the chromosomal DNA with a restriction endonuclease that cuts DNA at a constant position within a specific recognition site, usually composed of 4 to 6 bp. Because of the high specificity of restriction endonucleases, complete digestion of a given genomic DNA with a specific restriction endonuclease provides a reproducible array of fragments. Separation of the fragments by agarose gel electrophoresis and staining with ethidium bromide provide a restriction pattern that can be compared with that of related strains.