Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases.

There are numerous restriction endonucleases (ENases) which are known never to achieve total cleavage of certain unmethylated target DNAs. In addition to EcoRII we found seven ENases (AtuBI, Cfr9I, Eco57I, Ksp632I, NaeI, NarI, and SauBMKI) that were stimulated by oligodeoxyribonucleotide (oligo) duplexes containing enzyme-specific recognition sequences to cut the target DNAs much more efficiently and in most cases even to completion. These enzymes are class-II and class-IIS Enases isolated from different bacterial species and possess a varying number of specific sites in the refractory DNA substrates. For DNA analysis and large-scale preparation of certain restriction fragments where complete digestions are essential we recommend taking into account the fact that various ENases can be activated by specific oligo duplexes to drive restriction digestions to completion.