Multienzyme nested deletion (MEND) technique for creation of unidirectional deletions in cloned DNA

An alternative method to construct nested unidirectional deletions of cloned DNA is presented. The multi-enzyme nested deletion technique exploits the fact that the 6-base recognition enzyme ClaI produces termini compatible with at least five restriction enzymes that have different 4-base recognition sequences. A plasmid deletion cloning vector with a ClaI site in the polylinker is linearized with ClaI and then cut with a second enzyme to yield incompatible ends. Directionally cloned insert DNA (in this example SfiI and NotI) is partially digested with one or several of the 4-base recognition enzymes AciI, HinP1I, MaeII, MspI, or TaqI. An aliquot of this partial digest is ligated into SfiI/ClaI cut vector to yield deletions from the NotI end of the insert. Another aliquot is ligated into ClaI/NotI cut vector to yield deletions from the SfiI end of the insert. In general, one of these 4-base cutters will yield deletion clones useful for manual DNA sequencing (i.e., deletions of 300-400 bp or less). It is also simple to perform partial digestion with two enzymes simultaneously if required.