Oligonucleotide Activation of the Type IIe Restriction Enzyme Nael for Digestion of Refractory Sites

摘要

Certain restriction endonucleases previously shown to exhibit DNA site preferences have a two-site DNA cleavage mechanism. These type IIe restriction endonucleases include NaeI, NaiI, EcoRII, HpaII and SacII. Because of this two-site mechanism, it isoften difficult or impossible to achieve complete digestion of DNA substrate. Inasmuch as these enzymes are commonly used in molecular biology, a method for enzyme activation to provide complete DNA digestion is useful We have commercialized such a method for NaeI using a double-stranded oligonucleotide containing a modified NaeI recognition sequence. Cleavage of resistant sites requires the presence of a DNA sequence that is more cleavable to bind the activator site. The regions flanking the recognition site on our NaeI oligonucleotide cause it to serve as this more cleavable sequence This activates the enzyme to cleave the resistant sequence in the catalytic site, while the oligonucleotide modification does not allow the activator to be depleted during the reaction. Turbo~(tm) Nael provides for rapid digestion of sites previously found difficult or impossible to completely cleave and does not interfere with subsequent molecular biology techniques that might be performed downstream on the substrate DNA, such as ligation, end-labeling or nick translation.