Methyltransferases as tools to alter the specificity of restriction endonucleases

Pulsed-field gel electrophoresis (PFGE) has allowed the resolution of very large DNA fragments from any organism. To apply PFGE to practical problems, such as genetic mapping and map-based gene cloning, it is necessary to specifically generate large DNA fragments that can then be separated by PFGE. Ideally, restriction enzymes would exist that could generate DNA fragments of desired sizes. Other factors being equal, and supposing that DNA sequences were random, then enzymes with larger target sequences should produce larger DNA fragments. In practice, no restriction enzymes are known to have larger than 8-bp-long target sites and, of course, DNA sequences are not random. These realities severely limit the observed sizes of DNA fragments produced by restriction enzymes acting on genomic DNA, particularly in the case of eukaryotic genomes, which are large and complex. This chapter describes enzymatic strategies to generate large DNA fragments and statistical tools that can aid researchers in choosing the restriction enzymes that are most likely to generate large fragments in the genome in question, if a sequence data base can be investigated. A recent review by McClelland and Nelson (1) focuses on the same technology as described here.