48] A general method for defining restriction enzyme cleavage and recognition sites

Abstract

This chapter discusses a general method for defining restriction enzyme cleavage and recognition sites. Class II restriction endonucleases cleave double-stranded DNA into specific fragments. These enzymes are powerful tools for the dissection of DNA; and they are essential for the construction of recombinant DNA molecules and for DNA sequence determination. These recognize specific sequences of nucleotides, four to six nucleotide pairs long. The wide variety in types of fragment termini has resulted in a number of strategies for characterizing the cleavage sites. These are based on the principle of 32 P-labeling the ends of a mixture of fragments produced by a given enzyme followed by determination of the nucleotide sequence at the labeled ends. The characterization of one cleavage site is sufficient to define a restriction enzyme recognition site, especially if the prediction is confirmed by the fragments produced using a DNA substrate whose sequence is completely known. In other cases, where the cleavage site is offset from the recognition site, a small number of sites must be compared.