Constructing nested deletions for use in DNA sequencing

Nested deletions useful for dideoxy DNA sequencing are a set of deletions originating at one end of a target DNA fragment and extending various lengths along the target DNA. Each successively longer deletion brings "new" regions of the target DNA into sequencing range (about 300 bp for normal sequencing gels) of the primer site for a general discussion of nested deletions in DNA sequencing). Two protocols for generating nested subclones via enzymatic digestion are included in this unit. In the first, a set of nested deletions is generated by exonuclease III. The primary advantage of this method is that the deletion products generated from the original clone can be recircularized to generate functional plasmids and thus do not require subcloning into another vector. An alternate method utilizes Bal 31 nuclease to generate the deletions. This method requires subcloning of the deletion fragments into a separate vector for subsequent use. Both methods require the presence of unique restriction sites in the vector that are not present in the insert DNA. Bal 31 can also be used to generate nested deletions for chemical sequencing in conjunction with specialized chemical sequencing vectors.