Generation of a nested set of deletions using exonuclease III.

Abstract

AbstractExonuclease III (Exo III) will digest double-stranded DNA in a 3′ to 5′ direction if the DNA is blunt-ended or possesses a 5′ overhang. It will not digest if there is a 3′ overhang of three or more bases, or if the 3’ end has had thiophosphate-containing bases incorporated into it. In order to generate a set of insert deletions using Exo III it is necessary to cut the polylinker twice with different restriction enzymes so that the cut end nearest the primer site possesses a 3′ overhang, or has thiophosphate residues at the 3′ end, and the end of the polylinker attached to the insert possesses a 5′ overhang or blunt end. If this can be achieved, digestion with Exo III will result in progressive deletion of the 3′ end of the insert, leaving a single-stranded 5′ overhang that can be removed by treatment with mung bean nuclease (1) or exonuclease VII (Exo VII). The blunt ends thus formed are ligated, a suitable host transformed, and colonies are picked at random and screened for insert size (2), following which a suitable range of inserts is then sequenced.KeywordsInsert SizeDigestion RateMung Bean NucleaseProgressive DeletionLonge InsertThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.