Expanding the potential of restriction endonucleases: use of hapaxoterministic enzymes

A new class of restriction endonucleases called hapaxoterministic enzymes, hapaxomers for short, has been defined. A hapaxomer cleaves DNA outside the recognition site or within an interrupted "palindrome" at bases which are not specified producing fragments with asymmetric, staggered ends. Such termini are unique; in the general case, the protrusions of a fragment obtained with the aid of a hapaxomer cannot self-hybridize, nor can the fragment be ligated to the vast majority of other fragments produced by the same enzyme. When the fragments generated by a hapaxoterministic enzyme are mixed, they can reassemble in only one configuration--that of the original structure from which they were derived. Hapaxomers, then, are characterized not by their recognition sites, which may be symmetric or asymmetric, but by their cleavage sites. The ability to reunite once-contiguous fragments efficiently means that hapaxomers cleaving DNA at many locations are virtually equivalent to restriction enzymes cutting at unique sites. These properties can be exploited for applications such as site-specific mutagenesis or the isolation of large intact DNA fragments.