Multienzyme Nested Deletion (MEND) Technique for Creation of Unidirectional Deletions in Cloned DNA

Abstract

An alternative method to construct nested unidirectional deletions of cloned DNA is presented. The multienzyme 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.